Methods of detecting contaminants of glucose polymers

FIELD: medicine.

SUBSTANCE: claimed invention relates to medicine, namely to immunology, and can be applied for detection of contaminants in glucose polymers. For this purpose analysis of inflammatory response is carried out in vitro with application of cell line, which represents cell line of either macrophages or differentiable in macrophages, or cell, expressing one or several toll-like receptors (TLR) or NOD-like receptors, selected from TLR2, TLR4 and NOD2. Analysis contains the following stages: (a) placing macrophages in presence of preparation of glucose polymers, which can contain anti-inflammatory contaminants, and measuring cytokine RANTES production, with production of RANTES cytokine indicating to the fact that preparation contains contaminants capable of initiating inflammatory reaction, and (b) placing cell line, which makes it possible to detect activity of innate immunity receptor or several receptors of innate immunity, selected from TLR2 and NOD2, in presence of preparation and detection of signal of reporter gene, bound to said receptor, with detection of said activity or said signal indicates presence in preparation of contaminant, which represents receptor agonist.

EFFECT: application of claimed method makes it possible to detect contaminants of glucose polymers, and addition of components, such as MDP or LPS, in tested sample makes it possible to act synergistically with contaminants, which increases sensitivity and reduces threshold of detection, with synergetic response being registered for RANTES.

28 cl, 5 ex, 23 dwg, 5 tbl

 

The present invention relates to methods for detecting contaminants of glucose polymers, in particular, lines of producing polymers of glucose, more specifically, polymers for peritoneal dialysis.

In a broader sense, this method also allows to detect contaminants of glucose polymers, in particular, lines of producing polymers of glucose for enteral and parenteral nutrition, or even for feeding of newborns.

The object of the present invention is also the need for identification of Pro-inflammatory contaminants.

Technical background of the invention

The applicant company has chosen to develop his invention scope, the known risk of contaminants that can be introduced by means of glucose polymers, these contaminants are responsible for inflammatory reactions, which are extremely harmful to human health, the area of peritoneal dialysis.

Peritoneal dialysis is a type of dialysis, which is the removal of waste products such as urea, creatinine, excess potassium or excess water that the kidneys can not cope or not cope, to clear the blood plasma. This medical treatment is indicated in case of end-stage chronic renal failure.

PR� such intracorporeal cleaning the peritoneum is used as a dialysis membrane. Toxic waste products from the blood pass through the semipermeable membrane of the peritoneum into the solution known as dialysate. The dialysate is introduced into the abdominal cavity through an indwelling catheter. There are two types of peritoneal dialysis:

- CAPD (continuous ambulatory peritoneal dialysis), treatment based on the transmission of four packets of dialysate per day for medical purpose,

- APD (automated peritoneal dialysis), continuous night treatment, which corresponds to approximately 15 liters of dialysate for 8 hours for a medical appointment.

The most commonly used based dialysates consist of a buffer solution (lactate or bicarbonate) with an acidic pH of 5.2 to 5.5) or the physiological pH of 7.4), to which is added electrolytes (sodium, calcium, magnesium, chlorine) and osmotic acid (glucose or polymer of glucose, such as "Icodextrin" that exists in the solution for ambulatory peritoneal dialysis Extraneal®, manufactured by Baxter).

Polymer of glucose, such as Icodextrin, mentioned above, it is preferable to glucose as osmotic agents, since due to its small size, glucose, which quickly crosses the peritoneum, causes loss of osmotic gradient within 2-4 hours of infusion.

Standard polymers of glucose produced by acid or fermentative� hydrolysis of starch from cereals or tubers.

Acid hydrolysis of starch, which is completely random, or enzymatic hydrolysis, which is a bit more orderly, provides a mixture of glucose (monomer) and chains of glucose, which include a very short molecules (oligomers) with a low degree of polymerization (DP) and very long molecules (polymers) with high DP. In addition, polymers of glucose are extremely varied molecular weight.

In a more specific application of glucose polymers for continuous ambulatory peritoneal dialysis quickly became obvious that these starch hydrolysates (a mixture of glucose and oligomers and polymers of glucose) cannot be applied as such.

In European patent application EP 207676 reported that glucose polymers, forming a transparent and colorless solution at 10% concentration in water, with a weighted mean molecular weight (Mw) from 5,000 to 100,000 daltons and the molecular weight (Mn) of less than 8000 daltons, are preferred.

These glucose polymers are also preferably contain at least 80% of glucose polymers of molecular weight ranging from 5000 to 50,000 daltons, slightly or not at all contain glucose or glucose polymers with a DP of less than or equal to 3 (molecular weight 504) and little or no contain polymers g�uCoz with a molecular weight of more than 100,000 (approximately 600 DP).

In other words, the preferred polymers of glucose are glucose polymers with low polydispersity index (the value obtained by calculating the relationship Mw/Mn).

Proposed in the patent application EP 207676 methods for such of glucose polymers with low polydispersity index from hydrolysates of starch are:

or in fractional deposition of maltodextrin with soluble solvent,

in conducting molecular filtration of the same product through different membranes with the corresponding cutoff or exception.

In these two cases, these methods aim to remove simultaneously polymers with very high molecular weight monomers and or oligomers with low molecular weight.

However, these methods are not satisfactory from the point of view of their implementation, and from the point of view of the efficiency and quality of the products that they provide.

In order to develop a method of obtaining a fully water-soluble glucose polymer with a low polydispersity index, preferably less than 2.5, preferably with Mn of less than 8000 daltons and Mw from 12,000 to 20,000 daltons, in this method, there are no disadvantages of the prior art, the applicant company was trying to solve this problem in its Pat�NTE ER 667356 based on hydrolyzed starch, instead of maltodextrin.

Polymer of glucose, obtained by chromatographic fractionation, preferably contains less than 3% of glucose and glucose polymers with a DP of less than or equal to 3 and less than 0.5% of glucose polymers with a DP of more than 600.

In the end, since experts in the field of peritoneal dialysis, it is assumed that these glucose polymers used for their osmotic power, are quite satisfactory.

However, the risks of microbial contamination of these products intended for peritoneal dialysis, are subject to criticism.

Actually it is known that the line of producing polymers of glucose can be contaminated by pathogens or Pro-inflammatory substances in these microorganisms.

Contamination of corn starch or wheat by microorganisms of different types of yeast, mold and bacteria, and more specifically, acid-thermophilic bacterium Alicyclobacillus acidocaldarius (extremophilic bacteria that grow in hot and acidic zones of the line) is described, for example, upon receipt of the starch.

The main risk for the patient receiving such contaminated products, is peritonitis.

Clinical suspicion of peritonitis is diagnosed when there is a cloudy dialysate with different clinical manifestations, namely the pain vividly�e, nausea, vomiting, diarrhea and fever.

Such episodes of peritonitis caused by intraperitoneal bacterial infection, and the diagnosis is usually easily installed through positive dialysate cultures.

"Sterile peritonitis, which is also described as aseptic, chemical or culture-negative peritonitis, in turn, is usually caused by a chemical irritant or foreign body.

After the introduction of Icodextrin for receiving peritoneal dialysis (PD) have been reported cases of aseptic peritonitis, which can be associated with several causes, in particular, with the induction of potentially present proinflammatory substances.

Thus, aseptic inflammatory episodes are the main complications observed after injections of dialysis solutions.

Although some of these inflammatory episodes associated with the problem of the chemical nature (accidental injection of chemical contaminants or incorrect doses of some compounds), most cases are directly related to the presence of contaminants of microbial origin that are present in the solutions used for preparation of dialysis solutions.

Lipopolysaccharides (LPS) and peptidoglycans (PGN) are the main contaminants microbial origin: Jiangsu�I, which pose a high risk of initiation of inflammation, when present in trace amounts.

Standard analyses theoretically allow one to reject the party that contains contaminants of this type and thus pose a risk to health. However, such analyses are not satisfactory, as is still reported as aseptic inflammatory episodes, despite the fact that solutions were announced as safe.

Thus, despite the constant attention from those involved in this area, based on reducing the risk of contamination, in particular by improving their detection, there still remains a need to improve levels of performance in the detection of contaminants that can induce inflammation.

Detailed description of the invention

To the credit of the applicant company was the consideration of the presence of molecules that could exacerbate the inflammatory response induced by other contaminants, in particular, LPS or PGN, especially through the mechanism of the interaction between TLR (toll-like receptors) and NOD (nucleotide-binding protein containing the oligomerization domain)-like receptors. In fact, the only consideration of the effect of selected contaminants in inflammation is simplified.

Unlike LPS, which is with�fight ligand, recognizable type receptors TLR4 (toll-like receptor), PGN (as well as numerous glycolipids and lipopeptides) is a ligand recognized by TLR2 receptor type that induce a weak inflammatory response in models of in vitro and in vivo, thereby implying that these molecules must be present in higher concentrations to be detected.

Thus, on models with the use of mononuclear cells (RVMS, primary monocytes/macrophages or lines of monocytes) LPS induces a significant response at concentrations of approximately 1 ng/ml, whereas it required at least 100-fold higher concentrations of PGN to get a similar response (ratio weight/weight).

In addition, although soluble PGN (Mw≈125 kDa) induce an inflammatory response via activation of TLR2, their products of depolymerization, the minimum structure which remains biologically active, representing muramyl dipeptide (MDP), interact with NOD-like intracellular receptors.

Such derivatives, taken alone, do not show clearly on inflammation in vitro and give a significant answer for values >1 mcg/ml.

On the other hand, the presence of these molecules has a synergistic effect on the inflammatory response through the mechanism of interaction between TLR and NOD-p�like receptors independently of the applied experimental models (mouse, lines of monocytes/macrophages, mononuclear blood cells).

In addition to the products of depolymerization PGN formulated microbial peptides, the prototype of which is the f-MLP (Tripeptide formyl-Met-Leu-Phe), also have significant synergistic activity. Initially these peptides were identified by their chemoattractant activity against leukocytes, although they are not able to induce cytokine response per se.

However, in combination with TLR agonists they also help increase the production of cytokines by sensitization of target cells.

It is therefore important not to ignore these "small molecules" as they may indirectly be responsible for aseptic inflammatory episodes, reinforcing the effects of trace quantities of PGN and/or LPS.

Over the past few years we developed a lot of tests with the use of embryonic cells in order to replace animal models in the analysis of the inflammatory response.

However, these in vitro models are subject to considerable individual variability, which may be responsible for systematic experimental errors.

On the other hand, cell lines of monocytes give a permanent answer, which explains so why in the analysis, currently under development, are increasingly used in cells of this type in culture. About�however, these analyses have the disadvantage of supposing a General inflammatory response to all the contaminants that are present in the form of a mixture in solution, and therefore do not allow to characterize the nature of contaminant.

It is also important to note that the enhanced inflammatory response is visible in respect of cytokines acute phase of inflammation, such as TNF-α (tumor necrosis factor alpha), IL-1β (interleukin-1β) and chemokines such as CCL5 (chemokinesis (C-C motif) ligand 5)/RANTES (regulated upon activation, normal murine T-cells and secreted), but did not see this in IL-6 (interleukin-6). Thus, methods based on the production of the latter (US 2009/0239819 and US 2007/0184496) are not suitable for the detection of contaminants in the form of a mixture in solution.

Thus, the applicant company has reached the following conclusions:

(i) difficult to detect bacterial contaminants present in trace amounts in biological fluids,

(ii) it is important not to limit the detection of PGN and LPS due to synergistic effects,

(iii) the need to develop new methods of detection that are sensitive and reproducible, and

(iv) it is advantageous to apply a sensitive and reproducible detection techniques that are able to characterize the nature of the contaminants.

Thus, the merit of the applicant company was the development �justicelink and effective methods of detection of microbial contaminants with proinflammatory effects, below the threshold of sensitivity of the methods currently used and/or described in the literature, and then to identify the family or even the nature of Pro-inflammatory molecules present in trace quantities in shipments received on the production lines.

In fact, a very sensitive method of measurement of inflammatory responses in vitro will allow us to detain or not to detain the party on the basis of pollution levels that are "not significant" in the sense that these levels will be lower than the levels measured in real time using standard tests.

It will be possible to offer such parties for the manufacture of compositions of solutions for therapeutic use in humans (e.g., peritoneal dialysis (PD).

In addition, identification of the molecules responsible for inflammatory responses, should allow to detect the sources of pollution during production processes and to introduce corrective modification in order to reduce the levels of contaminants or even eliminate them.

