Method and set for immunoenzymometric determination of functional activity of c1 inhibitor on alternative path
SUBSTANCE: pharmaceutical preparation CIP (complex immunoglobulin preparation, consisting of IgG, IgA and IgM) is bound in cavities of a micropanel, said preparation containing a C3b complement component, samples containing the determined functionally active human C1 inhibitor are incubated in the cavities, and the bound C1 inhibitor is determined using a conjugate of an antibody against the human C1 inhibitor with an enzyme and a substrate for that enzyme based on the amount of the formed product of the enzymatic reaction. The set contains flat-bottomed micropanel with the sorbed CIP, the enzyme conjugate with antibodies against the human C1 inhibitor, a substrate buffer of that enzyme and a standard for the active C1 inhibitor.
EFFECT: use of the method enables to determine activity of the C1 inhibitor which regulates the complement alternative path.
2 cl, 1 dwg, 1 tbl, 2 ex
The invention relates to medical immunology, and in particular to methods for determining the functional activity of complement components in the serum of human blood in the diagnosis of several diseases and biological agents.
C1 inhibitor of the complement system as an inhibitor of serine proteases involved in the regulation of many enzymes in the blood plasma, such as C1r, C1s components of the classical pathway of complement, kallikrein kallickrein-kinin system, as well as factors of coagulation and anticoagulation systems - XIa, Hu, XIIf and plasmin (fibrinolizina) . It was also discovered its ability to inhibit serine proteases pectinophora way of complement . However, recently opened a new function C1 inhibitor is its ability to regulate the alternative pathway of complement by interacting with a component 3b complement and inhibiting the binding factor In complement with 3b . This function is chemically different from the already known at the time of its implementation of the C1 inhibitor does not block covalently bound to the active center serine proteases, as it was in all the above cases, and is associated ecovalence component 3b, inhibiting the formation or destabilizing the final factor In .
The deficiency of this inhibitor in the patient's blood leads to periodic edema, setrag the living mainly the limbs, the face, throat and mucous membrane of the gastrointestinal tract.
Inherited deficiency of C1 inhibitor can be of two types. Type I is caused by the low concentrations of C1 inhibitor. When type II is produced by C1 inhibitor with reduced or absent functional activity, but normal or elevated concentrations . Because type II is caused by mutations of the gene C1 inhibitor, is currently not known, are affected by these mutations on both the functional activity of C1 inhibitor: inhibition of the classical pathway and regulation of the alternative pathway of complement. Immunnofermentye methods for determining the activity of C1 inhibitor regulating the alternative pathway of complement, is not known.
Objective of the claimed invention is a method and kit for immunoassay determination of the functional activity of C1 inhibitor on an alternative path.
This object is achieved by developing a method for determining the functional activity of C1 inhibitor, which provides for binding in the hole microarrays component 3b, incubation in the wells samples containing defined functionally active C1 inhibitor man, and determination of bound peroxidase C1 inhibitor using conjugate antibodies against C1 inhibitor of the human complement with the enzyme and substra the ohms for this enzyme. For fixing in the hole microarrays 3b spend sorption pharmacy drug KIP (complex immunoglobulin preparation, consisting of immunoglobulins G, a and M people). It has been shown (see table 1)that in the samples of the commercial product KIP contains component C3 of the human complement in trace amounts that were sufficient for specific binding of C1 inhibitor.
|The content of component C3 in various series commercial complex immunoglobulin preparation preparation|
|Sample||Protein, mg/ml||C3, µg/mg protein|
|6||61,6||The set contains a flat-bottomed to something called a microarray with an associated drug KIP, the enzyme conjugate with antibodies to C1 the inhibitor man, substrate buffer and the standard for active C1 inhibitor.|
The technical result of the claimed invention is to develop a method and kit for determining the functional activity of C1 inhibitor of the human complement by the alternative pathway of complement by using as source component 3b complement pharmacy drug KIP.
