Recombinant polypeptide a2 selectively binding hsa, recombinant dna pa2 coding hsa-binding part of polypeptide a2, producer thereof - recombinant strain of escherichia coli m15-a2, containing recombinant plasmid dna pqe 32-pa2, facilitating production of polypeptide a2 and use of polypeptide a2 for diagnosis of microalbuminuria and separating hsa from blood serum
SUBSTANCE: invention relates to microbiology and molecular genetics and pertains to a recombinant polypeptide A2, DNA coding said polypeptide, a strain which produces polypeptide A2 and methods of using such a recombinant polypeptide. The disclosed recombinant polypeptide A2 is characterised by an amino acid sequence of 346 amino acids in which the first 13 amino acids are coded by the plasmid sequence pQE 32 and are covalently bonded with the next 333 amino acids which are coded by a sequence of the HSA-binding fragment of chromosomal DNA of the strain DG 13 of streptococci of the group G-CFG.
EFFECT: group of inventions can be used in diagnosis, eg, when making a test system for determining microalbuminuria based on a laboratory criterion of the preclinical phase of diabetic nephropathy, as well as a reagent for separating human serum albumin by affinity chromatography and for freeing serum from HAS, which enables to determine other proteins present in the serum in lower concentrations.
9 cl, 11 dwg, 1 tbl, 9 ex
The invention relates to the field of Microbiology and molecular genetics and can be used in the medical industry, in particular, in test systems for determination of microalbuminuria main laboratory criterion for pre-clinical stages of diabetic nephropathy. The invention can also be applied in biotechnology available as a reagent for isolation of human serum albumin (HSA) by affinity chromatography and liberation of the blood serum from the HSA. This allows you to identify other proteins present in serum at low concentrations.
One of the most important medical and social problems is diabetes mellitus (DM). This disease plays a significant role in the structure of chronic diseases in all age groups, leads to early disability patients because of complications, as well as a significant increase in mortality.
The leading cause of mortality in patients with diabetes type I is a worldwide chronic renal failure (CRF) due to the progression of diabetic nephropathy (DN) - specific kidney damage in diabetic patients. In the United States and Japan NAM occupies the first place in popularity among all kidney diseases (35-40%), edging out second-third position such renal diseases, such as glomerulonephritis, Palo efrit, polycystic and other European countries DN is less threatening, but is kept at the level of 20-25% on the needs of in-vitro treatment. In Russia, mortality from renal failure in diabetic patients type I according to the State register does not exceed 18%, which is three times below the level recorded in the world for the past 30 years. In DM type II mortality ESRD in Russia is 1.5%, which is 2 times lower than the world. Such significant differences in the prevalence of NAM and mortality of patients with diabetes from the CRF does not reflect the true epidemiological situation in Russia. The most likely causes of such discrepancies with these global statistics are: the lack of widespread implementation of screening programmes NAM in diabetes institutions of Russia; the lack of a unified methodology of screening days; unavailability of dialysis treatments for ESRD patients with diabetes, which leads to their death in unsafe conditions; registration no death from kidney failure, and cardiovascular complications or other reasons.
In 1999-2000, under the leadership of the Federal diabetes centre of the Russian Ministry of health has organized expeditions to various regions of Russia, equipped with the most necessary and accessible methods of screening NAM. It was shown that the actual most the pursuit of NAM in the cities of Russia in 2-8 times higher than registered.
Timely diagnosis NAM is an important task of the diabetologists as to detect even the earliest stages NAM requires urgent treatment.
The Ministry of health approved a new classification of NAM, including 3 stages of development:
1) the stage of microalbuminuria,
2) stage of proteinuria saved with the filter function of the kidneys,
3) the stage of chronic renal failure.
The earliest and most reliable method for diagnosing NAM is a test for microalbuminuria.
The term "microalbuminuria" understand the excretion of albumin in urine at low concentrations (from 30 to 300 mg/day). This amount of protein is very difficult to determine in practice it is not defined in the traditional routine urinalysis, and therefore the early stage of DN may not be diagnosed. But this stage is only reversible with timely appointment of pathogenetic therapy.
It is the detection of microalbuminuria may prevent the development of DN.
The purpose of angiotensin converting enzyme (ACE) at the stage of microalbuminuria even normal values of AD systems can prevent the onset of proteinuria in 55% of patients with DM. The purpose of ACE inhibitors on stage proteinuria prevents the development of ESRD in 50-55% of patients with DM. The purpose inhib the tori ACE on stage CRF allows you to extend docility period of 4-5 years.
According to the survey of regional, regional and urban endocrinologists different regions of Russia, at the stage of microalbuminuria therapy with an ACE inhibitor is prescribed only in 20% of the regions where the study of microalbuminuria is conducted on an ongoing basis. In other regions of Russia the absence of screening patients for the presence of microalbuminuria is not allows to assign therapy, resulting in kidney disease continues to progress rapidly, moving into the stage of proteinuria and ESRD.
The most promising and cost-effective for the national health direction in the development of modern diabetes care is the prevention of DN. Such prevention is only possible with excellent metabolic control of diabetes, starting from the beginning of disease occurrence; in the early diagnosis of diabetic kidney disease, based on mandatory regulated screening patients with diabetes for the presence of microalbuminuria; when timely appointment of pathogenetic therapy based on the use of ACE inhibitors.
Double definition of microalbuminuria during the year is a required study for each patient DM, documented in the Federal program "Diabetes".
All the currently used diagnostic test with the system for detection of microalbuminuria are foreign and are designed for immunological semi-quantitative and quantitative determination of albumin concentration in urine. Semiquantitative is test strips or latex systems that use different reagents with albumin. The General principle is to register a positive result only when detecting in the sample of urine albumin concentrations, corresponding to the range of microalbuminuria (20-200 mg/ml).
Known diagnostic kit "Micro-Bumin test" (Mile's Ins Ekkhart, FIN). It is based on the use of albumin blue Bromphenol, fixed on the alkaline matrix. Staining occurs when the concentration of albumin in the sample exceeds 40 mg/l Low specificity of this test (<80%) due to the ability of the reagent to contact with other plasma proteins.
Known diagnostic kits "Albu Screen" and "Albusure" ("Cambridge Life Siences", Cambridge, UK), which are latex-agglutinating inhibition test based on the use of antisera to human albumin and latex particles with albumin. If the concentration of albumin in the urine sample is less than 30 mg/l, there is a latex-agglutination. The specificity and sensitivity of this test is 90%.
Known diagnostic kit "Micral-test (Boehringer Mannheim, Indiana-polis, Germany), which is based on the use of monoclonal antialbumin antibodies labeled with galactosidase, and fixed on the test strip. Assessment of response is made after 5 minutes by visual CPA is in the coloring of the reaction zone of the test strip supplied with a colour scale division which correspond to the albumin concentration of 0, 10, 20, 50, 100 µg/L.
The use of test strips is quite acceptable as a screening test for semi-quantitative detection of microalbuminuria. However, the test needs to quantify the excretion of albumin in the urine.
For the quantitative determination of the level of excretion of albumin used radioimmunoassay, enzyme immunoassay and immunoturbidimetric methods. They are all immunohistochemistry using antibodies to albumin.
A known method of radial immunodiffusion (RID). It is based on the incorporation antialbumin antibodies in agar gel with subsequent immunodiffusion albumin sample (Turro F.V. Clin.Chem. 28: 1359-1361? (1982)). Being a reliable and inexpensive, it is, however, not widely used in practice due to the fact that the reaction requires a very long incubation period. In addition, REED cannot be automated.
Known radioimmunoassay method (RIA), which is based on the application of isotope of iodine and analbuminemia antisera, is performed in the liquid phase with an excess of antigen. This method is sensitive and accurate. Disadvantages of RIA consist in the use of radioactive reagents that have a short shelf life, the method requires expensive equipment and waste disposal system.
Known methods IMM is naturalisatie and nephelometry, based on the property of urine albumin to form immune complexes in the interaction with antibodies (Watts G. Clin.Chem. 32: 1544-1548, (1986)). The turbidity of the sample, due to their education, measured spectrophotometrically at a wavelength of 340 nm (turbidimetry). The method is simple, accurate, easy to automate. It allows you to determine the albumin in a concentration of 5-40 mg/l, and at high concentrations of albumin required additional dilution of the sample. The sensitivity of the method can be improved by adding in the test system latex particles. The method requires the use of expensive equipment. A prerequisite for the implementation study is a preliminary centrifugation of the samples, as any contamination of the urine, i.e., any level of turbidity leads to false-positive results. Complexes of albumin urine antibodies is the basis of these diagnostic methods for determining whether microalbuminuria.