The method according to the present invention, thus, relates to a method for detecting Pro-inflammatory contaminants of glucose polymers, in particular polymers for the preparation of a peritoneal dialysis solution comprising �analysis of inflammatory response in vitro.

These glucose polymers can be used for peritoneal dialysis, enteral and parenteral nutrition and feeding of newborns. In one preferred embodiment of the polymers of glucose that can be tested using methods of the present invention, represent Icodextrin or maltodextrins. They can be tested in one or more stages of their production and, in particular, at the level of raw materials, at any stage of their preparation and/or at the final product of the method. They can also be tested in a sample of peritoneal dialysis solution.

As mentioned previously, some molecules of bacterial origin, such as MDP and f-MLP, are weak inducers of inflammation, but they may act in combination or in synergy and increase the response induced by other contaminants.

This property is based on the fact that molecules act through the intervention of the receptor, distinct from the TLR.

In addition to LPS, which reacts with TLR4, the majority of inflammatory molecules with potential that may be present in parties are TLR2 agonists.

These contaminants are difficult to detect, because they are present in low concentrations, and inflammatory response that they will stimulate, often close to background noise�.

Consequently, the presence of molecules with synergistic activity may exacerbate the inflammatory response induced by TLR2 ligands that may be used to detect low doses of contaminants.

Samples contaminated with MDP (NOD2 agonist), f-MLP (microbial peptide receptor ligand) or LPS (to initiate synergies TLR4/TLR2), will result in stimulating the inflammatory response in vitro.

Thus, Pro-inflammatory contaminants, detected using the method according to the present invention, is able to stimulate separately or in combination inflammatory response. In particular, these contaminants may be weak inducers of inflammation, when considered separately, but can induce a significant inflammatory response when they are in combination. Method according to the present invention allows to take into account the effect of the contaminants present in the preparation of polymer of glucose, and not just the specific effect of each of them.

Method according to the present invention includes at least one analysis of the inflammatory response in vitro using cell lines, which can detect at least one factor of the inflammatory response. Preferably the cell line is a cell line of macrophages or differentiate�Yu in macrophages, or cells expressing one or more TLR or NOD-like receptors such as TLR2, TLR4 or NOD2, or a combination thereof.

According to the first embodiment of the cell line used in the analysis of the inflammatory response is the cell line of macrophages or differentiated into macrophages. In particular, this cell line produces TNF-α and the chemokine CCL5/RANTES. Preferably, the analysis is carried out on cells THP-1 differentiated into macrophages.

In one preferred embodiment of the macrophages or cells differentiated into macrophages, in particular cells of the THP-1 differentiated into macrophages, is used at a density of 0.5 to 1×106cells/ml culture medium, preferably from 0.7 to 0.8×106cells/ml, and even more preferably approximately 0.75×106cells/ml.

Analysis of the inflammatory response in vitro may be based on the production of TNF-α and/or of the chemokine CCL5/RANTES by macrophages, in particular, cells of the THP-1 differentiated into macrophages, provided that a synergistic effect (an effect obtained by the combination of these various contaminants) is noticeable mainly in respect of cytokines acute phase of inflammation (TNF-α, IL-1β, chemokines), but not against cytokine slow phase, such as IL-6.

Thus, according to one specific embodiment osushestvlenie� analysis of the inflammatory response is to place the cells of the cell line, preferably macrophages, in the presence of a preparation of glucose polymers that may contain Pro-inflammatory contaminants, and to measure the production of cytokines acute phase of inflammation, in particular, TNF-α, IL-1β and/or chemokines, in particular, CCL5/RANTES, the production of these cytokines indicates that the preparation contains contaminants that can stimulate an inflammatory response. In one particularly preferred embodiment of the analysis involves measuring the production of TNF-α and/or CCL5/RANTES, preferably CCL5/RANTES. Analysis of the cytokines may be performed by any method well known to those skilled in the art, and, in particular, by using ELISA. In one preferred embodiment of the analysis involves measuring the production of TNF-α after 8 h of stimulation. In another preferred embodiment of the analysis includes a measurement of the RANTES production after 20 h of stimulation, in particular, by using ELISA.

To enhance the cell response induced proinflammatory contaminants, for example, LPS and/or PGN, a component that allows to act in synergy with contaminants, can be added to the tested sample. In fact, this may allow to detect low doses of contaminants. Preferably, this component may represent from�Oh MDP or related molecule (N-glycolyl-MDP, L18-MDP), formulated microbial peptide (f-MLP) or LPS. Preferably this component is MDP, f-MLP or LPS. Even more preferably, this component is MDP or LPS. In particular, LPS represents LPS from E. coli.

In one preferred embodiment, the implementation of the MDP, in particular, MDP from S. aureus, was added to the sample. Preferably, the MDP is added to the sample in concentrations greater than 1 μg/ml, preferably in a concentration of from 1 to 100 μg/ml. In one of the most preferred embodiments MDP is added to the sample at a concentration of 10 μg/ml.

In another preferred embodiment, the implementation of f-MLP is added to the sample at a concentration of more than 10 nm, preferably at least 50, 100, 150, 200, 300, 400 or 500 nm.

In yet another preferred embodiment of the LPS, in particular LPS from E. coli, can add to the sample at a concentration of at least 10 PG/ml, e.g., at a concentration of 25 PG/ml.

In one preferred embodiment of the test drug polymer of glucose has a concentration of polymer of glucose from 5 to 50 mg/ml, preferably from 5 to 10, 20, 30, or 40 mg/ml. In one specific embodiment of the test drug glucose polymer has a polymer concentration of glucose of about 5 mg/ml. In one preferred embodiment of the test drug polymer of glucose has �Concentratio polymer of glucose is approximately 25 mg/ml.

Optional sample preparation of polymer of glucose can be processed by mutanolysin before analysis. This enzyme is due to its muramidase activity capable of depolymerization PGN. For example, the enzyme at a concentration of approximately 2500 Units/ml can be placed in the presence of the sample, optionally diluted so that the concentration of polymer of glucose ranged from 7.5 to 37.5% (weight/volume), for 6 to 16 hours, preferably about 16 h the thus Treated sample is then subjected to analysis by macrophages according to the present invention. Not necessarily the result, i.e. the production of cytokines, obtained with the sample treated with mutanolysin, can be compared with the result obtained without treatment.

Not necessarily in another alternative sample preparation of polymer of glucose can be filtered before analysis. The purpose of this filter is essentially the removal of molecules with high molecular weight, such as high molecular weight PGN, and the analysis is performed on the filtrate in order to analyze contaminants in a special way small size. The cutoff for filtering can be, for example, from 30 kDa to 150 kDa, preferably from 30 to 100 kDa, or from 30 to 50 kDa, and in particular approximately 30 kDa. Preferably the filtration is carried out by ultrafine�the radio. It can also be by any method known to those skilled in the art. Thus, filtered in this way the sample, the filtrate is subjected to the analysis with the help of macrophages according to the present invention. Not necessarily the result, i.e. the production of cytokines obtained filtrate, can be compared with the result obtained without filtering or prior to filtration. This will allow you to set specific contribution to the inflammation of molecules of small size.

In one preferred specific embodiment of the method has one or more of the following characteristics:

cell line used in density from 0.5 to 1×106cells/ml, preferably from 0.7 to 0.8×106cells/ml, and even more preferably approximately 0.75×106cells/ml; and/or

the polymer concentration of glucose less than 50 mg/ml, preferably about 25 mg/ml; and/or

- production of cytokines was measured after 20 h of stimulation for RANTES and/or 8 h of stimulation for TNF-α; and/or

- MDP added to the drug glucose polymer in a concentration of from 1 to 100 μg/ml, preferably about 10 μg/ml.

Preferably the method includes all features.

Thus, in one of the most concrete mode of the present method includes:

- placement of macrophages in presence�Wii for about 20 h of drug polymer of glucose, which may contain Pro-inflammatory contaminants, in the presence of MDP, preferably in a concentration of from 1 to 100 μg/ml, the concentration of the polymer of glucose of less than 50 mg/ml, preferably about 25 mg/ml, and macrophages with a density of 0.5 to 1×106cells/ml, preferably from 0.7 to 0.8×106cells/ml, and even more preferably approximately 0.75×106cells/ml; and

- measurement of the production of CCL5/RANTES, producing CCL5/RANTES, indicating that the preparation contains contaminants that can stimulate an inflammatory response.

Quite specifically, this first variant of implementation can detect contamination of the polymer of glucose PGN and/or LPS, preferably PGN average size (in particular, approximately 120 kDa) and/or LPS, more particularly LPS.

In particular, the method may include quantification of the contaminants. For example, a quantitative determination can be made using a curve dose-response. This curve dose-response may, in particular, to be obtained on the same cells under the same conditions with increasing doses of contaminants. Preferably such a curve dose-response can be obtained with increasing doses of LPS.

This first variant of implementation can be implemented in methods according to the present invention individually or in Combi�tion of the second embodiment of the implementation.

According to a second embodiment of the cell line used for analysis of inflammation in vitro, is a line that allows you to detect the activity of one or more receptors of innate immunity.

In particular, this cell line can be obtained by stable transfection with one or more vectors encoding one or more receptors of innate immunity.

The activity of the receptor of innate immunity can be detected, e.g., using a reporter gene that is under the direct control of signaling pathways associated with the specified receptor. Preferably, the reporter gene encodes a colored or fluorescent protein, or encodes a protein whose activity can be measured using a substrate or without it. In particular, the reporter gene encodes alkaline phosphatase.

Thus, the method includes placing one or more cell lines expressing one or more TLR or NOD-like receptors, in the presence of a preparation of glucose polymers and measuring the activity of receptors, in particular, by means of a signal of reporter gene. This signal of reporter gene indicates the presence in the drug contaminant, which is an agonist of this receptor.

Preferred�flax cell line to detect the activity of one or more TLR or NOD-like receptors, such receptors like TLR2, 3, 4, 5, 7, 8, 9 or NOD2. Preferably the cell line to detect the activity of one or more receptors selected from TLR2, TLR4 and NOD2. In one specific embodiment of the cell line expresses the receptors TLR2, TLR4 and NOD2, as well as to detect their activity.

Used cell line can be, for example, the line of HEK-Blue™ (manufactured by the company InvivoGen), modified by stable transfection with vectors encoding the receptors of innate immunity. However, it should be noted that specialists in the art can use other commercially available lines (Imgenex) or they can prepare them.

Such cells may also be simultaneously transfected by a reporter gene, producing, for example, secreterial form of alkaline phosphatase (SEAP: secretiruema embryonic alkaline phosphatase), synthesis of which is under the direct control of the signaling pathway associated with the receptor (the receptor), expressed in the same cell line. In one preferred embodiment of the enzymatic reaction is carried out with the use of the relationship 1:3 analyzed environment to the reagent SEAP (for example, 50 μl of the medium and 150 μl of reagent SEAP). In addition, the reaction time is at least 60 minutes is preferred.

p> Cell lines, for example, be selected from the group consisting of:

a - line HEK-Blue™ hTLR2 (the line that specifically responds to TLR2 agonists),

a - line HEK-Blue™ hNOD2 (line that effectively responds to the products of depolymerization PGN and related molecules (MDP, L18-MDP, etc.) and

a - line Raw-Blue™ line of mouse macrophages, kyota line transfected in such a way that expresses alkaline phosphatase). Line Raw-Blue™ expresses receptors of innate immunity, namely the receptors TLR2, TLR4 and NOD2.

These lines are described in detail below in this description.

In one preferred embodiment, the implementation will be used line expressing TLR2 and allows to detect its activity, and/or line expressing receptors TLR2, TLR4 and NOD2, as well as detecting their activity. As an example, will be used cell lines HEK-Blue™ hTLR2 and/or Raw-Blue™. More specifically, by using method will carry out the analysis using cell lines HEK-Blue™ hTLR2 and Raw-Blue™.

In another preferred embodiment, the implementation will be used two lines, expressing, respectively, TLR2 and NOD2, as well as detecting their activity (separately), and one line expressing receptors TLR2, TLR4 and NOD2, as well as detecting their activity. As an example, will be used cell lines HEK-Blue�� hTLR2, HEK-Blue™ hNOD2 and Raw-Blue™. More specifically, by using method will carry out the analysis using cell lines HEK-Blue™ hTLR2, HEK-Blue™ hNOD2 and Raw-Blue™.

The use of such lines, therefore, allows to replace the analysis of cytokines enzymatic analysis (fosfataza activity), and to identify some of these families of molecules of bacterial origin in accordance with the receptor(s) expressed by a line.