Example 1. Determination of the functional activity of C1 inhibitor in alternative ways. Dissolve Pharmacopeia drug KIP at a concentration of 40 µg/ml in 0.05 M sodium carbonate buffer, pH of 9.5, and contribute 100 ál of the solution into each well of flat-bottomed polystyrene 96-well microarrays. Close the lid and leave overnight at 4°C. washed Three times to something called a microarray veronalum buffer solution, pH 7.4, containing 0.15 M NaCl and 50 mm EDTA (ethylenediaminetetraacetate sodium), filling in 150 ál into each well, and then to something called a microarray dry by shaking out the remaining liquid. In wells microarrays make the sample containing C1 inhibitor with unknown activity. After incubation in an incubator for 1 h at 37°C., washing twice with phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.05% tween-20, and drying Microban is whether in each well contribute 100 μl of peroxidase conjugate with antibodies against C1 inhibitor man in the same buffer at selected breeding. After incubation in an incubator for 1 h at 37°C, five times washing with detergent and drying the tablet into each hole making 100 μl of substrate buffer (3,3',5,5'-tetramethylbenzidine in 15 ml of citrate-phosphate buffer, pH 5.0, and 50 μl of 3% hydrogen peroxide). After 15-30 min incubation in the dark, the reaction is stopped by the introduction to each well 50 μl of 14% sulfuric acid. The results of the reaction consider using a spectrophotometer with a vertical beam of measuring light absorption at 450 nm. The functional activity of the component C1 inhibitor is calculated by the standard curve (figure 1).
Example 2. Kit for determination of the functional activity of C1 inhibitor in alternative ways. The set contains a flat-bottomed to something called a microarray adsorbed drug KIP, peroxidase conjugate with antibodies to C1 the inhibitor man, substrate buffer and a standard with a known activity of C1 inhibitor man. This set is used in accordance with example 1.
From the above figure results, it follows that the measured optical density is linearly dependent on the concentration of active C1 inhibitor with correlation coefficient R2=0,999, which allows to reliably determine the functional activity of C1 inhibitor in alternative ways in concentrations from 1 ng/ml in the described manner by using the described set.
Cu is IU, carried out a determination of the functional activity of C1 inhibitor in the serum of a patient with hereditary angioneurotic edema type II, i.e. with a functional deficiency of this protein. The methods developed previously [4, 5], it was found that the content of C1 inhibitor in the serum of this patient was 204 mg/ml, and the activity of the classical pathway inhibition corresponded to 93 µg/ml active. Determination of the functional activity of C1 inhibitor on alternative ways of the blood of this patient gave a value of 95 mg/ml active. This implies that the proposed method represents the activity of C1 inhibitor, and does not quantify as protein, and that the activity shown by C1 inhibitor of the alternative pathway of complement, in this particular case corresponds to its activity in the classical pathway.
1. Davis AE 3rd. CI inhibitor and hereditary angioneurotic edema. Ann. Rev. Immunol. 1988. V.66. P.595-628.
2. Matsushita M., Thiel, S., Jensenius J.C., Terai L, Fujita T. Proteolytic activities of two types of mannosebinding lectin-associated serine protease. J. Immunol. 2000. V.165. P.2637-2642.
3. Jiang H., Wagner, E., Zhang H., Frank, M.M. Complement 1 inhibitor is a regulator of the alternative complement pathway. J Exp Med. 2001. V.194. P.1609-1616.
4. Andina C.C., Kozlov, L.V., Dyakov V.L. Determination of functional activity, the number of C1 inhibitor and autoantibodies to it as a tool for differential diagnosis of edema. Biomedical chemistry 2004. T. No. 1. Pp.86-91.
5. Kozlov, L.V., Andina S., Husova VA, Dyakov V.L., Batalova so-CALLED. The method of determining the functional activity of C1-inhibitor of the human complement. RF patent №2195662. Bull. No. 36. 27.12.2002.