Thus, to date there is no unified methodology for the determination of microalbuminuria. Available on the domestic market diagnostics of various Western manufacturers ("Albu Screen", "Albusure" - "Cambrige Life Science, UK; "Micral-test, Boehringer Mannheim, Germany and others) are test systems, mainly for semi-quantitative determination of albumin in the urine and are the Xia expensive to perform large-scale screening programmes and monitoring of the NAM in full. Domestic counterparts, have been implemented.
Creating a sensitive and cost-effective diagnostic method for the determination of microalbuminuria is an urgent problem.
The essence of the invention consists in that for the determination of microalbuminuria are encouraged to use recombinant albumin receptor-binding polypeptide derived from streptococci.
Albumin is the most abundant protein in blood plasma of humans and mammals. In the last decade identified and studied proteins of bacteria possessing receptor activity against HSA.
Streptococci (Streptococcus group a, C and G Express surface proteins that bind HSA. (Myhre E. and Kronvall G. Infect.Immun. 27: 6-14 (1980)), Wideback K., and others, Acta Pathol. Environ. Immunol. Scand. Sect. B. 91: 373-382 (1983)).
Among these surface proteins of Streptococcus group G interest is a G protein, a full-sized molecule which has the ability to bind immunoglobulin G (IgG) humans and various mammals, as well as HSA human and animal (Bjorck and other Mol.Immunol. 24: 1113-1122 (1987)).
From one of the strains of Peptostreptococcus magnus selected RAV binding protein HSA (de Chateau, M., Bjorck L.J. of Biol. Chem. 269: 12147-12151 (1994)). Analysis of its amino acid sequence revealed the plot of the 45 amino acid residues responsible for binding to HSA. This serial is lnost, named GA module has homology with HSA-binding region of protein G (Akerstrom and others J of Biol. Chem. 262: 13388-13391 (1987), Sjobring U, etc. J Immun. 140: 1595-1599. (1988), R. Nygren And other J.Mol. Recognit. 1: 69-74 (1988)).
Unlike RAV protein, which is not homologous repeats, protein G, isolated from strain G 148, contains three homologous repeated sequences, i.e. three GA module.
The emergence of new receptor proteins in bacteria often associated with gene transfer, their parts or parts of the genome. It is believed that these fragments encode products that are modular blocks of proteins. Thus, it is assumed that the HSA-binding GA module protein RAV occurred during the transfer of the corresponding element of protein G. the structure of the GA module of the G protein and the GA module in the protein RAV is the same and consists of three helices (Johansson M. and others J.Mol.Biol. 316: 1083-1099 (2002)).
From a strain of Streptococcus canis DG12 isolated from cow's milk obtained HSA-binding protein that contains two of the GA module. It does not bind IgG, but by the ability to bind HSA exceeds protein G (U. Sjobring Infection and Immun. 60: 3601-3608 (1992)). This property makes it attractive for practical application.
The study of the structure of the HSA-binding proteins is of great importance to the technology of protein reagents that are relevant in immunochemistry, proteomics, biotechnology and clinical diagnostics.
Replacement in enzyme-linked immunosorbent analysis op is adelene microalbuminuria commonly used antibodies on receptor recombinant HSA-binding polypeptide creates certain advantages, because it allows you to:
1) to eliminate the time consuming process of preparation of specific antibodies (for which use of laboratory animals),
2) to standardize used albumen receptor, and thereby to stabilize the whole system analysis.
Created by the authors of the method receptordependent analysis (XRF) is an inexpensive, easy to use, sensitive and specific method for determination of microalbuminuria in patients with diabetes.
Created test system differs from foreign analogues that do not require immune sera, vsokospecificno, sensitive enough, does not give the background of false reactions available to use and economical.
The use of microarray technology in conjunction with the HSA-binding polypeptide for the determination of microalbuminuria can give rapid quantitative assessment of the content of albumin in the urine at the same time from a large number of patients and thereby to facilitate the screening and monitoring of diabetic nephropathy.
Almost all strains CTG, both human and animal produce on their cell surface protein G, which binds to HSA.
For the first time recombinant HSA receptor, was obtained by the Swedish authors by cloning a fragment of the gene HSA receptor in non-pathogenic host. (R. Nygren and others J. Mo. Recognition. 1: 69-74. (1988)). In this work, for cloning AB region (region responsible for binding to HSA) was restrictively plasmid pSPG2 enzyme EcoRI, followed by processing the fragment maple, adding synthetic linker Sail and legirovaniem. When enzyme SalI > PST and was received fragment 640 n / a, which was isolated from agarose and inserted between the same sites in the vector pEML8. The gene fragment was cut out from the obtained plasmid using EcoRI and HindIII sites and inserted into plasmid pEG, the construction of which is described in the work of Eliasson (Eliasson, M. And others J. Biol. Chem. 263: 4323-4327 (1988)).
As a result of this modification was obtained plasmid pB1B2responsible for the HSA-binding activity. From subclone producing HSA-binding protein, was isolated protein. It was purified by affinity chromatography.
Described by the authors of protein was heterogeneous degradation of the pure product. Cause of heterogeneity, obviously, was a quite complicated way of their genetic design. Practical application because of its shortcomings, this protein does not find.
In 1996 was prolongirovanne fragment of the gene encoding the third GA module G protein strain G148. The obtained protein G148-GA3 was used to study the structure of the HSA-binding module. (Kraulis, P. and others, FEBS Lett. 378: 190-194 (1996)), i.e. only for research purposes.
In 1992, the CTG alive the private origin, strain DG 12, was prolongirovanne fragment of the gene encoding two HSA-binding GA module, using a plasmid vector RK-2 and determined its nucleotide sequence. (U. Sjobring Infect, and Immun. 60: 3601-3608. (1992)).
The prototype of the invention is recombinant HSA-binding receptor polypeptide A1. It was obtained from CTG, strain G148 isolated from humans in the Department of molecular Microbiology in 1996 (Orlov S.N. and other Biotechnology. 8: 13-21.(1996)). The strain producing the polypeptide A1, deposited in the collection of epidemiology and Microbiology NWB RAMS, No. 357.
The peculiarity of this polypeptide is that it contains HSA-binding region, covering three GA module. Was studied fragment of the gene that encodes the polypeptide A1.
The study of this receptor polypeptide has proven his ability to communicate exclusively with the main protein of human plasma - HSA. This property of the polypeptide tried to use a diagnostic test systems to determine the concentration of HSA in various biological fluids, in particular urine.
However, when the separation and purification of the recombinant polypeptide A1 of the active reagent in sufficient quantity was difficult, which has led to the need to create a new HSA-binding drug that has greater activity than the polypeptide A1 of protein G.
The objective of this from the retene received recombinant polypeptide A2, having a high ability to bind HSA and its use in diagnosis when creating a sensitive and cost-effective test system for the determination of microalbuminuria in patients with diabetes type I and II.
The invention was the creation of producer strain recombinant polypeptide A2.
The problem is solved by designing primers directed to areas of the gene encoding HSA-binding polypeptide A2, which consists of two GA module. Their use in polymerase chain reaction (PCR) allows to obtain the desired DNA fragment, legirovannye later with plasmid vector pQE 32 (The QIAexpress System, Qiagen, USA). This way made it possible to facilitate isolation and purification, created by the authors of the recombinant polypeptide.
The creation of the producer strain, designated as Escherichia coli (E. coli) M 15-A2, was carried out by genetic transformation on the basis of E. coli strains M 15.
It is known that the strain of E.coli M 15 used for the preparation of recombinant polypeptides by transforming bacteria plasmid DNA (Ustinovich I.A. Biotechnology. 1: 3-10. (2002)), (Duplic NV Medical immunology, 11: 7-14. (2009)), (Mandel M. J.Mol.Biol. 53: 154-157. (1970)).
Obtained by the authors of the invention producing strains of E. coli M 15-A2 includes a plasmid containing the sequence of the gene encoding HSA-binding region is t (sequence published Sjobring (U. Sjobring Infect. Immun. 60: 3601-3608. (1992)), associated with DNA vector pQE 32. Earlier this variant modification of the producer strain was not implemented.
It is this transformation of E.coli bacteria M 15 led to the emergence of the ability under certain conditions to induce the synthesis of recombinant polypeptide A2. The strain is deposited in the collection of epidemiology and Microbiology NWB RAMS, No. 593.
The invention is a recombinant DNA (labeled RA2), the resulting panorama, using chromosomal DNA of strain DG 13 CTG isolated from cow's milk, primers RA1 and RA2 and the subsequent cloning using the expression plasmid pQE 32.
Also the invention is a recombinant DNA (labeled pQE 32-RA2)carrying the recombinant Dncr.
A fragment of the gene encoding the polypeptide A2 of size 999 n obtained in the course of Poland on the basis of chromosomal DNA of strain DG 13 CTG using primers RA1 and RA2. The authors carried out the cloning of this fragment using the expression plasmid pQE 32 and the subsequent transformation of recombinant plasmid DNA in the heterologous system of E.coli M 15.