In addition, such lines can detect contaminants at very low thresholds, in particular, agonists for TLR2 (PGN, LTA (lipoteichoic acid), LM (lipomannan), etc.) and NOD2 agonists (products of depolymerization PGN and MDP). Thus, the line expressing NOD2, in particular, HEK-Blue™ hNOD2, allows you to more specifically detect the contamination by the products of depolymerization PGN and MDP, preferably MDP. Line expressing TLR2, in particular, HEK-Blue™ hTLR2 and/or Raw-Blue™, allows most specifically to detect contamination of PGN.

According to this second embodiment of the analysis of the inflammatory response in vitro is to place cells cell line that can detect the activity of one or more receptors of innate immunity, in the presence of a preparation of glucose polymers that may contain Pro-inflammatory contaminants, and measuring the activity of the receptor or the signal of reporter gene�, which is associated with it.

The detection of this activity or this signal indicates that the preparation contains contaminants that are able to activate one or more receptors of innate immunity and the initiation of the inflammatory response.

In one preferred embodiment of the test drug polymer of glucose has a concentration of polymer of glucose from 5 to 50 mg/ml, preferably from 5 to 10, 20, 30, or 40 mg/ml. In one specific embodiment of the test drug glucose polymer has a polymer concentration of glucose of about 5 mg/ml. In a preferred embodiment of the test drug polymer of glucose has a concentration of polymer of glucose approximately 37.5 mg/ml when using cells HEK-Blue™ hTLR2 and/or HEK-Blue™ hNOD2. In another preferred embodiment, the implementation of the test drug polymer of glucose has a concentration of polymer of glucose approximately 50 mg/ml when using cells Raw-Blue™.

Optional sample preparation of polymer of glucose can be processed by mutanolysin before analysis. This enzyme is due to its muramidase activity capable of depolymerization PGN. For example, the enzyme at a concentration of approximately 2500 Units/ml can be placed in the presence of the sample, optionally diluted so that the concentration of p�therefore polymer of glucose ranged from 7.5% to 37.5% (weight/volume), within 6 to 16 hours, preferably about 16 h. the thus Treated sample is then subjected to the methods according to the second embodiment of the. Not necessarily the result obtained in the sample treated with mutanolysin, can be compared with the result obtained without treatment.

Not necessarily in another alternative, the sample preparation of polymer of glucose can be filtered before analysis. The purpose of this filter is essentially the removal of molecules with high molecular weight, such as high molecular weight PGN, and analysis on the filtrate in order to analyze the most detailed small size contaminants. The cutoff for filtering can be, for example, from 30 kDa to 150 kDa, preferably from 30 to 100 kDa, or from 30 to 50 kDa, and in particular approximately 30 kDa. Preferably the filtration is carried out by ultrafiltration. It can also be performed by any method known to those skilled in the art. Thus, filtered in this way the sample, the filtrate will be subjected to the methods according to the second embodiment of the. Not necessarily the result obtained with the filtrate, can be compared with the result obtained without or before filtration. This will allow you to set specific contribution to the inflammation of molecules of small size.

In one p�edocfile and specific variant of the implementation of this second embodiment of the method has one or more of the following characteristics:

cell line used at a density of approximately 50,000 cells/well of 96-hole tablet for HEK-Blue™ hTLR2 or Raw-Blue™ and 10000 cells/well of 96-hole tablet for HEK-Blue™ hNOD2; and/or

- analyzed the drug polymer of glucose has a concentration of polymer of glucose from 5 to 50 mg/ml, preferably a concentration of polymer of glucose approximately 37.5 mg/ml when using cells HEK-Blue™ hTLR2 and/or HEK-Blue™ hNOD2, as well as the concentration of polymer of glucose approximately 50 mg/ml when using cells Raw-Blue™; and/or

- bringing the drug polymer of glucose into contact with cells of approximately 16 to 24 hours and/or

- the signal of reporter gene SEAP detected with respect to the culture supernatant:the SEAP substrate 20:180, preferably 50:150, preferably through at least 60 minutes of incubation, ideally 60 minutes.

In one specific embodiment of the method includes all these characteristics.

In particular, the method may include quantification of the contaminants. For example, this quantification can be carried out with the use of the curve dose-response. This curve dose-response may, in particular, to be obtained on the same cells under the same conditions with increasing doses of contaminants. Preferably such a curve dose-response can be obtained for cells expressing TLR2 (�reamer, HEK-Blue™ hTLR2 and Raw-Blue™), with increasing doses of PGN and for cells expressing NOD2 (for example, HEK-Blue™ hNOD2), with increasing doses of MDP.

Method according to the present invention may also include the step consisting in identifying contaminant(s) capable of initiating an inflammatory response.

For this cell line, which allows to detect the activity of the receptor of innate immunity or more receptors of innate immunity, as described above, is placed in the presence of the drug polymer of glucose, is subject to analysis. Measure the activity of this receptor or the signal of reporter gene associated with this receptor. The detection of this activity or this signal indicates the presence in the drug contaminant, which is an agonist of this receptor.

Thus, the line expressing NOD2, in particular HEK-Blue™ hNOD2, most specifically allows to detect the contamination by the products of depolymerization PGN and MDP, preferably MDP. Line expressing TLR2, in particular HEK-Blue™ hTLR2 and/or Raw-Blue™, allows most specifically to detect contamination of PGN. In addition, the macrophages, in particular macrophages THP-1, most specifically allow to detect contamination of LPS.

According to one embodiment of the method according to the present invention includes the steps consisting in

(a) �ispolnenii at least one analysis of the inflammatory response in vitro as described above in the first variant of implementation, which consists in placing the cells of the cell line, preferably macrophages, in the presence of a preparation of glucose polymers that may contain Pro-inflammatory contaminants, and to measure the production of cytokines acute phase of inflammation, in particular, TNF-α, IL-1β and/or chemokines such as CCL5/RANTES, the production of these cytokines indicates that the preparation contains contaminants capable of initiation of the inflammatory response, and/or

(b) placement cell line, which allows to detect the activity of the receptor of innate immunity or more receptors of innate immunity, as described above in the second variant of implementation, in the presence of the drug and the detection of the activity of the receptor or the signal of reporter gene associated with this receptor, the detection of this activity or the signal indicating the presence in the drug contaminant, which is an agonist of this receptor.

According to one preferred embodiment of the method according to the present invention includes the steps consisting in

(a) performing at least one analysis of the inflammatory response in vitro, which consists in placing the cells of the cell line, preferably macrophages, in the presence of the preparation of polymers �lukosi, which may contain Pro-inflammatory contaminants, and to measure the production of cytokines acute phase of inflammation, in particular, TNF-α, IL-1β, and/or chemokines such as CCL5/RANTES, the production of these cytokines indicates that the preparation contains contaminants capable of initiation of the inflammatory response, and,

(b) when the preparation contains contaminants, the placement cell line, which allows to detect the activity of the receptor of innate immunity or more receptors of innate immunity, as described above, in the presence of the drug and the detection of the activity of the receptor or the signal of reporter gene associated with this receptor, the detection of this activity or of the signal, indicates the presence in the drug contaminant, which is an agonist of this receptor.

Stages a) and b) this method is performed according to the details provided above. Preferably the present method comprises two stages.

In one preferred embodiment, the implementation will be applied to the cell line expressing the receptor TLR2 and allows to detect its activity, such as HEK-Blue™ hTLR2 and/or line expressing multiple receptors of innate immunity, as described above, in particular, TLR2, TLR4 and NOD2, such as Raw-Blue™. More specifically, by using method will carry out the analysis with �rimeneniem cell lines HEK-Blue™ hTLR2 and Raw-Blue™.

In another preferred embodiment, the implementation will be applied to the cell line expressing the receptor TLR2 and allows to detect its activity, such as HEK-Blue™ hTLR2, the cell line expressing the receptor NOD2 and allows to detect its activity, such as HEK-Blue™ hNOD2, and/or line expressing multiple receptors of innate immunity, as described above, in particular, TLR2, TLR4 and NOD2, such as Raw-Blue™. More specifically, the method will carry out the analysis using cell lines HEK-Blue™ hTLR2, HEK-Blue™ hNOD2 and Raw-Blue™.

This method, therefore, allows not only to detect the presence of Pro-inflammatory contaminants in the product polymer of glucose, but also to obtain information about the nature of these contaminants. He, in particular, to determine whether these contaminants agonists (TLR) or NOD-like receptors such as TLR2, TLR4 or NOD2. According to one preferred embodiment of the multiple lines can be used to provide additional information to that obtained, for example, in the analysis of the cytokine response to differentiated cells THP-1:

a - line HEK-Blue™ hTLR4: this line is specifically respond to TLR4 agonists. It allows, in particular, to analyze LPS;

a - line HEK-Blue™ hTLR2: this line is specifically respond to TLR2 agonists. It allows you, in private�ti, to analyze PGN.

Their application, therefore, allows to determine the contribution of these contaminants in the initiation of inflammatory responses.

In one specific embodiment of the sample preparation of polymer of glucose can be filtered, as described above, preferably with a cutoff of 30 kDa, in particular by ultrafiltration, to remove PGN, in particular, PGN large size. Thus, lipopeptides and glycopeptides small size, which are also agonists of TLR2, stored in the filtrate, and only their answers will be measured in the analysis of the filtrate.

In addition, the processing solutions of lysozyme and/or β-glucanase eliminates PGN and/or β-glucan, and thus to determine the significance of other TLR2 agonists that may be present in the contaminated batches (glycolipids and lipopeptides). In addition, the sample preparation of polymer of glucose can be processed by mutanolysin before analysis, in particular, as described above;

a - line HEK-Blue™ hNOD2: this line is specifically respond to agonists NODS. It allows, in particular, to analyze the MDP.

The use of this line, thus, allows to detect them at low concentrations. This analysis is the most favorable, because the presence of PGN means that the products of its degradation are also present and that they can �to astolat synergistically with TLR agonists;

a - line HEK-Blue™ Null2: this reference line, the application of which is necessary in order to verify that the solution of the polymer of glucose does not induce the production of the enzyme through an internal mechanism;

- line the Raw-Blue™: a line of mouse macrophages, kyota line transfected with the SEAP.

The advantage of this line is that it naturally expresses almost all the receptors of innate immunity.

It serves as a positive control in the analysis, so as to respond to microbial contaminants of any type.

The object of the present invention is also a method for the identification of Pro-inflammatory contaminants.

Analyses of LPS and PGN can carry out by any method known to those skilled in the art. For example, the content of peptidoglycan can be defined according to two tests:

standard analysis of SLP, manufactured by WAKO Pure Chemical Industries Ltd.,

- analysis of SLP-HS, high-sensitivity analysis developed and approved by the applicant company, as described in patent application WO 2010/125315.

These tests have various reagents (reagents SLP, standards PGN) exhibiting biological reactivity to significantly different impurities PGN, and, thus, different performance criteria:

Standard analysis SLP: re�Gent SLP n°297-51501-Micrococcus luteus, the PGN standard n°162-18101.

- Analysis of SLP-HS (high sensitive): a set of SLP-HS n°293-58301 (including standard PGN from Staphylococcus aureus).

Method SLP-HS, developed by the applicant company, is more sensitive. It has a limit of detection (LD) and limit of quantification (LQ) is lower than that of the standard method SLP:

in the analysis of SLP-HS LD is 1 ng/g and LQ 3 ng/g;

in the standard analysis of SLP LD is 20 ng/g and LQ 40 ng/g.

Thus, unless otherwise noted, the quantitative analysis of PGN in accordance with generally accepted methods carried out in the present application using the analysis SLP-HS, since, as will be shown in subsequent examples described below, the analysis of SLP-*HS is more sensitive than the standard analysis of the SLP.

Analyses "inflammatory response", as described above, for example, using cells HEK-Blue™, allow to identify the nature of the main contaminant (LPS, PGN, β-glucans, MDP and related molecules) and to assess their contribution to the induction of the inflammatory response.

Other contaminants that may be present in the tested parties who are essentially glycolipids and lipopeptides, which are TLR2 agonists, or microbial peptides;

- for glycolipids and lipopeptides perform the procedure of fractionation on the basis of their amphiphilic nature, in order to extract and concentrate them.

About�a job with a mixture of chloroform/methanol allows to extract these molecules from solutions of the polymer of glucose and to concentrate after evaporation of chloroform.