1. The method of determining the functional activity of C1-inhibitor of the human complement via the alternative pathway, characterized by the fact that the holes microarrays for enzyme immunoassay absorb pharmacy drug KIP, then in wells microarrays make a solution of the sample containing C1-inhibitor of the human complement of unknown activity, carry out incubation and after drying the tablet and washing the wells, making the enzyme conjugate with antibodies against C1-inhibitor and substrate of this enzyme, followed by calculation of the content of active C1-inhibitor on the amount of the formed product of the enzymatic reaction.
2. Kit for determination of the functional activity of C1-inhibitor on the alternative path, characterized in that it contains a flat-bottomed to something called a microarray adsorbed drug KIP, the enzyme conjugate with antibodies to C1-inhibitor man, substrate buffer and the standard for active C1-inhibitor.
Complex antigen of measles virus used as a component of immunoenzymometric test system for diagnostics of antibodies to measles virus // 2441666
SUBSTANCE: complex antigen of measles virus used as a component of immunoenzymometric test system for diagnostics of antibodies to measles virus was produced from culture liquid containing measles virus stain Leningrad 16 with titer no less than 105.0 50% tissue cytopathic dose/ml, Edmonston - no less than 106.0 50% tissue cytopathic dose/ml, NovO/96 no less than 108.0 50% tissue cytopathic dose/ml by inactivation of its infectious activity by detergent and separation of protein from cellular lysate by chromatographic purification in amount no less than 2 mcg/ml with the purity no less than 70%. Culture virus-containing liquid was processed using separate cultivation of measles virus stains Leningrad 16, Edmonston and NovO/96 on monolayer of Vero cells culture with subsequent mixing of culture virus containing liquid in proportion 1:1:1 v/v.
EFFECT: Usage of invention provides for increased sensitivity and specificity of a complex antigen possessing the property to detect antibodies in blood serum.
2 cl, 4 tbl, 6 ex
Dragoon strain of parotitis virus for obtaining antigene component of test system, and immunoenzyme test system for parotitis virus antibody diagnostics // 2348691
FIELD: chemistry; biochemistry.
SUBSTANCE: invention concerns medical virology and microbiology. Strain is deposited in culture collection of Federal State Research Institution State Research Centre of Virology and Microbiology "Vektor" of Rospotrebnadzor, under registration No VB-05. Strain features higher productivity. More sensitive immunoenzyme test system for hepatitis virus antibody diagnostics is created on the basis of this strain. Invention can be applied in virology.
EFFECT: production of more sensitive immunoenzyme test system for hepatitis virus antibody diagnostics.
2 cl, 1 dwg, 1 tbl, 4 ex
FIELD: medicine; ophthalmology.
SUBSTANCE: invention is intended for simultaneous verification of uveal melanoma and forecast of metastasis development. Immunohistochemical analysis of protein S-100 and melanin-A expression within tumour cell is accompanied with simultaneous estimation of S-100-positive cells number within visual field. Uveal melanoma is verified if reaction of S-100 and melanin-A is positive with forecasted high possibility of tumour dissemination in case number of S-100-positive cells number is less than 50.
EFFECT: simplified examination associated with high specificity and sensibility of simultaneously verified uveal melanoma and forecasted metastasis.
1 tbl, 1 dwg, 2 ex
FIELD: medicine, veterinary.
SUBSTANCE: specific antibodies to virus of goose enteritis are determined by IEA where virus received following injection of epizootic strain "P-75" of enteritis virus, is used as an antigen, and macro porous glass with diameter not less than 700 is used for purification of virus-containing material, the goose Ig G specific immunoperoxidase conjugate is used as an anti-specific agent, and the IEA results are calculated by use of the formula: titer = antiIg[2.02(lg S/P)+3.51], where S is for optical density of tested serum; P is for optical density of the positive control.
EFFECT: method reduces the test time and economical costs.