Recombinant polypeptide A2 obtained by the inducible expression of strain-producing E.coli M 15-A2 was purified single-stage affinity chromatography on Ni-NTA-agarose (Qiagen, USA). The yield of recombinant poly is eptide A2, obtained from one liter of culture medium for growing strain of E.coli M 15-A2, amounted to 40 mg, while the output of the recombinant polypeptide A1 was 8 mg, i.e. the output A2 5 times higher.
In contrast to known polypeptides to bind HSA polypeptide A2 stable, has a high HSA-binding activity, which is not reduced during long-term storage.
The authors performed a characterization of the interaction of HSA with the polypeptide A2 in comparison with the interaction of HSA with previously obtained (known) a polypeptide A1.
It is established that the ability to bind HSA in recombinant polypeptide A2 is more pronounced than in the polypeptide A1. The polypeptide A2 can bind a number of HSA in two times more than the polypeptide A1.
The technical result of the invention is obvious: obtained and characterized recombinant polypeptide A2 suitable for use in the test system to determine the level of albumin in the urine, i.e. for the diagnosis of microalbuminuria in diabetic nephropathy, a complication of diabetes.
The properties of the new recombinant polypeptide A2 allows to use it also for affinity separation of HSA, and release of blood serum from HSA.
Obtaining recombinant polypeptide A2.
The source of chromosomal DNA was strain DG 13 SGAs (isolated from to the Rovigo milk). DNA purified phenol-chloroform extraction was used as template in polymerase chain reaction (NDP) to obtain a fragment genera (999 BP) Oligonucleotide primers shown in table 1.
The underlined parts of the nucleotide sequences indicate restriction sites.
The analysis of the results of Poland carried out by separation of DNA fragments in a 1% agarose gel using horizontal electrophoresis (figure 1).
Highlight search of the amplified DNA were carried out using a set of "QIAquick Gel Extraction Kit" (Qiagen, USA).
The resulting fragment was cloned using the expression plasmid pQE 32. In preparation for the cloning was carried out restriction isolated from the agarose slice (999 n) and the plasmid pQE 32 enzymes BamHI and kpni restriction sites with the formation of sticky ends. Food restriction were separated by horizontal electrophoresis in 1% agarose gel, and then ligated.
The products of ligation were performed calcium transformation of E.coli strain M 15. The transformation process was carried out according to the method of transformation of Escherichia coli using dense selective medium containing 50 μg/ml ampicillin, 25 μg/ml kanamycin. (Maniatis T., Fritsch E.F., Sambrook J. Molecular cloning: a laboratory manual. - Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 867. (1982)).
Antibiot causticity transformants were split into two parallel Petri dishes with antibiotics. Colonies with one Cup were transferred to nitrocellulose membrane and exposed with a solution containing 0.2 N NaOH, 0.1% of SDS and 0.5% β-mercaptoethanol. The membrane was incubated 1 hour at room temperature in blocking solution (2 parts 3% milk and 1 part phosphate-saline buffer (FSB)) to remove nonspecific binding and then in blocking solution containing HSA labeled with horseradish peroxidase (Sigma, USA).
Conjugation of horseradish peroxidase with HSA was performed controllable periodic destruction method (Primal, Immunological methods: M. Medicine: 438-439. (1987)).
Then the membrane was sequentially washed with the blocking solution and the FSB. Peroxidase activity showed a solution paraphenylenediamine at a concentration of 1 mg/ml with 0.15% of hydrogen peroxide. Clones-transformed with the expression of HSA-binding activity, are presented in figure 2.
From the transformants with the most pronounced expression of HSA-binding activity were isolated recombinant plasmids using Mini-prep plasmid DNA purification kit (Qiagen, USA). The presence of recombinant DNA was confirmed in PCR with the original primers RA1 and RA2. Plasmid DNA pQE 32-RA2 was used as the matrix in Poland with primers pQE1 and pQE2. Amplificat was isolated after electrophoresis in 1% agarose gel and sequenced.
The resulting data coincided with the known nucleotide sequence encoding HSA-binding polyp is Ted with two GA modules. (U. Sjobring Infect, and Immun. 60: 3601-3608. (1992)).
Figure 3 presents the nucleotide sequence consisting of 999 BP recombinant Dncr and 39 BP plasmid pQE 32
4 shows the amino acid sequence consisting of 346 amino acid residues, in which 333 amino acids of the polypeptide A2 of the encoded recombinant DNA RA2 and the first 13 amino acids of the plasmid pQE 32.
Culture of E. coli strain M 15 containing recombinant plasmid pQE 32-RA2, designated as E. coli M 15-A2, were cultured in liquid medium BHI (Brain Heart Infusion Broth, Gibco, USA) supplemented with antibiotics (ampicillin (100 μg/ml) and kanamycin (25 μg/ml)) to late logarithmic growth phase (OD600=0.7 to 0.9). Then the expression of recombinant protein was induced by adding isopropyl-beta-D-thiogalactopyranoside (IPTG), and cells were cultured for another 4 hours. After that, the cells were besieged by centrifugation, washed lytic buffer A (20 mm Na2HPO4, 20 mm NaH2PO4, 500 mm NaCl, 20 mm imidazole, pH 8.0) and suspended in the same buffer, adding a protease inhibitor, phenylmethylsulfonyl (PMSF), to a concentration of 1 mm. Suspension cells were exposed to ultrasound and centrifuged. The supernatant was applied to a column Packed with Ni-NTA-agarose (Qiagen, USA). After the protein was associated with Ni-NTA-agarose column was washed with buffer to remove respecof the Cesky bound peroxidase proteins. Recombinant protein was suirable buffer B (20 Na2HPO4, 20 mm NaH2PO4500 mm NaCl, 250 mm imidazole, pH 8.0). After affinity chromatography protein were dialyzed for 18 hours against distilled water.
Selected recombinant polypeptide was analyzed by the method of SDS-polyacrylamide gel electrophoresis, which allowed us to make a conclusion about a satisfactory purification of the polypeptide and also about its molecular weight by comparing the mileage of the polypeptide A2 with mileage proteins of known molecular weight (Precision Plus Protein standards (161-0373), Bio-Rad, USA). The molecular mass of the polypeptide A2 is equal to (48,0±0,5) kDa (figure 5).
Thus, the recombinant polypeptide A2 contains the amino acid sequence of HSA-binding polypeptide CTG strain DG 13 containing 333 amino acids, covalently linked with a 13 amino acid residues encoded by pQE 32.
The study of the interaction of the recombinant polypeptide A2 with HSA when compared to the same reaction with a polypeptide A1.
The authors have obtained immunochemical characterization of recombinant molecules A2, representing the HSA-binding polypeptide. The results allow to extend the scope of the polypeptide A2, adding the use of diagnostics, for affinity separation of HSA and to release syvorotkina from HSA.
Structural differences of recombinant polypeptides A1 and A2 make recombinant polypeptide A2 preferred in all use cases.
Comparison of the receptor activity of recombinant polypeptides A1 and A2 in relation to the HSA, is represented by the following samples: 1) HSA - polyclonal serum albumin human labeled with horseradish peroxidase. 2) Polyclonal unlabeled HSA.
It is established that the polypeptide A2, as well as polypeptide A1, has the ability to selectively bind to HSA in any of the used options setting ELISA direct ELISA, indirect ELISA, inhibition ELISA, competitive inhibition). Structural differences between the two polypeptides that are implemented by methods of molecular genetics, contribute substantial differentiation of recombinant polypeptides A1 and A2 of the HSA-binding properties.
In the case of a direct ELISA polypeptides A1 and A2 were immobilized on the solid phase. It was estimated the number of contacting them labeled HSA. Figure 6 shows a histogram that reflects the mapping of the HSA-binding activity of the polypeptide A1 and A2. The study polypeptides A1 and A2 have the HSA-binding activity in respect of labeled HSA, and the number of HSA bound by the polypeptide A2 is higher than the polypeptide A1 twice. For the analysis conducted in Odinak the new conditions, the increase in binding activity 2 times, is quite significant.
Polypeptides A1 and A2, created on the basis of different types of streptococcal undoubtedly have structural differences that define HSA-receptor activity of each of them. We can assume that the implementation of this ability is affected by conditions and performances of ELISA, namely the presence of the polypeptide on the solid phase or in solution. The influence of this factor is explained by the presence of peptides spatial configuration, which is different for molecules that are freely circulating in solution or fixed on the solid phase.
The change in conformation may theoretically lead to an increase or decrease in the ability of molecules to bind HSA. To test this assumption was used indirect and inhibition ELISA.