Once these molecules are extracted, they are taken in a minimum volume of DMSO (or other solvent, which is non-toxic to cells) and then analyzed in the analysis of the inflammatory response described earlier.

Thus, the use of cell lines, which can detect the activity of the receptor, such as TLR2, for example, lines HEK-Blue™ hTLR2, allows to confirm the presence or absence of trace amounts of TLR2 agonists, non PGN files in batches to be analyzed.

These compounds can also be tested on the model with the use of cells expressing all types of receptors, such as cells Raw-Blue™, but in this case a solution of the polymer of glucose pre-treated with Detoxi-gel to remove trace amounts of LPS.

Depending on the extracted quantities perform a more subtle analysis of contaminants.

In particular, the sample can be filtered. For example, the sample preparation of polymer of glucose can be filtered with a cutoff of 30 kDa, in particular by ultrafiltration. This allows you to remove PGN, in particular PGN large size. Thus, lipopeptides and glycopeptides small size, which are TLR2 agonists, stored in the filtrate, and the filtrate can be analyzed in order to determine the nature of contaminants.

Besides�, treatment with a mixture of chloroform/methanol extraction allows to obtain products, which are then fractionary in a column with a C18 resin and/or carbon column. Then the compounds are eluted using a gradient of water/acetonitrile. Depending on the purity of the fractions and the amount of material finer analysis allows to determine the biochemical nature of these compounds, or even their structure.

For microbial peptides stage ultrafiltration filter, 5 kDa allows to extract them from solutions of the polymer of glucose. If necessary, the coal passing through the column isolates the filtrate from the more hydrophobic compounds by size <5 kDa.

These solutions are further concentrated and then tested in respect to their biological properties. Since these peptides are chemoattractants for leukocytes, their presence can be detected in the analysis of cell migration in vitro, is available in the laboratory.

It is possible that the glucose polymers contaminated with other molecules known as initiators of the inflammatory responses, such as flagellin, which is a protein TLR5 agonist, and derivatives of nucleic acids and related molecules, agonists of TLR3, 7, 8 and 9 (the first three are involved in reactions with compounds of viral origin, while TLR9 is activated by bacterial DNA origin).

In this �inappropriate cell line, which can detect the activity of different TLR, in particular, the line of HEK-Blue™, are available and can be used to analyze the effect of these molecules on the inflammatory response.

Moreover, the presence of oligonucleotide compounds can be confirmed or excluded by biochemical analysis.

A method according to the present invention can detect contaminants of glucose polymers for peritoneal dialysis, and these contaminants that are able to act in synergy with each other in order to stimulate an inflammatory response, characterized in that it includes at least one analysis of the inflammatory response in vitro using modified cell lines.

In one specific embodiment of the present invention also provides a method for quantifying PGN in the sample, in particular of glucose polymers for peritoneal dialysis, which includes incubation of the sample with the cell line that can detect the activity of the receptor TLR2 and measure the activation of the signaling pathway associated with TLR2, thus allowing to determine the number PGN contained in the sample.

In particular, this line is a line, modified by transfection (preferably stable transfection) vector encoding the receptor TLR2. Preferred�to accept this line expresses not other receptors of innate immunity. In addition, this line may contain a reporter gene which is under the direct control of the signaling pathway associated with the receptor TLR2. Preferably, the reporter gene encodes a colored or fluorescent protein or encodes a protein whose activity can be measured using a substrate or without it. In particular, the reporter gene encodes alkaline phosphatase. These cells can be simultaneously transfected by a reporter gene, producing, for example, secreterial form of alkaline phosphatase (SEAP: secretiruema embryonic alkaline phosphatase), synthesis of which is under the direct control of the signaling pathway associated with the receptor TLR2. This line can be, for example, a line of HEK-Blue™ hTLR2.

To determine the number PGN contained in the sample, by measuring activation of the signaling pathway associated with TLR2, while curve dose-response calibration range containing increasing concentrations of PGN, preferably PGN from S. aureus.

Before analysis the sample may optionally be partially purified in order to remove, for example, any adverse contaminants. Glycopeptides and lipopeptides can be removed from the sample by extraction with chloroform. After centrifugation, the analysis will wire�tsya in the aqueous phase, from which lipophilic contaminants are removed in the usual way. Can be performed fractionation on microconcentrators with the filters cut-off from 30 to 50 kDa, after which the analysis is performed on the UF-retentate. Pretreatment β-glucanases may improve the quality of the analysis by eliminating related molecules.

Alternative and preferably, the test sample is treated prior to analysis by mutanolysin. For example, an enzyme at a concentration of approximately 2500 Units/ml can be placed in the presence of the sample is preferably diluted so that the concentration of polymer of glucose ranged from 7.5% to 37.5% (weight/volume) if necessary. Treatment can last from 6 to 16 hours, preferably for about 16 h. In a preferred embodiment of the treatment is carried out for about 16 h at about 37°C in the sample with a polymer concentration of glucose of about 7.5% (weight/volume). The thus treated samples are then brought into contact with cells expressing TLR2, in particular, cells HEK-Blue™ hTLR2. Not necessarily the result obtained in the sample treated with mutanolysin, can be compared with the result obtained without treatment.

A similar method can also be carried out to detect the contaminant of polymers CH�goats for enteral and parenteral nutrition, or even for feeding of newborns.

The present invention would be better understood through the following examples, which are intended to be illustrative and non-limiting.

Brief description of graphic materials

Figure 1: Production of RANTES in response to PGN and LPS in cells THP-1 sensitized MDP at a concentration of 10 μg/ml.

Figure 2: Production of TNF-α in response to PGN and LPS in cells THP-1 sensitized MDP at a concentration of 10 μg/ml.

Figure 3: Production of RANTES in response to PGN in cells THP-1 sensitized to f-MLP (10 nm).

Figure 4: Production of RANTES in response to PGN in cells THP-1 sensitized LPS (25 ng/ml).

Figure 5: Effect of polymer concentration of glucose on the production of RANTES, PGN induced in cells THP-1.

Figure 6: Effect of concentration of cells THP-1 on the production of RANTES induced by PGN.

Figure 7: Production of RANTES sensitized cells THP-1 in response to various samples of polymer of glucose.

Figure 8: Analysis of the response of cells HEK-Blue. Cells stimulated with PGN, MDP and TNF-α, which is a positive stimulation control cells HEK-Blue™.

Figure 9: Response SEAP depending on the volume of culture supernatant added to the reaction medium. Activation of the cells (HEK-TLR2) performed with the use of TNF-α.

Figure 10: Optimization of reaction time between SEAP � its substrate. Cells HEK-TLR2 (Fig.10A) and Raw-Blue (Fig.10B) stimulated in the presence of increasing concentrations of PGN. After 20 h of stimulation, the supernatants containing the SEAP, were incubated in the presence of a solution of Quanti-blue in one of these times and then measured the optical density at 620 nm.

Figure 11: Effect of polymer concentration of glucose on the production of SEAP cells HEK-TLR2 (Fig.11A) and Raw-Blue (Fig.11B) in response to increasing concentrations of PGN.

Figure 12: Comparison of responses SEAP cells HEK-NOD2, cultivated according to the method provided by the supplier as compared with advanced stage procedure without subculturing prior to stimulation.

Figure 13: Production of SEAP in response to PGN in cells HEK-TLR2 and HEK-Null.

Figure 14: Production of SEAP in response to LTA in the cells of the HEK-TLR2 and HEK-Null.

Figure 15: Production of SEAP in response to PGN and LPS in cells Raw-Blue.

Figure 16: Production of SEAP in response to MDP in the cells of the HEK-NOD2.

Figure 17: the SEAP Activity in the cells of the HEK-TLR2 in response to various samples of polymer of glucose.

Figure 18: the SEAP Activity in the cells Raw-Blue in response to various samples of polymer of glucose.

Figure 19: the SEAP Activity in the cells of the HEK-NOD2 in response to various samples of polymer of glucose.

Figure 20: Production of SEAP in the cells Raw-Blue and HEK-TLR2 in response to various samples of polymer of glucose before and after ultrafiltration at 30 kDa (filtrate).

Figure: Calibration curve cell response depending on the level of PGN from S. aureus. Fig 21A, theoretical curve. Fig 21V, the curve obtained on cells HEK-Blue™-hTLR2.

Figure 22: the SEAP Activity in the cells of the HEK-TLR2 in response to various samples of polymer of glucose before and after treatment with mutanolysin.

Figure 23: Production of SEAP cells HEK-TLR2 in response to PGN before and after treatment with mutanolysin.

Example 1: Preparation of glucose polymers for peritoneal dialysis

Raw materials for the production of glucose polymers according to the present invention is produced from waxy corn starch as follows:

- cleaning the corn in such a way as to retain only whole grains of corn,

- soaking the thus purified maize in the presence of lactic acid to soften the grain,

wet grinding and then the separation of the various components, i.e. the embryo, pulp and husk, protein and starch,

- cleaning of starch in a countercurrent mode with purified water to clean the starch as physico-chemically and bacteriologically,

- centrifugation and drying of starch,

- suspending starch in purified water at the end of the dry matter content of 40% and at temperatures from 45°C to 50°C,

- acidification of starch suspension by adding HCl at pH <2 and the temperature increase from 115 to 120°C for 6 to 8 minutes,

- flocculation of proteins and fats at a given pH,

- �atrelease suspension at pH 5,

filtration of the suspension through diatomaceous earth (to hold residual proteins, fats and cellulose),

- demineralization on a strong cationic resin and a weak anionic resin,

- treatment with activated carbon at a temperature of 70-80°C and at a pH of 4 to 4.5; which removes colored impurities and reduce the level of microbiological impurities.

Powder activated carbon, which is added in a concentration of from 0.2% to 0.5% on dry substance, hold for 10 micron ceramic filter, a pre-filled filter medium,

- concentration by passing through a falling film evaporator,

- spray drying of the concentrated solution in the spray dryer MSD manufactured by Niro.

This starch hydrolysate meets the requirements of the monographs of the European Pharmacopoeia (link Maltodextrins: 1542).

- pH: 4.0 to 7.0 for solution at 10%,

- I. d.: corresponds,

- Loss in drying: max. 6%,

- DE:<20

- Sulphated ash: max 0,5%

- SO2: max 20 ppm

- Heavy metals: < 10 ppm

- E. coli: absent/g

- Salmonella: absent/10 g

- The total content of viable microorganisms: max. 100 CFU/g (ER 1000 CFU/g)

- Fungi: max. 100 CFU/g

In addition to that received shipments analyzed based on the values:

- contamination with yeast + Plyos�the commodity mushrooms: max. 150 CFU/10 g, i.e., max 15/g

- aerobic microorganisms: max 500 CFU/10 g, i.e. max. 50/g

- endotoxin (gel clot LAL-analysis of final results): 20 max. EE/g

- peptidoglycans: max. of 2700 ng/g

Conditions for obtaining glucose polymers in accordance with the present invention from the starch hydrolysate thus obtained are as follows:

1) Water treatment/water quality

- water purification by filtration through 3 μm, treatment with activated carbon, demineralisation on cation and anion exchange resins and filtering (UA),

- use two containers:

- 10 m3for the dissolution of starch hydrolysate and stages of washing with the spray drying and cleaning,

- 60 m3for the main method (cleaning of tanks, slurries of activated carbon and chromatography).

3) Chromatography

- solubilisate hydrolyzate of starch purified water so as to achieve a solids content of 35-45% at a temperature of from 60 to 85°C

- sterilizing grade filtration of the hydrolysate of starch by passing it through 0.45 μm and 0.22 μm is carried out at ΔP<3 bar

- separation by size exclusion chromatography (SEC) is carried out using a continuous system consisting of six series of double plates, each of 1 m3resin. Apply resin PCR145K manufactured by the Purolite company.

The solution, which is cool�dit through this resin, has a temperature of from 75 to 85°C solids content of 35-45%.

The duration of each sequence defines the method.

In this case, the duration of each sequence is 15 minutes.

The control is carried out using analysis of molecular weight distribution and chromatographic analysis of output as follows: (number of desired dry matter fraction) / (number of dry raw material).

The lowest molecular weight interact with the resin and the high molecular weight of the eluted purified water.

Concentration is carried out by evaporation of falling film in the dry matter content of 35-45%.

The heat treatment is conducted at a temperature of 120°C for 2 minutes.

Activated carbon is added from 0.5% to 1.5% of the total weight of hydrolyzed starch at 75°C with cationic resins (1 to 3 l) to adjust pH (4-4,5) and anionic resins (5 to 10 l) to adjust pH to 5.5-6).