2 tbl, 2 ex
Immunoenzyme method and kit for determining iga1 and iga2 using polyclonal iga antibodies // 2310854
SUBSTANCE: method involves determining total IgA quantity using immunoenzyme method based on polyclonal IgA antibodies as binding antibodies on the micropanel and the same antibodies in conjugate with peroxidase for detecting bound IgA in sample before and after treatment with specific IgA1-protease. Total IgA1 and IgA2 content is determined before being treated with enzyme. IgA2 content only is determined after being treated with enzyme. IgA1 is not determined after being disintegrated with IgA1-protease. Kit has microplate containing absorbed polyclonal IgA antibodies, conjugate of the same antibodies with peroxidase, reference sample having given IgA concentration, IgA1-protease and substrate buffer.
EFFECT: simplified method and kit for determining IgA1 and IgA2; improved results reproducibility; no specific monoclonal antibodies being used.
2 cl, 1 tbl
SUBSTANCE: method involves absorbing myeloma IgA1 on micro panel, incubating solutions containing IgA1-protease and determining enzyme activity from IgA1 sorbate quantity reduction determined by conjugate with polyclonal IgA antibodies peroxidase. Determining IgA1-protease activity with inhibitors available in various concentrations enables one to calculate its inhibition constants. Kit has microplate containing absorbed myeloma IgA1 preparation, peroxidase conjugate with antibodies against human IgA and substrate buffer.
EFFECT: simplified method and kit for determining IgA1-protease activity and studying its inhibition process.
2 cl, 1 dwg, 1tbl
Method for differential diagnostics of viral gastro-intestinal infections in cattle due to immunoenzymatic assay // 2306567
FIELD: veterinary virology.
SUBSTANCE: the present innovation deals with interaction of antibodies with an antigen, with antibodies labeled with horseradish peroxidase, addition of substrate mixture and registration of reaction results. Moreover, one should apply plotting boards with presorbed antibodies and general immunoenzymatic conjugate. The innovation enables to shorten terms for diagnosis and obtain more significant results of diagnostics.
EFFECT: higher efficiency.
FIELD: biotechnology, analytical chemistry.
SUBSTANCE: claimed method includes providing of complexes between antigen molecules and specific antibodies on carrier surface, wherein said complexes are disclosed by addition of enzyme label thereto followed detection thereof based on formation of enzyme reaction product. Enzyme label addition is carried out by two protein interaction, namely bacterial ribonucleaze barnase and barstare, which is inhibitor thereof, wherein either of the two is added to immunoreagent, and the other one is added to enzyme label. Abovementioned complexes have high affinity, specificity, and binding constant of 1014 M-1.
EFFECT: new method for antigen detection.
6 dwg, 2 ex
Method for differential diagnostics of bovine brucellosis and a method of preparing a preparation for implementation thereof // 2300107
FIELD: veterinary and medicine.
SUBSTANCE: invention, in particular, relates to production and use of biological preparations intended for differential diagnostics of brucellosis and to a method of differentially diagnosing brucellosis. Method involves serologic analysis of sera using antigenic "IFK", which is horse-radish peroxidase-labeled electrophoretically purified polypeptide fraction of virulent strain B.arborus 54. Diagnosis of brucellosis is stated when anti-brucellosis antibodies in sick animal sera diluted to 1/100 and higher are revealed at CSP reaction intensity 2.1 and higher.
EFFECT: elaborated method increasing immuno-enzymatic test specificity and allowing performing differentiation of postvaccinal immunological reactions and postinfectious reactions induced by microorganisms having antigenic affinity with brucellas, especially Yersinia enteriocolitica.
2 cl, 4 ex
Antibodies specifically reacting with prionic protein or its fragment, and their application // 2281510
FIELD: medicine, biotechnology.
SUBSTANCE: the present innovation deals with elaborating diagnostic reagents for testing prionic protein in mammalian cerebral tissue due to IEA technique and refers to antibodies specifically reacting with prionic protein PrP or its fragment. The innovation includes polyclonal antibodies obtained to synthetic peptides including amino acid sequences of bovine prionic protein 143-168, 101-134 and 211-241, their conjugates with horseradish peroxidase and diagnostic reagents obtained upon their basis that enable to detect prionic protein in mammalian cerebral tissues.