When setting the indirect ELISA results showed that the polypeptides in solution bind HSA. However, A2 binds to HSA with activity in two times greater than A1. (Fig.7).
Inhibition ELISA showed that the polypeptide A1 and A2, while in solution, prevented the interaction of labeled HSA with adsorbed molecules A1 and A2 within 70-94% (Fig).
The peculiarities in the behavior of proteins under conditions of ELISA. Polypeptides A1 and A2 are able to interact with HSA in solution (inhibitory and indirect ELISA), and in the adsorbed state (direct the FA). This property allows you to use them as immunochemical reagents in ELISA, and, as in immobilized form, as well as detecting molecules in the presence of the enzyme label.
The greatest degree of binding observed in protein A2 in terms of direct and indirect ELISA, although the polypeptide A1 has three, and the polypeptide A2 - only two HSA-binding GA module. This can be explained by the fact that all three of the GA module of the polypeptide A1 have almost the same sequence, and GA modules polypeptide A2 contain sequences with less homology between themselves and due to this difference more actively bind HSA.
The position of the binding sites of antibodies to HSA and recombinant protein on the HSA molecule was studied in inhibitory variant of ELISA. Antibodies to HSA was adsorbing on the tablet, and then thereto was added a pre-prepared mixture of labeled HSA and polypeptides A1 or A2 (in the ratio 1:1, incubation at room temperature for two hours). The incubation of the immobilized antibody with a mixture of polypeptides and labeled HSA was carried out for 1 hour at 37°C.
On the influence of recombinant polypeptides to interact labeled HSA antibody was tried on peroxidase activity of the enzyme label HSA, contacting the adsorbed antibodies.
Preliminary interaction of HSA with polypeptides is e prevented subsequent binding of antibodies, from which it follows that the binding sites of antibodies and recombinant polypeptide A1 or A2 on the HSA molecule is completely independent.
Simultaneous binding to HSA molecule two reagents opens the possibility of creating multi-test systems.
The method of determining the concentration of HSA in urine, i.e. the creation of test systems for the determination of microalbuminuria.
Studies on the interaction of polypeptides A1 and A2 with HSA showed that recombinant molecules have HSA-binding ability. This property has served as the basis for establishing a method for determining the concentration of HSA in urine.
Given the greater yield of the recombinant polypeptide A2 in the process of protein expression in E. coli and its higher ability in comparison with the polypeptide A1 to bind HSA, he was selected to create a diagnostic test kits for the qualitative detection and quantitative determination of microalbuminuria.
To create a quantitative test systems preferred competitive ELISA method, because this method contains the minimum number of operations, requires little consumption of reagents and can easily be automated.
Commonly used in ELISA antibodies to albumin were replaced by recombinant receptor polypeptide A2, and the immunoassay principle was transformed into the ARF. This change created the certain advantages, because allowed to separate from the laborious process of preparation of specific immune sera, used to standardize the recombinant polypeptide and, thus, to unify the whole system analysis. In addition, the use of recombinant receptor polypeptide is possible to avoid the background reactions, possible immunological reactions.
Previously this approach for analysis of albumin was not applied.
The principle of XRF method is as follows: on a hard surface tablet immobilities polypeptide A2, which are applied analyzed urine samples simultaneously with labeled HSA. When this formulation albumin samples and labeled HSA compete for the HSA-binding polypeptide immobilized on a solid phase. The concentration of albumin in the analyzed sample is inversely proportional to the enzymatic activity of the solid phase.
When developing test systems were selected conditions analysis, providing the necessary sensitivity and specificity. As a result of the experiments was selected working dilution of labeled HSA 1:8000 at a concentration of immobilized polypeptide A2 from 0.1 to 0.25 µg/ml According to a serial dilution of unlabeled HSA is constructed calibration curve.
After the selection of the optimum conditions were building a calibration curve according to optionscollection on the concentration of HSA in the standard samples (3,125; 6,25; 12,5; 25; 50 and 100 µg/ml) (Fig.9). The concentration of HSA in urine were found in the values of optical density calibration curve.
Thus, were able to show that recombinant polypeptide A2 can be used not only for qualitative analysis, but also as a highly specific reagent for the quantitative determination of albumin in urine. The minimum concentration of HSA, which can be defined in the urine amounted to 1-2 μg/ml and a maximum of 100 mcg/ml This is a very sensitive method of detection. Urine with a higher concentration of albumin dilute solution of the FSB. As a result, the sensitivity of this detection method allows to detect the amount of albumin in the urine within, including albumin excretion with urine in norm, i.e. up to 20 mcg/ml and within microalbuminuria from 20 to 200 µg/ml Method is highly accurate.
For screening and monitoring patients with diabetes mellitus requires the simultaneous analysis of several dozens of samples. To solve this problem, the possibility of creating a method which combines the use of stable high-affinity recombinant HSA-binding polypeptide and technology of microchips. As a matrix for the deposition of urine samples of patients with diabetic nephropathy was selected nitrocellulose membrane. On a nitrocellulose membrane, once the leader in 1 cm 2were applied 30 urine samples, was used automatic calibrator. The first row on the membrane corresponded to the dilutions of the standard preparation HSA to construct a calibration curve(100; 50; 25; 12,5; 6,25; 3,125 µg/ml). The membrane was preincubation in blocking solution (2% milk dissolved in FSB) with peroxidase labeled HSA-binding polypeptide A2. Then the membrane was washed and stained with a solution of paraphenylenediamine with hydrogen peroxide. On the membrane clearly manifested spots, different degrees of staining which corresponded to different concentrations of albumin in the standard samples and urine samples (Fig. 10). Having a reading device and a corresponding computer program, you can obtain a quantitative estimation of albumin in the urine, and simultaneously in a large number of samples.
Obtaining HSA from serum by affinity chromatography and liberation of the blood serum from the HSA.
Sorbent served as Affi-Gel 10 (Bio-Rad, USA). Was prepared column, in which the sorbent contained attached recombinant polypeptide A2. The column was washed first with bicarbonate buffer with 0.5 % NaCl, pH 8.0, and then 0.1 M glycine buffer, pH 2.2 and again bicarbonate buffer with 0.5 % NaCl. The column was balanced by the FSB, pH 7.4 and kept at 4°.
A sample of human serum was applied to a column of Affi-Gel 10 is a recombinant polypeptide A2. Netwas Chiesa proteins were collected in the washing of the column with a solution of the FSB. The elution of contacting the sorbent HSA conducted OD M glycine buffer, pH of 2.2. The eluate was collected fractions of 500 ál, the concentration of protein was determined spectrophotometrically by the values of optical density (OD) at a wavelength of 280 nm. Fractions with the highest OD value were combined and the pH was brought to 7.5 with 2 N caustic soda solution. Samples of the original serum samples of serum proteins, not contacting the sorbent in the column and the sample, elyuirovaniya from the column were tested for the presence of HSA by the method of polyacrylamide gel electrophoresis (SDS-PAGE).
SDS-PAGE showed that the serum after passing through the column were exempted from the HSA. The release of serum from the HSA allows you to detect proteins, previously screened HSA. At the same time in the eluate was HSA, which was obtained in a highly purified state (Fig. 11).
Thus, the obtained sorbent with the recombinant polypeptide A2 allows to identify proteins that are located in low concentrations, after the liberation of the serum from the HSA and get in one stage, the purified HSA drug.
Thus, the invention is:
1. Recombinant DNA RA2, the nucleotide sequence of which is 999 BP
2. Recombinant DNA pQE 32-RA2 represents a plasmid pQE 32, carrying the recombinant DNA RA2 and providing the polypeptide A2.
3. The strain E. coli M 15-A2, trance is formirovanii recombinant plasmid DNA pQE 32-RA2 and expressing the recombinant polypeptide A2.
4. Recombinant polypeptide A2, having the amino acid sequence represented in figure 4 and having sposobnostyami to bind HSA, and the polypeptide A2 binds to HSA in solution and in the adsorbed state.
5. The ability of the polypeptide A2 selectively bind HSA allowed to use it in the test system for the qualitative detection and quantitative determination of microalbuminuria.
6. The ability of the polypeptide A2 selectively bind HSA allowed us to obtain highly purified preparation of HSA from serum by affinity chromatography and release the serum from the HSA, thereby to define other blood proteins present in lower concentrations.
Following are specific examples illustrating some versions of the invention, but not limiting it.
Example 1. Obtaining a DNA fragment/RA2 PCR.