Spend filtering through a polypropylene batch filters with ΔP<5 bar 5 to 6 hours per batch.

Spend the second and third filtering through 1.5 and 0.45 μm, and then through 0.22, and 0.1 µm and ultrafiltration through a membrane with a cutoff of approximately 40000 Yes.

For spray drying: the raw material is supplied at 500 kg/h in the solution with a dry matter content of 40% and �ri 250°C the spray dryer MSD manufactured by Niro.

With spray-dried product is at the output of the moisture content less than 6%.

The product is then cooled in a fluidized air layer, containing three of the cooling zone supplied with air at 40, 30 and 20°C. Then the resulting product is sieved through an 800 μm to remove aggregates.

Approximately 500 kg of the final product is prepared from 800 kg of maltodextrins, i.e. the output is approximately 60%.

Determination of possible contamination of the line is carried out by content analysis of peptidoglycans and endotoxin levels in the final product.

For example, the content, usually observed and measured in batches of the final product (expressed in grams of polymer of glucose) is, for the criteria listed above, the following:

- yeasts and moulds: 0/g

- aerobic microorganisms: 0/g

- endotoxin (gel clot LAL-analysis of final results): ≤0,3 EE/g

- peptidoglycans: <3 ng/g

- B. acidocaldarius: 1/g

Example 2: Use "sensitized" cell line THP-1 for the detection of Pro-inflammatory contaminants

Materials and methods

Cells THP-1 (88081201, ESASS) cultivated in the laboratory in the prescribed manner.

For experiments on Pro-inflammatory activation of cells THP-1 differentiated for 3 days in the presence forblog ester (PMA). An hour�ness, cells are cultured in 200 μl complete medium in the presence of 20 nm PMA for 72 h (final cell density: 0,75×106cells/ml).

Samples of polymer of glucose receive according to Example 1.

Table 1
Samples of polymer of glucose
I-10.01I-10.02I-10.03I-11.12MM-10.04MM-10.05MP-10.06MP-10.07
LAL-analysis (EE/g)<0,30,3<0,30,61,29,6<0,338,4
Standard analysis of SLP (ng/g)<2075527530<202755<204613
Analysis of SLP-HS (ng/g)<3 25312393<3501<3645

Standard molecules for the establishment of calibration ranges are:

- LAL analysis: LPS from E. coli O55B5

- Standard analysis SLP:PGN from M. luteus - Wako

- Analysis of SLP-HS:PGN from S. aureus - Wako.

Analyzed values PGN differ from a single test Wako to another because of differences in reactivity and sensitivity of these tests, perhaps because of their limited and relative specificity (in particular, a possible response to β-glucan).

Optimization studies were performed with solutions of glucose polymer (I-10.01 (table 1), artificially supporting standard inflammatory molecules: PGN and MDP (source: S. aureus), LPS (source: E. coif), f-MLP (synthetic peptide).

Solution for diluting standards is I-10-01<0,3 EE/g LPS (LAL test), <20 ng/g PGN (standard analysis SLP) and <3 ng/g (analysis of SLP-HS), used at a final concentration of 5 mg/ml.

Then carry out the analysis with the first series of samples corresponding to the different parties, selected on the basis of the levels of contamination of impurities, measured by the LAL assays and SLP (PGN, LPS and β-glucan).

The ELISA kits for the quantitative analysis of TNF-α and CCL5/RANTES acquire from AbCys standard agonists (PGN, LPS, f-MLP and MDP) from Sigma Aldrich and InvivoGen.

Differentiable cells THP-1 (0,75×106cells/ml) are cultured in 200 μl of complete medium and then incubated in the presence of various test samples.

Each analysis is carried out in three replicates.

Cell supernatants are harvested in order to analyze the secretion of cytokines after 8 h of stimulation for TNF-α, and after 20 h for RANTES.

The ELISA tests performed in accordance with the instructions provided by the vendor.

Results

The first analyses was to explore the synergistic potential of MDP and f-MLP, as well as combinations PGN/LPS relative to the production of proinflammatory cytokines differentiable cells THP-1.

MDP was analyzed in doses of 1, 10 and 100 μg/ml. the Minimum dose induced a significant synergistic effect. On the other hand, doses of 10 and 100 μg/ml had similar synergistic activity relative to the production of RANTES and TNF-α in response to PGN and LPS.

The results presented in figures 1 and 2 clearly demonstrate the synergistic effect of MDP on the production of RANTES. On the other hand, this synergistic effect is not very pronounced for TNF-α. In addition, the detection threshold (the number of PGN or LPS, causing a response greater than three SD of background noise) for RANTES is lower (see table 2).

f-MLP was used in doses of 1 nm, 10 nm and 100 nm. However, no synergistic EF�EKTA on cells THP-1 were observed (figure 3).

the synergistic effect of LPS was analyzed by adding a sub-optimal dose (25 PG/ml) to increasing doses of PGN (figure 4). Synergistic effects of these two agonists reveal after quantitative analysis of the two cytokines. However, LPS induced a synergistic effect on RANTES production remains less than the effect induced MDP.

The answer is quantitatively determined by measuring the production of RANTES and TNF-α. In all cases, the synergies are clearly identified for the analysis of RANTES with high sensitivity. This analysis, therefore, will be preferred and will persist in the remaining experiments.

These results demonstrate that the addition of MDP at a concentration of 10 μg/ml to the cells THP-1, in turn, with the quantitative analysis of RANTES, is the most effective way of sensitization to detect low levels of PGN and LPS. Theoretically, these detection thresholds should allow to detect contaminants present in industrial products.

These first tests were carried out by diluting the standard of inflammatory molecules in the presence of polymer I-10.01. The solutions were added to the cells THP-1 thus, to obtain the final concentration of polymer of glucose 5 mg/ml final cell density of 0.75×106cells/ml in order to increase the sensitivity of the analysis, tests conducted from�emeniem these two parameters.

The presence of glucose polymer does not preclude the production of RANTES at concentrations less than or equal to 25 mg/ml. This increase in sensitivity is not associated with proinflammatory effect of the polymer on the cells, since the response is identical to the background noise in the absence of PGN (figure 5).

On the other hand, the increase of cell density higher than 0.75×106/ml reduces the production of RANTES in response to PGN (depletion of the culture medium or the modification of cells) (figure 6).

Sensitivity analyses with MDP at a concentration of 10 μg/ml reconstituted three times for LPS and six times for PGN cells THP-1, originating from various drugs. The data obtained allowed to determine the detection thresholds and / EC50 (table 2). To assess the sensitivity values are given in g of polymer to the concentration of 25 mg/ml.

The synergistic effect induced MDP, identical to LPS and PGN, because it allows to increase the sensitivity of cells THP-1 5-fold in both cases.

Table 2
Detection thresholds and / EC50 for PGN and LPS on the model of the sensitized THP-1.
PGNPGN+MDPLPS LPS+MDP
Detection threshold14,5±8.5 ng/mlOf 2.8±1.2 ng/ml10±7 PG/ml2±1 PG/ml
EU501,2±0,7 mg/ml0,4±0,2 mg/mlOf 0.9±0.1 ng/mlOf 0.3±0.1 ng/ml
Sensitivity580±340 ng/g112±48 ng/g400±280 ng/g80±40 PG/g

These studies show that sensitized cells THP-1 can be used to develop a sensitive analysis of the inflammatory response to the detection of trace quantities of contaminants in preparations of polymer of glucose.

The following experimental conditions:

- the final concentration of differentiable cells THP-1: 0,75×106cells/ml,

- sensitizing agent: MDP (S. aureus) at a concentration of 10 μg/ml,

- the final concentration of polymer of glucose: 25 mg/ml

- a: ELISA analysis of RANTES production after 20 h of stimulation.

To test this method tests for MDP-sensitized cells THP-1 was performed with the samples presented in table 1.

The analysis based nepodozirana RANTES sensitized cells THP-1, has revealed the polymer samples contaminated LPS: polymers, designated I-11.12, MP-10.07 and, to a lesser extent, MM-10.04 and MM-10.05 (figure 7).

On the other hand, samples contaminated with only PGN (e.g., I10-02) does not give the answer, indicating that this cell-based assay is more suitable for the detection of endotoxins. However, the amplitude of the answers is directly proportional to the level of LPS, measured by LAL analysis (see, for example, the responses received from the I-11.12 compared to MM-10.05), thereby suggesting that other contaminants in addition to PGN may operate with LPS on the response of cells THP-1.

Cells THP-1 was responsible for the control solutions PGN, but weakly or not at all answered on samples from batches that are contaminated only natural PGN.

The size of the PGN is very heterogeneous, and some of these molecules can reach impressive weight (>106Yes). The latter have low solubility, which affects their proinflammatory capacity, but facilitates their removal by filtration. On the other hand, PGN, which are soluble and, accordingly, active, have an average weight of 120 kDa and are not removed by filtration. Thus, we can assume that these two analyses SLP Wako capable General quantitative analysis of all PGN, whereas cell-based assay detects only active PN.

Example 3: Use of cell lines HEK-Blue™ (hTLR2, hNOD2, Null2) and Raw-Blue™ (InvivoGen) for detecting contaminants

Materials and methods

Cell lines HEK-Blue™ (InvivoGen) are lines, modified by stable transfection with vectors encoding the receptors of innate immunity. These cells simultaneously transfected by a reporter gene that produces secreterial form of alkaline phosphatase (SEAP: secretiruema embryonic alkaline phosphatase), synthesis of which is under the direct control of the signaling pathway associated with the receptor(s) expressed in the same cell line.

For experiments involving the detection of inflammatory contaminants, use four lines:

a - line HEK-Blue™ hTLR2 (HEK-TLR2): this line is specifically respond to TLR2 agonists (in particular, PGN and most of glycolipids and lipopeptides),

a - line HEK-Blue™ hNOD2 (HEK-NOD2): this line is specifically responds to MDP and related molecules such as monomers PGN,

a - line HEK-Blue™ Null2 (HEK-Null): this control line, the application of which is necessary in order to verify that the solution of the polymer of glucose does not induce the production of the enzyme through an internal mechanism,

- line the Raw-Blue™: a line of mouse macrophages, kyota line transfected with the SEAP. This line, which naturally expresses almost all prescription�ry innate immunity, used as a positive control in the tests.

Cells Raw-Blue™ or HEK-Blue™ are cultured according to the supplier's recommendations. In particular, the selection pressure for the plasmid encoding the receptors of inflammatory molecules (TLR2 or NOD2) and encoding SEAP, provide the addition to the culture medium of antibiotics HEK-Blue Selection/blasticidin. At 75% confluence of cells was resuspended at a density of cells of 0.28×106cells/ml Before stimulation with 180 ál of the cell suspension is distributed into the wells for cultivation (96 well plate), i.e. 50,000 cells/well. Then the cells are stimulated for 24 h by adding 20 ál of the test samples (ten times concentrated form). Stimulation lasts from 16 to 24 h.

Production of SEAP in response to contaminated molecules evaluated by measuring phosphatase activity according to the Protocol supplied by the manufacturer: 20 µl of culture supernatant was diluted in 180 μl of Quanti-blue. Staining develops at 37°C and read perform at various time points at 620 nm. The results are expressed as the absorption after subtraction of the background noise, obtained by adding the same amount acondicionadores culture medium in the reaction environment of the enzyme.

Optimization studies performed with solutions of glucose polymer (I-10.01 (table 1), artificially supporting standard�nye inflammatory molecules: PGN, LTA and MDP (source: S. aureus) and LPS (source: E. coli). Then carry out the analysis on the series of samples corresponding to the different parties, selected on the basis of pollution levels of PGN and LPS (table 1).

Results

Cells HEK-Blue get in the form of frozen vials. Before beginning the analyses of the inflammatory response necessary to ensure the resumption of cell growth and in their ability to respond to inflammatory molecules. In addition, these transfetsirovannyh cells rapidly degenerate after several passages, which can lead to loss of expression vectors for the receptors of innate immunity, or even vector encoding SEAP.

Thus, it is recommended to check the ability of these cells to respond to inflammatory factors at the beginning of the cultivation and then through several stages of re-planting. These assays were performed with PGN for HEK-TLR2, MDP for HEK-NOD2 and TNF-α, the latter is a potent activator of the way NF-κ. In fact, HEK cells naturally possess the receptor for this cytokine, and thus, will produce SEAP (expression vector which is under the control path NF-κ) in response to TNF-α.