EFFECT: higher specificity of detection.
15 cl, 8 ex, 8 tbl
Monoclonal antibodies and one-chain fragments of cell-surface prostate-specific membrane antigen antibodies // 2458073
SUBSTANCE: there are offered versions of: a SSMA monoclonal antibody and its antigen-binding fragment which are bound with a mark or a cytotoxic agent. There are described versions of a pharmaceutical composition and a diagnostic kit based on such antibodies. There are disclosed methods for identification in vitro of tumour cells, as well as for diagnostic identification of tumour cells based on such antibodies. What is described is an isolated polynucleotide for producing monoclonal antibodies.
EFFECT: using the invention provides the antibodies which can bind SSMA in its native form on the surface of tumour cells, are bound with LNCAP, but are not bound with cells with lost SSMA expression that can find further application in prostate cancer therapy.
15 cl, 21 dwg, 3 tbl, 18 ex
SUBSTANCE: method for prediction of a risk of chronic dust bronchitis in coal mine workers consists in blood examination for genetic markers. It involves the genetic markers of systems: haptoglobin (HP), group-specific component (GC), fluorescent esterase (EsD), erythrocyte acid phosphatase (AcP). A prognostic coefficient (PC) score is calculated. If the marker 1-1 is found in the HP system, the PC is specified to be equal to (+1.5); the marker 1-2 makes the PC to be equal to (+0.1), while the marker 2-2 shows the PC (-1.1). If the marker 1-1 is found in the GC system, the PC is specified to be equal to (-1.9); the marker 1-2 makes the PC to be equal to (+1.7), while the marker 2-2 shows the PC (+5.5). If the marker 1-1 is found in the EsD system, the PC is specified to be equal to (-1.2); the marker 1-2 makes the PC to be equal to (+2.3), while the marker 2-2 shows the PC (+3). If the AcP system comprises the marker (aa), the PC is equal to (-5); the marker (ab) shows the PC equal to (0); the marker (bb) provides the PC to be equal to (+3.6); the marker (ac) - the PC equal to (+2.2), the marker (bc) - the PC equal to (-2.4), the marker (ee) - the PC equal to (-3.5). The PC for all the markers are summed up; total PC being (+5) and more enables predicting the propensity for chronic dust bronchitis, while total PC equal (-5) and less shows resistance to chronic dust bronchitis.
EFFECT: use of the declared method enables higher accuracy of prediction of chronic dust bronchitis in coal mine workers.
1 tbl, 4 ex
SUBSTANCE: positive portion of the panel comprises patient's plasma samples positive to RNA VHS and is presented in the form of 4 boxes accommodating NS5, NS4, NS3 and Core antigen antibodies respectively. A negative portion of the panel comprises donor's plasma samples containing other than HIV-1/2, HBV, HCV, syphilis, HBsAg antibodies, as well as from HCV-noninfected risk groups with additionally included heterophillic serums with rheumatoid factor (RF) and with autoimmune antibodies for strict control of specificity.
EFFECT: method improvement.
5 cl, 5 tbl, 1 dwg
SUBSTANCE: sequential staining by Romanowsky-Giemsa and conducting an immunocytochemistry reaction are used to analyse destructive processes in epitheliocytes nuclei, specifically changed lymphoid and epithelial cells, high colonisation activity of opportunistic and pathogenic microorganisms, and phagocytic activity. If observing brown-black inclusions in cells under optical microscopy ensured by specific staining of the viral antigens in the cell structures, mono- or mixed herpes viral infection Herpex simplex 1, 2, EBV, CMV are diagnosed.
EFFECT: using the technique enables higher accuracy of etiological diagnosis, expressivity and reduced injures of material sampling for research.