To 0.25 microgram of genomic DNA extracted phenolic-chromosome extraction from strain DG 13 CTG was added to 10 micromol each of the specific primers flanking the sequence under consideration, the buffer with magnesium for the polymerase, 0.2 mm four deoxyribonucleotides, the volume was diluted to 25 ml and was added to 0.5 μl of thermostable DNA polymerase. On the surface of the liquid was just added 40 μl of mineral oil. The tubes were placed in and plification and incubated at 94°C 2 minutes The PCR program consisted of: denaturation at 94°C - 30 sec, annealing of primers to 60°C - 1 min, and synthesis - 72°C - 1 min This cycle was repeated 30 times, after which the mixture was incubated at 72°C for 10 min In this work, we used oligonucleotide primers listed in Table 1. Poland products were separated in 1% agarose gel horizontal electrophoresis (figure 1). The selection of the amplified DNA was performed using a set of "QIAquick Gel Extraction Kit" (Qiagen, USA). The size analysis of the obtained DNA fragment was performed based on comparing its electrophoretic mobility electrophoretic mobility marker of molecular weights (100 BP DNA marker, the Helicon).
Figure 1 (Electrophoregram amplified DNA fragment RA2):
1 - product PCR
2 - 100 BP DNA marker (from top to bottom: 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 nucleotide pairs).
In the NDP with primers RA1 and RA2 was obtained fragment 999 P.N., as demonstrated in figure 1.
Example 2. Cloning of the DNA fragment RA2 using the expression plasmid pQE 32.
Plasmid pQE 32 and the fragment resulting from Poland were treated with two restriction enzymes BamHI and kpni restriction sites, which led to the formation of sticky ends. Food restriction were separated by horizontal electrophoresis in 1% agarose gel and then isolated from the agarose using a set of "QAquick Gel Extraction Kit" (Qiagen, USA). During cloning to 30 μl of the DNA fragment RA2, was added 2 μl of DNA fragment pQE 32, obtained after restriction and cut out from the agarose in a concentration ratio of 5:1, 5 μl of tenfold ligase buffer and 1 μl of ligase of phage T4. The volume was made with distilled water to 50 μl. The mixture is incubated at 10° for 12 hours. As a result of the cloning was obtained recombinant plasmid, pQE identified as 32-RA2, carrying recombinant DNA (labeled RA2), consisting of 999 NP and 39 i.e. the plasmid pQE 32.
Example 3. The transformation of a culture of E. coli M-15 plasmid DNA pQE 32-RA2.
The recombinant plasmid was transformed in the heterologous system of E. coli M 15. As a positive control in parallel carried out the transformation of E. coli M 15 original plasmid DNA pQE 32. Genetic marker plasmid DNA pQE 32 is a marker amp, encoding beta-lactamase that provides resistance to ampicillin cells carrying this plasmid. Culture of cells of E. coli M 15 were cultured in BHI broth (Brain Heart Infusion Broth, Gibco, USA) with kanamycin (25 μg/ml) for 12 hours at 37° and vigorous stirring. Then the inoculum was perseval the new volume of the same medium (10 ml of medium and 0.1 ml of supernatant) and incubated at 37° for 2-3 hours under stirring until OD600=0,3. Grown cells in a volume of 1.5 ml was centrifuged for minutes at 12000 rpm, and the precipitate suspended in 200 μl of a solution of 0.1 M CaCl2. Next, the mixture was stirred in ice for 1 hour. After centrifugation the precipitate resuspendable 187, 5 μl of 0.1 M CaCl2and 64.5 μl of distilled water. Then the tube was made of 0.2 μg of plasmid DNA and incubated the mixture in ice for 1 hour. Next, the mixture was stirred for 2 minutes at 42°C and again shifted the mixture in ice for 10 minutes thereafter, to the mixture was added 1 ml of BHI broth with kanamycin (25 μg/ml) and incubated 1 hour at 37°C. After sedimentation by centrifugation (8000 rpm for 1 min), the cells were sown on plates with 1% L-agar (Difco, USA)containing ampicillin (100 μg/ml) and kanamycin (25 μg/ml). After 18 hours of growth, cells at 37°C were selected clones of transformants. Antibiotic-resistant transformants were transferred to two parallel Petri dishes with antibiotics. Colonies with one Cup were transferred to nitrocellulose membrane and exposed with a solution containing 0.2 N NaOH, 0.1% of SDS and 0.5% β-mercaptoethanol. The membrane was incubated for 1 hour in blocking solution containing HSA labeled with horseradish peroxidase (Sigma, USA). Then it sequentially washed with the blocking solution and the solution of the FSB. Peroxidase activity showed a solution paraphenylenediamine at a concentration of 1 μg/ml with 0, 15 M hydrogen peroxide (figure 2).
Figure 2 (Expression of HSA-binding activity of the colonies, sod is rasih recombinant polypeptide A2) shows the pronounced expression of HSA-binding activity of individual clones.
Thus, the obtained data allow to conclude that as a result of cloning were obtained recombinant clones carrying the plasmid pQE 32-pa2 with recombinant DICK RA2.
Example 4. Purification of recombinant protein A2.
Culture of E. coli strain M 15-A2 were grown in BHI broth with the addition of ampicillin at a concentration of 100 μg/ml and kanamycin at a concentration of 25 μg/ml overnight under vigorous stirring. Then the cells were perseval 800 ml of the same medium and incubated at 37°C for 2-3 hours under vigorous stirring to OD600=0.7 to 0.9. The expression of recombinant protein was induced by addition of a solution of isopropyl-beta-D-thiogalactopyranoside to a final concentration of 2 mm, after which the cells were incubated under the same conditions for another 4 hours. The obtained cell suspension was centrifuged at 4000 rpm for 20 min the Supernatant was decanted, and the cells suspended in buffer A (20 mm Na2HPO4, 20 mm NaH2PO4, 500 mm NaCl, 20 mm imidazole, pH 8.0)by adding the inhibitor of the protease phenylmethylsulfonyl (PMSF) to a final concentration of 1 mm. For lizirovania cells was used three ultrasonic treatment at 4°C for 20 sec. with a break in 40 seconds. in an ultrasonic disintegrator (USDN-U, England). The cell lysate was centrifuged at 20,000 rpm and 4°C for 20 min. N is posadochkuju liquid was passed through 0.45 µm filter (Millipore, USA) and then it was applied on the column with Ni-separate (Qiagen, USA), pre-equilibrated with buffer A. Next, the column was washed with the same buffer as long as the OD values280facing solution were not more than 0.01. Protein was suirable solution of buffer B (20 mm Na2HPO4, 20 mm NaH2PO4, 500 mm NaCl, 250 mm imidazole, pH 8.0). The eluate was collected in fractions of 500 ál and measured their values OD280. Fractions with the highest OD values280that United and were dialyzed against distilled water for 12 hours. Figure 5 presents electrophoregram recombinant polypeptide A2.
Figure 5. (Electrophoregram recombinant polypeptide A2):
1 - molecular weight marker, (top to bottom: 113, 92, 52, 35, 28 and 21 kDa).
2 - preparation of purified protein A2
The molecular weight of the polypeptide A2 was determined by comparing the mileage protein A2 with mileage proteins of known molecular weight (Precision Plus Protein standards (161-0373), Bio-Rad, USA). The molecular mass of the protein A2 is equal to (48±0,5) kDa.
Example 5. Binding of serum labeled HSA adsorbed polypeptides A1 and A2.
Analysis of the HSA-binding activity carried out by direct ELISA. For conducting the ELISA used tablets NUNC MaxiSorb (Danemark).
Preparation of diluted drugs labeled HSA was carried out using a solution of the FSB. Removal of newaza is provided reagents was performed three times with washing tablets solution FSB with 0.05% tween-20 (FSBT).
Polypeptides A1 and A2, diluted in FSB, pH 8.0 to 2 µg/ml, was adsorbing onto the tablet for 18 hours at 4°C. After three laundering in tablets made of the diluted labeled HSA. Incubation was performed at 37°C for 1 hour. Next tablets 3 times washed from unbound reagents. The activity of the enzyme label is horseradish peroxidase was determined using the Chromogen - tetramethylbenzidine (TMB), dissolved in 0.1 M citrate-phosphate buffer, pH 5.0, with the addition of hydrogen peroxide. The reaction was stopped by adding 2N sulfuric acid. The optical density was determined at a wavelength of 450 nm multiscene.
It is established that the polypeptide A2 has a higher HSA-binding activity than the polypeptide A1, as demonstrated in figure 6. The difference in activity is: A2 activity higher than that of A1 2 times.
Figure 6. (Compare the efficiency of binding of the polypeptide A1 and A2 with HSA):
white column - polypeptide A1
a black bar - polypeptide A2
on the x - axis cultivation labeled HSA
on the y - axis optical density (OD450).
Example 6. Binding of serum labeled HSA polypeptides A1 and A2 in solution.
In the case of indirect ELISA solid phase was adsorbing unlabeled HSA to which mortar was joined by molecules of A1 or A2, in turn linking from a solution labeled pen is sidati HSA.