The results presented in figure 8, show the analyses carried out in order to check the resumption of growth of the three lines. As expected, cells HEK-TLR2 and HEK-Null answer of TNF-α is equivalent to the production of SEAP. Cu�IU, line HEK-Null is insensitive to PGN and MDP, whereas cells HEK-TLR2 effectively only respond to PGN. These two lines thus acquired their phenotypic characteristics, and these could be used for the rest of the experiments. In this example, the line HEK-NOD2 responds very poorly on TNF-α and MDP, thus indicating that it has not acquired the expected characteristics.

The response of cells HEK-Blue and Raw-Blue on proinflammatory molecules is directly connected with the production of SEAP. The supplier recommends adding 20 µl of culture supernatant to 180 μl of SEAP substrate. The experiments thus performed in these conditions and then optimize, increasing the volume of culture supernatant and, therefore, the amount of enzyme in solution (figure 9).

The results show that the ratio 50/150 gives higher response than the ratio recommended by the supplier. On the other hand, the ratio of 100/100 is not more effective, perhaps due to the lack of SEAP substrate in the reaction medium.

The intensity of staining (620 nm) is proportional to the amount of SEAP secreted by cells, as well as the time of hydrolysis of the substrate by the enzyme. Various time points for imaging were thus tested using the same supernatants of PGN-activated cells HEK-TLR2 and Raw-Blue (figure 10).

Optimal okra�Ivana is achieved, starting with 60 min reaction between SEAP and its substrate for cells HEK-TLR2. A slight increase is also observed after 90 min for the cells Raw-Blue, but this increase is probably due to the fact that these cells produce less SEAP.

The effect of polymer concentration of glucose on the responses of the cells was analyzed by stimulating cells HEK-TLR2 and Raw-Blue PGN standard, diluted in medium with addition of polymer (I-10.01. Then the solutions were added to the cells so as to obtain the final concentration of the polymer in the range from 5 to 50 mg/ml (figure 11).

Concentrations up to 37.5 mg/ml did not significantly alter the amplitude of the response to PGN in line HEK-TLR2. Improved response to low concentrations of PGN, celebrated for polymer concentrations above 25 mg/ml, probably due to better dispersion contaminant. As for cells Raw-Blue, producing the SEAP does not change regardless of the concentration of the polymer of glucose.

Line HEK-NOD2 shows low amplitude response to MDP and even on TNF-α, which, apparently, is associated with high background noise. In these cells SEAP gene is under the control of a weak promoter, which is more sensitive to cell stress than used in other cells HEK-Blue. In order to reduce background noise and increase the amplitude of the response to contaminants, culturing conditions modified in such a way as to reduce PT�ESS cells.

According to the supplier's recommendations cells at 75% confluence was resuspended at a density of 0.28×106cells/ml and then 180 μl of the cell suspension is distributed into the wells for cultivation (96 well plate), i.e. 50000 cells/well, before stimulation during the day.

In the new procedure, how the procedure without subculturing, the cells of the HEK-NOD2 handle in the hole (10000/well) and cultured for three days. Before stimulation, the culture medium is removed and replaced by medium containing 1% FCS and solutions for analysis. In this case, the answer SEAP 5-6 times more than the negative control, which corresponds to the analysis for the detection of MDP in the contaminated solutions (figure 12).

These early studies indicate that cells HEK-Blue and Raw-Blue can be used to develop sensitive assays inflammatory response to the detection of trace quantities of contaminants in preparations of polymer of glucose.

Chose the following experimental conditions:

- the final concentration of polymer of glucose: 37.5 mg/ml for cells HEK-Blue and 50 mg/ml for cells Raw-Blue,

- enzymatic reaction: 50 μl conditioned medium+150 µl of reagent SEAP,

- minimum reaction time: 60 min.

In the figures 13 to 16 show examples of analyses the activity of SEAP produced by the cells of HEK-TLR2 and Raw-Blue in response to various inflammatory molecules.

Cell� HEK-TLR2 very effectively respond to PGN, then as a response to LTA weaker. These responses, however, are more effective than that observed on the cell type monocytes/macrophages. In addition, a polymer of glucose does not have any direct effect on the production of SEAP, because response is not observed in the supernatants of cells HEK-Null.

Cells Raw-Blue well answered PGN, but less sensitive than cells HEK-TLR2. Response to LPS is weak and remains much lower than the observed in the measurement of RANTES production by cells THP-1.

Unlike other cell lines the cells of the HEK-NOD2 effectively responded to MDP that can be used to detect degradation products of PGN.

Experiments with standard PGN, LPS and MDP were reproduced on different cell preparations, which allowed us to determine the characteristics presented in table 3. To assess the sensitivity values are presented in grams of the polymer to the concentration of 37.5 mg/ml for cells HEK-Blue and 50 mg/ml for cells Raw-Blue.

Table 3
The thresholds of detection and / EC50 for PGN, LPS and MDP models HEK-Blue and Raw-Blue
Line HEK-TLR2Line HEK-NOD2Line Raw-Blue
PGNMDPPGNLPS
Detection threshold0,07±0,04 ng/mlOf 1.5±0.5 ng/ml2,1±1.5 ng/ml0,24±0,06 ng/ml
EU503,1±2.5 ng/ml12±6 ng/ml210±75 ng/ml>10 ng/ml
SensitivityOf 1.9±1.1 ng/g40±13 ng/g42±30 ng/g4,8±1.2 ng/g

Line HEK-TLR2 and Raw Blue are very effective for the detection of PGN in low concentrations. In particular, the line HEK-TLR2 detects the PGN levels below 2 ng/g of polymer of glucose, i.e. a sensitivity 50 times higher than those obtained on sensitized cells THP-1. Line Raw Blue effectively detect a small trace amounts of PGN, but not too reactive to LPS with a detection threshold of approximately 50 times higher than for sensitized cells THP-1.

To check these tests, run the tests with samples of polymer of glucose.

Line HEK-TLR2 on�allows to detect contamination in batches I-10.02, I-11.12 and MM-10.05, and, to a lesser extent, in parties MP-10.06 and MP-10.07 (response observed with MM-10.04, is negligible compared with the I-10.01, and is therefore not saved).

On the other hand, the sample I-10.03 found, which may be associated with a very low level PGN close to the detection limit in the analysis of SLP-Wako (figure 17).

Although the positive responses received from the I-10.02, I-11.12, MM-10.05 and MP-10.07, have been expected, given the high level PGN present in these four samples, it should be noted that there is no proportionality between the concentration obtained in the analysis of the SLP, and the response of the cells.

In fact, glucose polymers I-10.02 and I-11.12 give the highest responses, while the PGN levels are less than 1 ág/g. on the other hand, the sample MP-10.07, which has the highest load PGN, gives an answer close to the background noise. PGN are macromolecules with a very diverse lot, and it was shown that their reactivity is inversely proportional to molecular weight. Thus, we can assume that PGN from I-10.02 and I-11.12 smaller than PGN present in samples of MM and MP 10.05-10.07, which may explain their higher proinflammatory potential.

It's surprising to see the answer with the party MP 10-06 despite the fact that it does not contain PGN. However, cells HEK-TLR2 react with other inflammatory molecules such as lepape�tidy, LTA or LAM (lipoarabinomannan), all of which are TLR2 agonists. Thus, this result suggests that the sample is uncontaminated based on the absence of LPS and PGN, contains other Pro-inflammatory molecules that are agonists of TLR2.

Except for the samples I-10.01 and 10.03, all samples give a positive response with cells Raw-Blue (figure 18).

The level of LPS present in the sample I-10.02, close to the detection threshold LAL-analysis, indicating that the observed response is due to the presence of PGN. This result confirms that unlike cells THP-1 that do not respond to this sample, the cells Raw-Blue are effective for the detection of small contaminants PGN. Strong response parties I-11.12, MM-10.05 and MP-10.07, thus, apparently due to the presence of two contaminants (PGN and LPS).

Like cells HEK-TLR2 cells Raw-Blue positively answered Mr-10.06, which confirms the presence contaminant in addition to LPS and PGN in this sample.

Finally, cells HEK-NOD2 react strongly in the presence of MP-10.07 and give a weak but significant response with samples I-10.03, MM-10.04 and Mr-10.06, which indicates that these samples were contaminated PGN during the stage of production, and that the latter were partially degraded during the production of the product (figure 19).

Cells HEK-TLR2 and Raw Blue are effective d�I detect trace amounts of inflammatory contaminants in products of type I-10.01 and 10.03.

They even have features additional to those of the sensitized cells THP-1. In fact, analyses using three of them to find the most inflammatory contaminants that may be present in samples of polymer of glucose.

- THP-1: any inflammatory contaminant with high reactivity in relation to LPS.

Raw Blue: any inflammatory contaminant with high reactivity against PGN.

- HEK-TLR2: specific for TLR2 agonists with high reactivity against PGN.

- HEK-NOD2: specific for MDP and, respectively, for the products of the degradation of PGN. As in the case of cells THP-1, also noted the lack of proportionality between the levels of PGN and/or LPS and amplitudes cell responses.

This problem is undoubtedly related to the size of these macromolecules and/or their presence in the form of aggregates, which influence their reactivity against cells. Thus, it was found that the passage of the standard PGN through a 0.22 μm filter reduces approximately 50% of its reactivity on cells HEK-TLR2. Therefore, in all tests a solution of the polymer of glucose prepared in aseptic conditions, but without the filtration stage standard molecules.

Example 4: Investigation of contaminants using sensibili�yovanny cell lines THP-1, HEK-Blue™ (hTLR2, hNOD2) and Raw-Blue™

Examples 2 and 3 show that sensitized line THP-1, HEK-TLR2, HEK-NOD2 and Raw Blue are effective for the detection of trace quantities of inflammatory contaminants in samples of polymer of glucose. In addition to LPS and MDP, the presence of which is detected by receptors TLR4 and NOD2, and other contaminants that may be present in the samples are mainly TLR2 ligands (PGN, LTA and lipopeptides). Consequently, the line failed to establish the contribution of PGN in TER-specific response. However, PGN are macromolecules, the weight of which is in the range of ~100 kDa to several million Da. On the other hand, LTA and lipopeptides have low mass, less than 15 kDa. Thus, the introduction of a stage ultrafiltration (30 kDa) should allow to detain PGN and measure response to TLR2 ligands small size.

Experimental procedure

For experiments related to this example, used five cell lines are presented in examples 2 and 3:

- line of monocytes THP-1: response to any inflammatory contaminant with high reactivity against LPS,

- line the Raw-Blue™: the answer to any inflammatory contaminant with high reactivity against PGN,

a - line HEK-Blue™ hTLR2: specific for TLR2 agonists with high reactivity, in respect of� PGN,

a - line HEK-Blue™ hNOD2: specific products for complete depolymerization PGN (MDP),

a - line HEK-Blue™ Null: negative control response.

Samples of the polymer of glucose is shown in example 2 (table 1). These samples are prepared in solution at concentrations described in examples 2 and 3. Cell responses (production of RANTES to cell THP-1 and secretion of SEAP cells for Blue™) analyze or unfiltered samples: the General answer, either with the filtrate obtained by ultrafiltration on microconcentrators with a cutoff of 30 kDa (Sartorius): the Response of the filtrate.

Before applying microconcentrators treated with saline (150 mm NaCl), prepared in non-pyrogenic water. Retentate and the filtrate tested with cell lines, and only filters with a negative response in each trial, retained for analyses with samples of polymer of glucose.

Results

The results presented in figure 20, show the responses of cells HEK-TLR2 and Raw Blue, obtained in the presence of different samples before and after ultrafiltration.

General responses similar to those observed in example 3 (figures 17 and 18).

The responses of the filtrates significantly reduced for samples I-10.02 and I-11.12 on two types of cells with values close to or equal to the thresholds of detection. These data confirm that these two samples are contaminated with PGN, who was arrested �ultram.

The answer filtrate MM-10.05 reduced on cells HEK-TLR2, but not on cells Raw-Blue, indicating that this sample is contaminated by a combination of several molecules, with a large part submitted to PGN.

Samples MM-10.04, MP-10.06 and MP-10.07 slightly contaminated PGN, as the overall response and the response of the filtrate in the cells of the HEK-TLR2 identical. On the other hand, 50% reduction of the response of the filtrate can be noted for MP-10.07 on cells Raw-Blue. LAL-analysis showed that this sample carries LPS. Although the mass of endotoxins is less than 30 kDa, these molecules are able to form aggregates that may be the cause of loss of response after filtering.