SUBSTANCE: recipient of a cadaver kidney graft is examined to compare recipient's and donor's HLA alloepitopes. The alloepitopes found in the donor, but not found in the recipient are recovered. It is followed by comparing the recipient's HLA-DR antigens and the recovered alloepitopes, the derived combinations are compared with the following combinations: A9C-DR7; A10C- DR3; A10C-DR6; A28C-DR1; Bw4-DR6; B8C-DR3; B12C-DR2; B27C-DR5. Detecting said combinations enables stating an adequacy of the preventive application of monoclonal antibodies blocking an alpha-chain of an interleukin-2 receptor molecule. The preventive application of the monoclonal antibodies blocking an alpha-chain of the interleukin-2 receptor molecule shall be indicated to the patients with said combinations.
EFFECT: improved outcome of the cadaver kidney grafting that is combined with reducing the price of the method.
1 tbl, 2 ex
Method for estimating effectiveness of inhalation antibacterial therapy of hospital-aquired pneumonia caused by gram-negative flora // 2456614
SUBSTANCE: method is ensured by the double evaluation of total endotoxin (LPS) of gram-negative bacteria: prior to the beginning of the inhalation antibacterial therapy and one hour after the first antibiotic inhalation. It involves controlling the total endotoxin of the gram-negative bacteria in patient's blood serum by activated particle method. Double and higher endotoxin increase as compared with the reference testifies to a bactericidal action of the antibiotic and proves the effectiveness of the conducted antibacterial therapy.
EFFECT: using the method enables the early estimation of the effectiveness of the antibacterial therapy.
Method for prediction of reduced transport function of band-3 protein of red-cell membranes of newborns delivered by mothers suffered aggravated herpes viral infection in third trimester of pregnancy // 2456613
SUBSTANCE: invention describes a method for prediction of reduced transport function of band-3 protein of red-cell membranes of newborns delivered by mothers suffered aggravated herpes viral infection in the third trimester of pregnancy wherein newborn's peripheral blood is examined for the TNFa content; band-3 protein is detected in the newborn's red-cell membranes by disk electrophoresis in polyacryl amide gel (10%) with sodium dodecyl sulphate; a discriminant function is calculated by formula Df=(5.167×band-3)+(2.196×TNFα), wherein Df is the discriminant function, band-3 is red-cell membrane stromal protein, TNFα is newborn's blood interleukine, and if Df is more than 218.67, reduced transport anion function by protein band-3 through newborn's red-cell membranes is predicted.
EFFECT: invention provides detecting the effect of herpes viral infection on the content of protein band-3 in newborn's red-cell membranes.
SUBSTANCE: peripheral venous blood of the patients with autoimmune thyroiditis is examined for the absolute transferring receptor (CD71+) T-lymphocyte count and the interleukin-2 (IL-2) concentration. If the CD71+ T-lymphocyte count exceeds 0.3×109 cell/l, and the IL-2 concentration is less than 25 pg/ml, a favourable clinical course of the disease is predicted, while the CD71+ T-lymphocyte count less than 0.3×109 cell/l and the IL-2 concentration exceeding 25 pg/ml shows an unfavourable clinical course of autoimmune thyroiditis.
EFFECT: method provides more accurate and objective prediction with using the material, reagents and research methods widely applied in common clinical-laboratory practise.
SUBSTANCE: patient's peripheral blood is examined for the content of cytotoxic lymphocytes, %: (CD8/CD16), (CP8/GranzymeB), (CP16/GranzymeB), (CDS), (CD16/CD56) and (CD8/HLA DR). It is conmbined with determining cytotoxic activity (CTA), %. It is followed by calculating a RA activity index (AI) by formula: If observing the condition 11.8%<AI≤18.2%, a moderate degree of rheumatoid arthritis activity is diagnosed. If observing the condition AI>18.2%, a high degree of rheumatoid arthritis activity is diagnosed.
EFFECT: using the technique enables high-reliability differential diagnosis of the degree of rheumatoid arthritis activity.
Method for prediction of developing mixed tick-borne encephalitis and borreliosis infection // 2456603
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
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.