For conducting the ELISA used tablets NUNC MaxiSorb (Danemark).
Preparation of diluted drugs labeled HSA was carried out using a solution of the FSB. In this case I senzibilizirani HSA dissolved in the FSB, for 18 h at 4°C. Unbound HSA washed three times by adding 150 ál PBST to each well was added in two-fold dilution of the recombinant polypeptide, starting with 20 μg/ml, incubated 1 hour at 37°C and again three times washed PBST. Polypeptide, contacting the HSA was determined using the HSA conjugate with horseradish peroxidase. The results were recorded as a direct method.
In the experiments used in parallel at least four rows of titration. The results showed that the polypeptide A1 and A2 in the solution bind HSA. However, A2 binds to HSA with greater activity than A1 (Fig.7). Activity in A2 is higher than A1 2 times.
7. (Compare the efficiency of binding in solution polypeptide A1 and A2 with HSA):
the dashed line is the polypeptide A1
the solid line is the polypeptide A2
the abscissa shows the concentration of polypeptides, ng/ml
on the y - axis optical density (OD450).
For the production inhibitory ELISA polypeptides A1 and A2 at a concentration of 1 μg/ml) were immobilized on the tablet. To them were added in advance preincubating mixture of the corresponding polypeptide and diluted labeled HSA (ratio :1, incubation at room temperature for two hours) Incubation of the immobilized polypeptide with a mixture of polypeptides and labeled HSA was carried out for 1 hour at 37°C. the values of optical density expected percentage of inhibition of the interaction of labeled HSA with adsorbed molecules A1 and A2 each of the polypeptides in solution (Fig). Polypeptides A1 and A2, while in solution, prevented the interaction of labeled HSA with adsorbed molecules A1 and A2 within 70-94%.
On Fig. (Compare the efficiency of binding of HSA adsorbed polypeptides A1 and A2 and polypeptides A1 and A2 in the solution):
triangles - polypeptide A1
diamonds - polypeptide A2
on the x - axis cultivation labeled HSA
on the y - axis inhibition, %
Example 7. The method of determination of microalbuminuria.
Quantitative determination of microalbuminuria by the method of x-ray fluorescence analysis was carried out as follows: on a hard surface polystyrene tablet immobilizovana polypeptide A2, which inflicted analyzed urine samples simultaneously with labeled HSA. When this formulation albumin samples and labeled HSA compete for the HSA-binding polypeptide immobilized on a solid phase. The concentration of albumin in the analyzed sample is inversely proportional to the enzymatic activity of the solid phase. Conditions analysis, both the providing the necessary sensitivity and specificity, the following: as a result of the experiments was selected working dilution of labeled HSA 1:8000 at a concentration of immobilized polypeptide A2 from 0.1 to 0.25 microgram/ml After the selection of the optimum conditions were building a calibration curve of the dependence of optical density on the concentration of HSA in the standard samples(3,125; 6,25; 12,5; 25; 50 and 100 μg/ml) (Fig.9). The concentration of albumin in the urine found in the values of optical density calibration curve.
Figure 9. (Standard curve of optical density on the concentration of HSA):
the abscissa shows the concentration of HSA, ug/ml
on the y - axis optical density (OD450).
Example 8. The creation of the microchip for the determination of microalbuminuria
As matrix microchip for applying urine samples of patients with diabetic nephropathy was selected nitrocellulose membrane. On the nitrocellulose membrane, the size of 1 cm2were applied 30 urine samples using automatic calibrator. The first row on the membrane corresponded to the dilutions of the standard preparation HSA (6 double breeding HSA, starting with 100 μg/ml) to construct the calibration curve. The membrane was preincubation in blocking solution (2% milk dissolved in FSB) with peroxidase labeled HSA-binding polypeptide A2. Then the membrane was washed and stained with a solution parafine the diamine with hydrogen peroxide. On the membrane clearly manifested spots, different degrees of staining which corresponded to different concentrations of albumin in the standard samples and urine samples (figure 10).
Figure 10. Nitrocellulose membrane with manifest spots, which correspond to the concentration of HSA in urine samples of patients:
first row - breeding standard drug HSA
other numbers of urine samples of patients
Example 9. Obtaining a highly purified preparation of HSA from serum by affinity chromatography and liberation of the blood serum from the HSA. The simultaneous release of serum from HSA and obtaining highly purified preparation of HSA was carried out as follows: on a column Packed with the sorbent Affi-Gel 10 with attached recombinant polypeptide A2, inflicted a sample of blood serum. Unbound proteins were collected in the washing of the column with a solution of the FSB. The elution of contacting the sorbent HSA was performed with 0.1 M glycine buffer, pH of 2.2. The eluate was collected fractions of 500 ál, the concentration of protein was determined spectrophotometrically by the values of optical density (OD) at a wavelength of 280 nm. Fractions with the highest OD value were combined and the pH was brought to 7.5 with 2 N caustic soda solution. Samples of the original serum samples of serum proteins, not contacting the sorbent in the column and sample elyuirovaniya from the column were tested for the presence of HSA using meth is Yes electrophoresis SDS-PAGE. SDS-PAGE showed that the serum after passing it through the column, were exempted from the HSA. At the same time in the eluate was HSA, which was obtained in a highly purified state (Fig. 11). The release of serum from the HSA allows you to detect proteins, previously screened HSA. In Fig. 11. (SDS-PAGE purified preparation HSA):
1 - molecular weight marker, (top to bottom 97, 66, 36, 21 and 14
2 - purified preparation of HSA
3 - commercial preparation of HSA (Sigma)
1. Recombinant polypeptide A2, which has the ability to selectively bind human serum albumin HSA and characterized by the amino acid sequence of 346 amino acids presented in figure 4, in which the first 13 amino acids, is encoded by the sequence of the plasmid pQE 32, covalently associated with subsequent 333 amino acids encoded by the sequence of HSA - binding fragment of chromosomal DNA of strain DG 13 Streptococcus group G-G.
2. Recombinant DNA RA2, the nucleotide sequence of which is presented on figure 3, encodes 333 amino acids of the polypeptide A2 according to claim 1, beginning with 14 of its amino acid sequence, with the first 39 BP figure 3 belong to the sequence of the plasmid pQE 32, covalently linked to DNA RA2.
3. Recombinant plasmid DNA pQE 32-RA2 represents plasmid DNA pQE 32, carrying the recombinant DNA according to the .2 and providing the polypeptide A2.
4. The strain of E.coli M15 transformed with recombinant plasmid DNA pQE 32-RA2 according to claim 3 and expressing the polypeptide A2 according to claim 1.
5. The method of quantitative determination of HSA in urine, including the construction of a calibration curve of optical density on the concentration of HSA in the standard samples 100; 50; 25; 12,5; 6,25; 3,125 μg/ml, immobilization of the polypeptide A2 according to claim 1 on a solid phase tablet, incubation, drawing on it analyzed urine samples simultaneously with horseradish peroxidase HSA at a dilution of 1:8000, developing a tetramethylbenzidine (TMB), the measurement of optical density and establishing the amount of HSA in the samples by the values of the calibration curve, and for determining in the urine HSA at a concentration over 100 mcg/ml sample diluted a solution of phosphate-saline buffer.
6. The method of determination of HSA in urine according to claim 5, characterized in that the solid phase is polystyrene surface tablet.
7. Method qualitative determination of HSA in urine, characterized in that the determination is carried out by applying 30 urine samples on nitrocellulose membrane with a size of 1 cm2with the first number of dilutions of the standard HSA 100; 50; 25; 12,5; 6,25; 3,125 μg/ml, incubation of samples in a blocking solution of 2% milk in phosphate-buffered saline with peroxidase labeled the polypeptide A2 according to claim 1, washing the specified buffer solution, staining HSA dissolve the om paraphenylenediamine with hydrogen peroxide and a visual comparison of the degree of staining of the urine samples with staining dilutions of the standard HSA.
8. The method of determination of HSA in urine according to claim 7, characterized in that the solid phase is a nitrocellulose membrane.
9. The application of the recombinant polypeptide A2 according to claim 1 as a reagent for the removal of HSA serum column with affinity sorbent.
SUBSTANCE: invention refers to diagnostic techniques for hepatic fibrosis in an individual involving urokinase plasminogen, matrix metalloproteinase 9 and β-2-microglobulin expression tests to derive a score and to diagnose. Also, the present invention refers to a diagnostic kit for hepatic fibrosis comprising a first antibody specifically bound to an urokinase plasminogen activator (uPA), a second antibody specifically bound to matrix metalloproteinase 9 (MMP9) and a third antibody specifically bound to β-2-microglobulin (β-2-MG).
EFFECT: higher diagnostic accuracy.