General answer and a filtrate analyzed for THP-1, Raw Blue, HEK-TLR2 and HEK-NOD2 cell types. The results are shown in table 4.

For cells HEK-NOD2 General answer and a filtrate is identical to that expected, since MDP and related molecules have a mass much smaller than 30 kDa (Mw~500 Da). Table 4 shows a General answer.

Table 4
Study of contaminants through analysis of the cellular responses. Pollution levels are expressed depending on threshold limits of detection (LOD) and / EC50, defined in examples 2 and 3 for each cell type (tables 2 and 3), with curves dose-response relative� LPS for sensitized cells THP-1, about PGN lines for Raw Blue and HEK-TLR2, and relatively MDP line HEK-NOD2: (-): the level of <LOD; (±): LOD≤level<3×LOD; (+): 3×LOD≤level<0.3 x / EC50; (++): 0.3 X / EC50≤level<3× / EC50; (+++): level≥3×EU50
ResponseI-10.01I-10.02I-10.03I-11.12MM-10.04MM-10.05MP-10.06MP-10.07
THP-1--±++±±+
<30 kDa--±±±±±±
Raw Blue-+±+±+±+
<30 kDa ----±±±+
HEK-TLR2-++±+++±++±
<30 kDa--±-±±±
HEK-NOD2--±-+-+++
The main effectnegativePGNresiduesPGNThe remains, including MDPPGN and balancesThe remains, including MDP LPS and balances

These data allow us to characterize the types of contaminants present in each solution of polymer of glucose, and to establish their contribution to the inflammatory response:

I-10.01: the sample is not contaminated.

I-10.02: the sample is heavily contaminated PGN. The lack of response of cells THP-1 and HEK-NOD2 and filtrates indicates that the contribution of other contaminants immaterial.

I-10.03: a sample of weakly contaminated by residues, probably derived from degradation products of small size. In fact, the overall response and the response of the filtrate are at the limit of background noise in all tests.

I-11.12: the sample is heavily contaminated PGN. The weak response of the cells THP-1 also indicates trace amounts of LPS.

MM-10.04: sample weakly contaminated with LPS and TLR2-activating molecules of small size (LTA, lipopeptides). The presence of MDP also indicates the PGN degradation products.

MM-10.05: the sample is contaminated PGN and other molecules of small size. Weak responses in other tests indicate the presence of trace amounts of LPS and other ligands of TLR2.

MP-10.06: the sample is contaminated by numerous small molecules. On the other hand, the weak responses of THP-1 and HEK-TLR2 indicate the absence of PGN and LPS.

MP-10.07: the sample is contaminated by numerous small molecules with a high proportion of LPS and MDP.

It should be noted that the results are consistent with analyses Wako SLP-S for samples I-10.01, I-10.02, I-10.03 and I-11.12, which are the final products, and samples MM-10.04 and Mr-10.06, which were identified as not containing PGN.

On the other hand, two sample MM-10.05 and MP-10.07 responded PGN weaker-than-expected results SLP. However, it is possible that these samples gave false-positive result in the analysis of SLP due to cross reactions with β-glucan, for example. Another possibility is that these samples contain very large PGN, low solubility which prevents the initiation of the response in the test cells.

Example 5: Method of analysis of peptidoglycans

This analysis is based on the specific recognition of PGN line expressing the receptor TLR2, and on the production of enzymatic activity measured by activating a signaling pathway associated with TLR2.

Experimental procedure

For experiments related to this analysis used two lines:

a - line HEK-Blue™ hTLR2;

a - line HEK-Blue™ Null2.

These two lines shown in example 3.

The construction of the curve dose-response

Curve dose-response was obtained with a standard PGN from S. aureus (figure 21).

Cells HEK-Blue™ were incubated with increasing concentrations of the standard and measure cell response by quantifying enzymatic activity obtained.

The result is the usual sigmoid�th cell response curve:

- part (a) corresponds to the responses obtained with low concentrations of PGN, below those that give effective activation of TLR2. This non-linear zone, thus, corresponds to the threshold detection limit of this method. In order thus to enable the variability of the method, this detection threshold is estimated according to the value of the background noise at three points in time (the response obtained in the absence of stimulus);

- part (b) is the most interesting because there is a linear response. This zone allows effective response to determine the direct relationship between cell response and the level of PGN. She, thus, is the area of quantitative analysis;

part (C) corresponds to the saturation of the cellular response in the presence of PGN concentrations that are too high. This is actually the saturation of the receptors TLR2.

Standard curve for the response of cells HEK-TLR2 on PGN demonstrates the zone of linearity for concentrations from 0.07 to 10 ng/ml (i.e., from 2 to 267 ng/g).

In the case of samples that may be contaminated PGN, it will be necessary to perform multiple consecutive dilutions, so that they were always in the zone of linearity. Conversely, low concentrations of PGN require a stage of concentration of the sample, if it is desirable to increase the sensitivity of the analysis.

Preparation of sample�in

Analyses PGN performed on the polymer solutions of glucose. A sample requiring a quantitative assessment of PGN incubated with cells HEK-Blue™ hTLR2 and measure cell response by quantifying the produced enzyme activity. The number PGN contained in the sample can be determined by, referring to curve dose-response.

The first tests were conducted with contaminated samples originating from industrial batches of polymer of glucose (example 3, figure 15).

Cells HEK-TLR2 allow to detect the presence of PGN in most of the samples. On the other hand, there was no correlation between levels of PGN, measured by way of SLP-Wako, and the amplitudes of cellular responses to PGN. In fact, the samples with the highest load PGN fail are the ones that induce the strongest SEAP production.

As an illustration of the sample MP-10.07, which is one of the most loaded PGN (645 ng/mg), no significant cell response to cells HEK-TLR2. On the other hand, samples I-10.02 and I-11.12 are the least contaminated (253 and 393 ng/g, respectively), but the most reactive based on cell response.

PGN have a very heterogeneous sizes. Severe (>106Yeah) have a low solubility and, thus, is not very reactive in tests on cells. On the other hand, they are to�icestone are defined in the analysis of SLP-Wako. PGN intermediate size (~100 kDa) soluble and are potent inducers of TLR2. Despite the low levels, they are able to stimulate a strong inflammatory response. This variance in size and, thus, the variance of the activity, is the main drawback for the relationship between the level of pollution with the risk of inflammatory response using conventional quantitative analyses (LAL and SLP-Wako).

PGN present in samples I-10.02 and I-11.12, are soluble and, therefore, very reactive. Conversely, the sample MP-10.07 probably PGN contains a large size that will be removed in the process of obtaining the final product.

On the basis of these first results method of analysis PGN based on the use of cells HEK-TLR2, will allow for the quantification of biologically active PGN that can cause inflammatory responses in vivo.

Particularly advantageous procedure for the quantitative analysis PGN capable of initiation of the inflammatory response is to reduce their size and thus increase their solubility for analysis of the cellular response in vitro.

Mutanolysin is an enzyme which, thanks to its muramidase activity capable of depolymerization PGN. In order to test its activity, the standard solutions (S. aureus) PGN was prepared by diluting supposedly�molecules in culture medium in the absence or in the presence of polymer I-10.01 (uncontaminated polymer) in concentrations from 7.5% to 37.5% (weight/volume).

Samples processed in the presence of 2500 IU/ml of mutanolysin for 16 h at 37°C and then added to cells HEK-TLR2. Cell response measured by subsequently produced SEAP activity according to the conditions described in example 3.

In the absence of glucose polymer processing mutanolysin for 16 h reduces the response of cells HEK-TLR2 by more than 50%, which indicates that the depolymerization was too strong and reduced reactivity PGN regarding cells. In turn, the presence of a polymer of glucose decreases the activity of the enzyme, since the response of cells even improved in the presence of 7.5% of the polymer and is not modified in the presence of 37.5% of the polymer.

Mutanolysin itself induces a cellular response that indicates that he is not dirty and has no activating effect on the cells.

In order to verify the effect of this processing, the polymers I-10.01, (I-10.02, I-10.03, I-11.12, MM-10.05 and MP-10.07 diluted to a concentration of 7.5% and then treated for 16 h at 37°C in the absence or in the presence of 2500 IU/ml of mutanolysin.

Cell response was induced by the addition of 40 ál 160 ál of cell suspension (final polymer concentration: 15 mg/ml).

Results

The responses of cells HEK-TLR2 in the presence of untreated polymers are weak, which was expected, given the lower concentration of the polymer in comparison with when�'erom 3.

After treatment with mutanolysin answers to a solution of the polymer of glucose is clearly increased (figure 22). In addition, it can be noted that the polymer (I-10.03 gives a response equivalent to the polymers I-10.02 MM and-10.05, although he was not reactive in the previous tests.

These results indicate that mutanolysin partially depolymerized and, thus, solubilities PGN, which were wholly or partly insoluble because of their overly large size.

The absorption values presented on the calibration curve constructed under the same conditions with a standard PGN (figure 23) to determine the concentration of PGN that are present in the polymers of glucose.

The results, expressed in ng PGN/g of polymer of glucose, is presented in table 5. After treatment with mutanolysin the values obtained are lower than in the analysis of SLP-Wako, but they reflect the load of glucose polymers on the basis of PGN with Pro-inflammatory activity (table 1).

Polymer I-10.03 gives a high value close to I-10.02, after processing mutanolysin. This piece of data is interesting that the two polymer have been the subject of complaints due to episodes of aseptic peritonitis. Treatment mutanolysin I-10.03, thus, enabled to identify the active load PGN probably giving the polluter the opportunity to be soluble.

Values for samples MM-10.05 and MP-10.07 stauts� below than that obtained in the analysis of SLP. However, these samples are contaminated with other inflammatory molecules in addition to PGN. These molecules, in particular β-glucan, probably interfering with the SLP analyses, increasing the response in the SLP analyses.

Table 5
Quantification PGN present in the polymers of glucose before and after treatment with mutanolysin. Values are expressed as ng equivalents PGN of S. aureus/g of polymer.
I-10.01I-10.02I-10.03I-11.12MM-10.05MP-10.07
Without treatment<213,5<217,511,53,5
Processing<2Of 33.536,8113,53613,5

1. A method for the detection of Pro-inflammatory contaminants of glucose polymers, and these contaminants are capable and�iciale alone or in combination of inflammatory responses, characterized in that includes at least one analysis of the inflammatory response in vitro using cell lines, which can detect at least one factor of the inflammatory response, wherein the cell line is a cell line of macrophages or differentiated into macrophages or cell expressing one or more toll-like receptors (TLR) or NOD-like receptors selected from TLR2, TLR4 and NOD2, and also allows using a reporter gene to detect the responses of the receptor(s), or a combination thereof;
which includes the steps:
(a) performing at least one analysis of the inflammatory response in vitro, which consists in the placement of macrophages in the presence of a preparation of glucose polymers that may contain Pro-inflammatory contaminants, and to measure the production of the cytokine RANTES, and the production of the cytokine RANTES indicates that the preparation contains contaminants capable of initiation of the inflammatory response,
and
(b) placement cell line, which allows to detect the activity of the receptor of innate immunity or more receptors of innate immunity, selected from TLR2 and NOD2, in the presence of the drug and the signal detection of reporter gene associated with this receptor, and the detection of this activity or this signal �shows the presence in the drug contaminant, which is an agonist of the receptor.

2. A method according to claim 1, characterized in that the glucose polymers selected from the group of glucose polymers for peritoneal dialysis, enteral and parenteral nutrition and feeding of infants, and more particularly from glucose polymers for peritoneal dialysis.

3. A method according to claim 1, characterized in that the cell line is a cell line differentiated into macrophages THP-1.

4. A method according to claim 3, characterized in that the cell line used in density from 0.5 to 1×106cells/ml.

5. A method according to claim 3, characterized in that the cell line used in density from 0.7 to 0.8×106cells/ml.

6. A method according to claim 1, characterized in that the production of cytokines was measured after 20 h of stimulation for RANTES.

7. A method according to claim 1, characterized in that the molecule is selected from the muramyl dipeptide (MDP) and related molecules (L18-MDP, glycolyl-MDP) and lipopolysaccharide (LPS), is added to the drug glucose polymer for stage (a).

8. A method according to claim 7, wherein said molecule is selected from MDP and LPS.

9. A method according to claim 7, characterized in that the MDP is added to the drug glucose polymer in a concentration of from 1 to 100 μg/ml.