17 cl, 33 tbl, 3 ex
SUBSTANCE: immunoenzymometric analysis of blood serum TIMP-1 and TIMP-2 is combined with determining a primary tumour size according to the TNM classification, a degree of tumour differentiation, aging the patient and calculating a discriminant functions from the equations: Y1=-138.748+X1*15.963+X2*(-4.803)+X3*0.018+X4*2.319+X5*1.188 Y2=- 159.5454-X1*17.918+X2*(-4.266)+X3*0.028+X4*2.427+X5*1.242, wherein X1 is the primary tumour size according to the TNM classification, X2 is the degree of tumour differentiation, X3 is blood serum TIMP-1, ng/ml, X4 is a patient's age, years old; X5 is blood serum TIMP-2, ng/ml. If Y1>Y2, a high probability of the favourable outcome of the chemoradiation therapy is predicted. If Y1<Y2, a probability of no effect of the chemoradiation therapy is predicted.
EFFECT: more accurate, informative and effective treatment of the patients suffering malignant new growths of the head and neck.
SUBSTANCE: what is found is a pre-therapeutic rate of haemopoietic stem cells with immunophenotype CD34+CD45 low among peripheral lymphocytes at the stages T3 or T4 to be compared to its discrimination level of 6.0×10-4. If the value occurs to exceed 6.0×10-4, a high radiosensitivity of the tumour is predicted, while the value of 6.0×10-4 or less enables predicting a low radiosensitivity of the tumour.
EFFECT: using the presented method enables predicting the radiosensitivity of the malignant growths to irradiation for the purpose of indicating for the radiation therapy in the patients with the stage T3 or T4 of airway cancer.
SUBSTANCE: cartridge (100) for detecting target components in a liquid sample comprises: a sample chamber (SC); at least two reservoirs (131, 132) filled with magnetic particles(MP, MP'), which are specific for different target components; at least two sensitive zones (121, 122) for detecting magnetic particles and/or target components, wherein magnetic particles (MP, MP') of different reservoirs primarily reach different sensitive zones while migrating into the sample filling the sample chamber under the effect of an activation magnetic field (B). The group of inventions also relates to a method of detecting target components using said cartridge, a device for detecting target components having said cartridge, a magnetic field generator and a sensor unit for detection inside the cartridge and to use of said device for molecular diagnosis, biological analysis of samples or chemical analysis of samples.
EFFECT: group of inventions enables to conduct multiple analyses in a single chamber without cross-reactions.
14 cl, 7 dwg
SUBSTANCE: there proposed is an antibody specific to TENB2 containing light and heavy chains. Heavy chain contains substitution for free (reaction capable) cysteine A121C that corresponds to A114C (Kabat numbering) or A118C (Eu numbering). Conjugate versions are proposed for prostate cancer treatment containing antibody covalently bound to auristatin, also by means of linker. The following is described: pharmaceutical composition for prostate cancer treatment that uses as active beginning the antibody or its conjugate; product for prostate cancer treatment on the basis of such composition. The invention proposes: method for defining protein TENB2 in the sample - on the basis of antibody as well as analysis for revealing prostate cancer cells at mammal and method for cell proliferation inhibiting on the base of antibody conjugate and auristatin. There described is the method for obtaining antibody conjugate (Ab) and auristatin (D) with expression Ab-(L-D)p, where p is equal from 1 to 4, and L is linker.
EFFECT: invention application provides conjugates with increased stability in serum in comparison with the same conjugates without A121C substitution in antibody that can be used in medicine.
33 cl, 18 dwg, 2 tbl, 4 ex
SUBSTANCE: pulmonary tuberculosis with multiple drug resistance is predicted in the patients newly diagnosed with pulmonary tuberculosis (PT) by determining the pre-therapeutic blood CD45R0+ memory T-cell, CD3+CD4(CD25+hl and CD4+CD25+Foxp3- regulatory T-cell, CD3+CD4+CD25- T-helper counts. Linear classification functions are calculated by equations: d1=-14.0015-0,0626xx1+0,.5181xx2+2.4285xx3+0.2255xx4 and d2=-22.6954-0.1710xx1+0.6689xx2+3.1202xx3+0.3229xx4, wherein x1, x2, x3, x4 are the numbers of the CD45R0+ memory T-cell, CD3+CD4+CD25+hi and CD4+CD25+Foxp3- regulatory T-cell, CD3+CD4+CD25- T-helper subpopulations, respectively. If observing d1<d2, tuberculosis with agent's multiple drug resistance is predicted.
EFFECT: enabled high-probability prediction of a tuberculosis therapy option and applicability of therapeutic correction measures.
5 dwg, 2 tbl, 2 ex
SUBSTANCE: original urine sample is divided into two equal samples; particle size histogram is prepared and used to find a percentage of an oligomer form of Tamm-Horsfall protein T&HE(7); the sample histograms are compared. A Tamm-Horsfall protein identification protein is presented by monoclonal antibodies undergoing an immune-affine reaction with Tamm-Horsfall protein. The second sample is used to recover Tamm-Horsfall protein T&HE(28); and both urine samples are used to measure the oligomer form of Tamm-Horsfall protein T&HE(28) in the free condition and in aggregates with oxalate microcrystals (%). The relation of the derived oligomer forms is calculated by formulas: C1=T&HE(28)A/T&HE(7) and C2=T&HE(28)F/T&HE(7). If observing C1>1.5 and any C2, urolithiasis is diagnosed; the relations C1<0.1 and C2<1.81 enable diagnosing the absence of the disease, while the relations C1<1.5 and C2>1.81 make the patient be referred to a risk group of developing urolithiasis. The technique enables distinguishing between those suffering urolithiasis and healthy ones among the persons being tested, and also separating a risk group of developing urolithiasis.
EFFECT: technique provides high accuracy, specificity and sensitivity, requires no heavy expenses and continuous study time.
4 tbl, 3 dwg
SUBSTANCE: invention represents a new diagnostic technique based on the detection of protein molecules being antibodies to a specific and sensitive combination of tumour associated glycans with underlying the patient's individual characteristics. The given invention can find application in immunology and medicine for the study and diagnosis of breast cancer. The diagnostic technique for breast cancer involves detecting the human blood antibodies to the following glycans: Manβ1-4GlcNAcβ, Galβ1-4G1cNAcβ1-6(Galβ1-3)GalNAcα, HOCH2(HOCH)4CH2NH2, GlcAβ, Galα, 6-O-Su-GlcNAcβ, GalNAcβ1-3Galβ, 6-O-Su-Galβ1-4(6-O-Su)Glcβ, GlcAβ1-SGa1β, Neu5Acα2-3Gaiβ1-4Glcβ, GlcNAcβ1-3Galβ1-3GalNAcα, Neu5Acα2-3Galβ1-3GalNAcα, Neu5Acα2-3Galβ1-3(Fucα1l-4)GlcNAcβ, Gaiβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Gaiβ1-4GlcNAcβ, Neu5Acα2-6(Galβ1-3)GlcNAcβ1-3Galβ1-4Glcβ with using a microchip with applied glycans; if the blood antibody level turns out to be higher than a threshold value to the above glycans simultaneously, breast cancer is diagnosed.
EFFECT: method enables improving the diagnostic sensitivity and specificity in breast cancer.
1 dwg, 1 tbl, 1 ex
SUBSTANCE: prescribing the specific chemotherapy is preceded by the immunological examination of peripheral blood of the patients suffering pulmonary tuberculosis and the determination of cell, humoral and molecular-genetic parameters of the patient's immune status. The peripheral blood CD4+CD25-Foxp3+ regulatory T-cell count more than 2.6%, the peripheral blood CD4+CD25+Foxp3+ regulatory T-cell count more than 5.1%, the peripheral blood CD3+CD4- CD25+ regulatory T-cell count more than 0.2%, the peripheral blood CD4+CD25+Foxp3- regulatory T-cell/activated T-helper count less than 25.5%, the peripheral blood CD3+CD4+CD25- T-helper count less than 35.4%, the peripheral blood γδT-lymphocyte count less than 4.9%, the peripheral blood CD45R0+ memory T-cell count less than 8.9%; 1L-2 less than 22.3 pg/ml, IL-4 more than 40.0 pg/ml, IL-10 more than 25.3 pg/ml, TGFβ more than 1109.0 pg/ml in a combination with the carriage of the allele C and genotype GG T-330G of IL2 gene and the allele A and genotype AA C-592A of IL10 gene, of the allele T and genotype TT C-509T of TGFB gene, and the allele T and genotype TT C-590T of IL4 gene, secondary immunodeficient disease is diagnosed.
EFFECT: invention enables the well-timed diagnosing of the immune system defects in the patients with pulmonary tuberculosis and provides the measures of therapeutic modality correction with using the advanced method of immunomodulatory therapy.