10. A method according to claim 1, characterized in that it has at the stage a) of the following characteristics:
cell line used in density from 0.5 to 1×106 cells/ml;
the polymer concentration of glucose less than 50 mg/ml;
- production of cytokines was measured after 20 h of stimulation for RANTES;
- MDP added to the drug glucose polymer in a concentration of from 1 to 100 μg/ml.

11. A method according to claim 1, characterized in that it can detect contamination of the polymer of glucose by peptidoglycans (PGN) and/or LPS.

12. A method according to claim 1, characterized in that the reporter gene b-stage) encodes secreterial alkaline phosphatase.

13. A method according to claim 12, characterized in that the cell line can detect the activity of the receptor TLR2.

14. A method according to claim 13, characterized in that the cell line is a line of HEK-Blue™ hTLR2.

15. A method according to claim 12, characterized in that the cell line can detect the activity of the receptor NOD2.

16. A method according to claim 15, characterized in that the cell line is a line of HEK-Blue™ hNOD2.

17. A method according to claim 12, characterized in that the cell line can detect the activity of the receptor TLR4.

18. A method according to claim 17, characterized in that the cell line is a line of HEK-Blue™ hTLR4.

19. A method according to claim 12, characterized in that the cell line can detect the activity of the receptors TLR2, TLR4 and NOD2.

20. A method according to claim 19, characterized in that the cell line is a line of Raw-Blue™.

21. A method according to claim 15, characterized in that it can detect contamination of the polymer Glu�Uzzah MDP.

22. A method according to claim 13 or 19, characterized in that it can detect contamination of the polymer of glucose PGN.

23. A method according to claim 1, characterized in that the analyzed drug glucose polymer has a polymer concentration of from 5 to 50 mg/ml.

24. A method according to claim 1, characterized in that it includes a stage of processing of a preparation of a polymer of glucose mutanolysin to specified incubation of the drug with cells.

25. A method according to claim 1, characterized in that it further comprises a filtration stage of the preparation of polymer of glucose with a cutoff of 30 kDa to 150 kDa, and the fact that the filtrate was incubated with the cells.

26. A method according to claim 1, characterized in that it includes the analysis with the help of macrophages (a) and analysis (b) using cells that can detect the activity of the receptor TLR2, cells that can detect the activity of the receptor NOD2, and cells that can detect the activity of the receptors TLR2, TLR4 and NOD2.

27. A method according to claim 26, characterized in that it includes the analysis with the help of macrophages (a) and analysis (b) using cells HEK-Blue™ hTLR2, HEK-Blue™ hNOD2 and Raw-Blue™.

28. A method according to claim 1, characterized in that it further comprises a stage consisting in the identification or quantification contaminant(s) capable of initiating an inflammatory response.



 

Same patents:

FIELD: medicine.

SUBSTANCE: peripheral leukocyte blood values before and after a loading test are measured with the use of the gradual submaximal exercise. The method involves the differentiated recovery of different types of leukocytes from the blood to produce a preparation; a total T-cell (Tt) and active T-cell (Ta) count is determined; a Tt/Ta ratio is derived; mononuclear cells are incubated with granulocytes; a granulocyte-binding lymphocyte index (GLI) is determined in accordance with a granulocyte rosette formation (GRL - contact bound to three granulocytes) to granulocyte contact lymphocytes (GCL - contact bound to one granulocyte) ratio in the preparation; leukocyte indices: lymphocyte index (LI), immune reactivity index (IRI), adaptation index, ("CПHP") are determined; immune functional state adaptation coefficients (K) are derived for each value. Total immune functional body state TIFBS is calculated by formula TIFBS = K"спнр"+Kli+Kiri+Kgli+K"итл" before the exercise - TIFBS1 and after the exercise - TIFBS2. A specific immune functional state coefficient SIFSC is calculated by formula SIFSC = (TIFBS1+TIFBS2)/5, and a level of the immune functional SIFSC reserve is determined. If the SIFSC values are above +1.0, the level of the immune functional reserve is considered to be optimal; the SIFSC values falling within the range of 0 to 1.0 show the satisfactory reserve, whereas the SIFSC values below 0 shows the unsatisfactory reserve.

EFFECT: method enables assessing the functional body reserves with the use of combined characteristics including the adaptation body potential, immune reactivity and immune cell interaction.

1 ex

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood. Genetic polymorphisms of tumour necrosis factor α (-308 G/A TNFα), tumour necrosis factor 1 receptor (+36 A/G TNFR1), interferon-inducible T-cell chemoattractant (A/G I-TAC), interleukin 1A (-889 C/T IL-1A), lymphotoxin α (+250 A/G Ltα) are typed by polymerase chain reaction. A high risk of developing hyperplastic processes of endometrium is predicted if detecting a combination of alleles -308 G TNFα, +36 A TNFR1, A I-TAC, -889 T IL-1A and/or a combination of alleles +36 A TNFR1, A I-TAC, -889 T IL-1A and/or a combination of alleles -308 G TNFα,+250 G Ltα, -889 T IL-1A.

EFFECT: higher accuracy.

2 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: chronic infectious-inflammatory diseases (CIIDs) are diagnosed. Clinical blood analysis and bacteriological tests are conducted. A sensibilisation index (SI) and an immune responsiveness index (IRI) are calculated; total microbial count per 1 m3 of the working space air is measured, and the total microbial number (TMN) is derived. If the TMN is less than 500 CFU/m3 with no CIIDs diagnosed accompanied by the SI of less than 1.08 standard units and the IRI of less than 13 standard units, the immunoassay is considered to be inadvisable. If the TMN falls within the range of 500-2,500 CFU/m3 with one CIID diagnosed accompanied by the SI from 1.08 to 1.3 standard units and the IRI from 13.1 to 15.7 standard units, the immunoassay with the first-level tests seems advisable. Whereas the TMN exceeding 2,500 CFU/m3 with at least two CIIDs accompanied by the SI of 1.4-1.5 standard units and the IRI of 15.8-18.3 standard units, the immunoassay with the second-level tests is thought expedient.

EFFECT: invention enables detecting the workers in need of further examination for the purpose of timely immune correction in the setting of mass routine examinations.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: DNA from peripheral venous blood is extracted. An analysis of a combination of genetic versions of polymorphous markers of genes of cytokines of the gene regulator of the activity of normal expression and secretion of T-cells (-403 G/A RANTES), macrophage protein -1β (+1931 A/T MIP 1β), factor of stromal cells (-801 G/A SDF1), interleukin -1 (-511 C/T IL-1B), monocyte chemoattractant protein -1 (C/G MCP-1), interleukin -4 (-590 C/T IL-4) is performed. An increased risk of development of a combination of uterine myoma with endometriosis and hyperplastic processes of the endometrium is predicted if the combination of alleles 403 A RANTES, G MCP-1,+1931 A MIP 1β, -590 C IL-4 or the combination of alleles -403 A RANTES,+1931 A MIP 1β, -801 G SDF1, -511 C IL-1B is identified.

EFFECT: application of the claimed method makes it possible to detect a group of patients with a risk of developing a combination of proliferative reproductive system diseases, which makes it possible to prescribe an adequate therapy to prevent further progressing of the diseases.

3 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention deals with method of predicting level of arterial pressure in women of Russian nationality, born in Central Black Earth region of Russia. Method includes separation of DNA from lymphocytes of peripheral venous blood and analysis of genetic polymorphisms. +46G/A ADRB2 and 4a/4b eNOS by method of polymerase chain reaction Level of systolic arterial pressure in women in late pregnancy is predicted by results of multiple regression equation of the following type: Y1=15,455+2,544x1+9,946x2+0,736x3+4,716x4+0,185x5, where x1 is genetic variant in locus - 4a/4b eNOS, namely 4b4b=1; 4a4b=2; 4a4a=3; x2 is presence of preeclampsia in relatives: yes=0, no=1; x3 is level of systolic arterial pressure before pregnancy, mm Hg; x4 is presence of cardiovascular system pathology: yes=0, no=1; x5 is woman's weight before pregnancy, kg Level of diastolic arterial pressure in women in late pregnancy is predicted, for which purpose multiple regression equation of the following type is used: Y2=14,200+7,768x1-2,877x2+7,500x3+0,414x4+3,668x5, where x1 is genetic variant in locus - 4a4b eNOS, namely 4b4b+4a4b=1, 4a4a=0; x2 is genetic variant in locus +46G/A ADRB2, namely GG+GA=1, AA=0; x3 is presence of preeclapsia in relatives:yes=0, no=1; x4 is systolic arterial pressure before pregnancy, mm Hg; x5 is presence of cardiovascular system pathology: yes=0, no=1.

EFFECT: invention makes it possible to realise early prediction of increase of arterial pressure level in women in late pregnancy, will make it possible to form of women at the stage of pregravidal preparation and at early terms of pregnancy groups of high risk of developing hypertension in late pregnancy, as well as realise required therapeutic-preventive measures aimed at prevention of development of said pregnancy complication in due time.

2 dwg, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method is based on contacting a membrane test strip with an analysed fluid sample and initiating thereby a motion along the test strip membranes of reagents being parts of the sample or coating the membrane, and forming the immune complexes to be detected in the course of reactions in the membrane pores or on the surface thereof. A distinguishing feature of the presented method for antigen detection is that the test strip is coated additionally within the test sample contact area with a certain amount of specific antibodies, which react to the detected antigen expected to be found in the sample, when a fluid front moves and block a certain number of binding sites. The number of the coating free antibodies is specified so that the low content thereof in the analysed sample being of no diagnostic importance ensures blocking the binding sites completely that prevents the antigen from binding in the analysed area of the test strip and from developing a destructive staining in the analysed area.

EFFECT: presented approach enables reliable diagnosing based on the detection results of the antigens of gastrointestinal disorders, avoiding the achievement of positive test results for the low-antigen samples, which testifies to no development of the disease in an individual.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular hepatology and infectious diseases, and can be used for determination of stage of fibrous process in monitoring of patients with chronic hepatitis C. To realise method levels of blood serum cytokins are determined in patients with chronic hepatitis C with diagnosed by means of biopsy or other non-invasive method stage of fibrous process 2 times a year, with further calculation of cytokine profile integral index (CPII) on their basis, at initial stage F0 growth of CPII higher than -8 testifies to debut of fibrous changes in liver (transition to stage F1), at initial stage F1, drop of CPII below -10 testifies to transition to stage F2, at initial stage F2 growth of CPII higher than -3 testifies to transition of fibrosis to stage F3, at initial stage F3 drop of CPII below -3 testifies to development of cirrhosis.

EFFECT: determination of stage of fibrous process in monitoring of patients with chronic hepatitis C.

3 ex, 1 tbl, 5 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a method of predicting spontaneous onset of pregnancy in women with I and II stage of external genital endometriosis. The essence of the invention consists in the fact that before treatment in peripheral blood of women with infertility, associated with I and II stage of external genital endometriosis determined is a relative quantity of IL- IL-1β + monocytes, and if the value of the said index is 50.0% and higher in the monocytic gate the spontaneous onset of pregnancy within a year after carrying out the surgical treatment of endometriosis is predicted.

EFFECT: application of the claimed method makes it possible to predict with high accuracy the spontaneous onset of pregnancy in the women with infertility in case of I and II stage of external genital endometriosis within a year after therapeutic laparoscopy, which makes it possible to select optimal tactics of the patients' management and estimate the necessity of administering them methods of assisted reproductive technologies.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: peripheral blood serum interleukin-6 is measured by an immunoassay; if the measured level is more than 5 pg/ml, a biocontrol session of the heart rate variability is predicted to be effective and is expected to represent the total spectrum power gain of the heart rate variability combined with a drop in a regulatory system strain index in relation to references.

EFFECT: method enables the more accurate prediction of the length of the biocontrol course in order to intensify the vagal effect on the heart rate.

1 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of biotechnology and immunology. Described is antibody, which specifically binds denatured CD70. Claimed group of inventions can be used in medicine.

EFFECT: disclosed is method of diagnostics, prediction, prevention and treatment of malignant tumours of ovaries, pancreas and other malignant tumours with application of antibodies.

5 cl, 10 dwg, 2 tbl, 9 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, medicinal microbiology.

SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.

EFFECT: improved assay method.

3 tbl, 3 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

FIELD: medicine, cardiology.

SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.

EFFECT: higher accuracy of prediction.

FIELD: medicine, parasitology.

SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.

EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

FIELD: medicine.

SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.

EFFECT: enhanced accuracy of prediction.

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

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