6 dwg, 4 tbl, 1 ex
SUBSTANCE: diagnostic technique for the autoimmune versions of diabetes mellitus, with the autoimmune versions of diabetes mellitus being diagnosed by comparing the production of tumour necrosis factor alpha (TNFα) (pcg/ml) in supernatant after lymphocyte culture taken from patient's blood sample in the autoantigen insulin stimulation environment. The test is considered to be positive if the TNFα in the insulin post-stimulation sample increases its value in the no-stimulation environment.
EFFECT: invention enables diagnosing the autoimmune inflammatory process in the pancreatic tissue that enables assessing a degree of autoimmune aggression on the pancreatic tissue.
SUBSTANCE: oral load of 10% potassium chloride 0.55 mm per kg of body weight by pressure chamber immersion at a depth of 60 meters and staying there for 1.5 hours is followed by calculating a kaliuretic renal activity index (KRAI) by formula: KRAI=0.2·K1-0.1·H1-0.2·D2-0.2·K2+0.2·K3+0.1·X, wherein: K1 is renal potassium excretion 40minutes after the water load, mmole/h; H1 is sodium excretion 60 minutes after the water load, ml/min; D2 is diuresis 60 minutes after the water load, ml/min; K3 is renal potassium excretion 60 minutes after the water load, mmole/h; K3 is renal potassium excretion 90 minutes after the water load, mmole/h; X is chloride excretion 90 minutes after the water load, mmole/h; if the derived value is less than 0, the kaliuretic renal function is considered to be inadequate; the value falling within the range of 0 through 1.0 shows the adequate kaliuretic renal function, while the value of 0.1 and more makes the good function be stated.
EFFECT: more accurate estimation of the human kaliuretic renal function in high pressure gas medium by comparing the clinical findings.
SUBSTANCE: analysed sample is treated with a protein-depositing reagent which is acetone; the extract is separated from the precipitate by filtering; acetone from the filtrate is evaporated in an air current at room temperature; the aqueous residue is diluted with water; the formed solution is treated with ammonium sulphate, extracted with 0.9 mol/dm3 of dimethyl phthalate solution in a 1,4-dioxane-chloroform mixture, taken in volume ratio of 1:1, with ratio of the aqueous phase to the organic phase of 5:1 by volume; the organic extractant is separated, and the amount of the analysed compound transferred to the extract is determined from the optical density of the extract which is diluted tenfold with 1,4-dioxane while measuring at wavelength of 279 nm.
EFFECT: high degree of extraction of azathioprine from biological fluids.
2 ex, 3 tbl
SUBSTANCE: method for prediction of recurrent cervical cancer involves biochemical daily urine analysis to determine daily urine androsterone and etiocholanolone to be related; if the relation is 0.75 mg/day or less, the recurrent disease is predicted for the first 2 years, and if the relation exceeding 0.75 mg/day, a prolonged recurrence-free period up to 10 years or more is predicted.
EFFECT: method enables the early, preclinical detection of the recurrent disease, as well as an individual selection of therapeutic approaches to each patient.
SUBSTANCE: what is considered is child's urine 6-sulphatoxymelatonine, and if the derived value appears to exceed 41.0 ng/ml, the standard therapeutic course is stated to be inadequate and to be prolonged by prescribing melatonine, and essential surgical management is regarded.
EFFECT: method provides a rapid and non-invasive technique for specifying the optimal therapeutic approach to children suffering gastroesophageal reflux disease.
SUBSTANCE: invention concerns medicine, particularly surgical diagnostics. The method involves measuring and analysing the pre-, intra-, and postoperative clinical, laboratory, instrumental and operative parameters. Age and blood pressure are taken into consideration. There are also determined fibrinosis, proteinuria, leukocyturia, low hematocrit. Besides, leukocytosis, peripheral blood immature neutrophils, increased ESR are evaluated. The immunoregulatory index variations are studied. The mental status is described. Levels of receptor antagonist interleukin, anemia, stab or segmental shift are determined. Hyperglycemia, lymphocytopenia, hematuria are taken into account. The ECG shows the conduction process variations. Additional evaluation is to be done to cover hypotension, hypofibrinogenemia, interleukin-8 (IL-8), low negative T waves on the ECG. A laparostomic wound, a phlegmon of the anterior abdominal wall, hyperbilirubinemia are assessed within the operative findings. Each value derived is assigned with a percentage. A severity of the patient's condition is determined by the percentage ratio of each factor in the overall prediction of a relative risk of death of 10 to 82%. And the risk of death of 10-39% shows the degree I severity of the patients. And the risk of death of 40-56% shows the degree II severity of the patients. And the risk of death of 57-82% shows the degree III severity of the patients.
EFFECT: method provides higher prediction accuracy of these group of patients with respect to the risk of death.
1 ex, 1 tbl
SUBSTANCE: method involves successive freezing at temperature 0-4°C for one hour, filtering through glass fibre and centrifuging at 3000 g for 15 minutes of a urine sample obtained from a patient, diluting with water in volume ratio 1:25, measuring, in the obtained urine test solution, potential relative a comparison electrode and determining concentration of novocaine and lidocaine using a calibrated curve.
EFFECT: faster and simpler method, high reliability and safety of analysis.
1 ex, 3 dwg
SUBSTANCE: daily urine of the premenopausal patients suffering breast cancer is examined for a pre- and post-menopausal level of androsterone. The pre- and post-treatment androsterone contents are related to derive a ratio. If the ratio exceeds 1, breast cancer dissemination is expected for the first 2 years following the anti-tumour chemotherapy. The ratio below 1 testifies to prolonged remission.
EFFECT: use of the method enables objective patient control, planned individual therapeutic approach, with including the patients into a group of risk for intensive care and applying the measures for the well-timed treatment.
1 tbl, 2 ex
SUBSTANCE: what is described is a method for evaluating calcium reabsorption (CaR) in disturbed homeostasis which involves blood sampling, centrifugation to separate blood serum and analysed for creatinine; 1.5-2.0 ml of blood serum is added with 5% carbogene in nitrogen, and a blood serum ultrafiltrate is prepared by using a filter trapping molecules of molecular weight exceeding 50000 MW; the ultrafiltrate is examined for the calcium concentration in mole/l (CaUF); the blood sampling is combined with urine sampling after the intake of distilled water 300-500 ml; then urine is analysed for the calcium and creatinine concentrations; it is followed by creatinine clearance (GFR), evaluating calcium excretion from 1 litre of a glomerular filtrate (CaE/GFR), therefor the urine calcium concentration is multiplied by minute diuresis, and the derived value is divided on creatinine clearance; further CaUF is subtracted from CaE/GFR to derive CaR, to draw a graphical diagram of the values CaR on CaUF, to determine a tolerance range boundary specific for healthy people; provided the values calculated for the patient lay within the tolerance range, normal calcium reabsorption is stated; if the specified values are higher than the tolerance range, increased calcium reabsorption is stated, while the values laying lower than the tolerance range show decreased calcium reabsorption.
EFFECT: method enables the reliable evaluation of Ca reabsorption in the patients with higher or lower blood plasma Ca concentration.
1 ex, 1 dwg
SUBSTANCE: there are involved recording clinical and laboratory manifestations of a CNS injury during the first days of the disease that is followed by the calculation of total diagnostic coefficients related to the detected graduation levels of pathognomonic signs of the disease. Total (+)7 points and more is related to predicting the developing mixed tick-borne encephalitis and borreliosis infection. Total (-)8 points and less shows the developing potential monoinfection of tick-borne encephalitis. If deriving the intermediate values of total diagnostic coefficients when none of said limits is reached, the prognosis is uncertain.
EFFECT: using the method for stating basic tendencies of the developing pathological process at the early stages of the developing disease.
1 tbl, 2 ex
SUBSTANCE: fresh morning urine is analysed for an albumin level (H). The values H≥0.15 g/l enable predicting developing recurrent angina following myocardial revascularisation.
EFFECT: method shows no invasiveness and provides higher reliability of the prediction of developing recurrent angina following myocardial revascularisation.
SUBSTANCE: method is characterised in that the DNA of the structure RNAb indicated on Figure 1, which encodes the fused protein of three parts, where N-terminal position is green fluorescent protein GFP, central - peptide of 73 amino acid residues with the amino acid sequence of SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and C-terminal - light chain of double-stranded protein Kunitz-type inhibitor from potato tubers (PKPI-BI), are introduced into cells of E. coli. The cells transformed by this construction are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifugation. The product of expression in the form of inclusion bodies is solubilised with the denaturant. Chromatography is carried out under denaturing conditions. The resulting product is used for detection of specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.
EFFECT: invention enables to obtain the recombinant antigen G2 of Hantavirus Dobrava with increased yield.
6 dwg, 1 ex