RU2195662C1 - Method of determining functional activity of human complement c1 inhibitor - Google Patents

Abstract

FIELD: immunochemical assays. SUBSTANCE: invention relates to determining functional activity of complement C1 inhibitor in human serum for diagnostics of a series of diseases and in biological preparations. Sorption is performed in wells of micropanel with fibrinolysin (pharmacy preparation of plasmin), after which solution to be analyzed containing aforesaid C1 inhibitor with unknown activity is placed in wells and the total is incubated. During incubation, inhibitor is covalently bound to fibrinolysin. After drying and washing of micropanel, conjugate of enzyme with anti-C1-inhibitor antibodies and enzyme substrate are placed into wells. Amount of bound C1 inhibitor is found on the basis of enzymatic reaction product by the aid of anti-C1- inhibitor antibodies, in particular fraction of immunoglobulins F covalently bound to enzyme. Method is based on ability of functionally active C1 inhibitor of binding to fibrinolysin. EFFECT: simplified procedure and reduced expenses. 1 tbl

Description

 The invention relates to medical immunology, and in particular to methods for determining the functional activity of complement components in human serum in the diagnosis of a number of diseases and in biological preparations.

 The most promising of modern methods for the determination of blood serum proteins is the enzyme-linked immunosorbent assay due to its sensitivity, selectivity and the ability to automate the analytical process. The US patent [1] used a modified method for determining the activity of a Cl inhibitor based on a test system manufactured by Cycotech (San Diego, California, USA). The method involves the following steps: 1 - avidin is adsorbed on the micropanels, 2 - a highly purified enzymatically active preparation of the Сls subcomponent is obtained, 3 - the biotinylated preparation of Сls is synthesized, 4 - samples of samples containing a functionally determined Cl-inhibitor are incubated, incubated with the biotinylated preparation of Сls, 5 - the resulting complex of a functionally active Cl inhibitor with Cl is adsorbed in the wells of a micropanel coated with avidin, 6 - the amount of Cl inhibitor bound in the wells is determined using a goat antibody conjugate otiv Cl-inhibitor with horseradish peroxidase and a chromogenic peroxidase substrate.

 The disadvantages of the method include its multistage nature (the use of an additional stage of biotin-avidin interaction, as well as the need to obtain biotinylated Cls) and the difficult availability of enzymatically active Cls (present in serum in proenzyme form in an amount of 30 mg / l, not commercially available).

 The objective of the claimed invention is to reduce the cost of determining the functional activity of the Cl-inhibitor of human complement based on the enzyme-linked immunosorbent assay and the simplification of the method by reducing the number of stages and the use of available drugs.

 The task is achieved by developing a determination method that provides for: sorption of fibrinolysin micropanel in the wells (a pharmaceutical drug of plasmin), samples of samples containing a functionally determined CL inhibitor are introduced into the micropanel wells and incubation is performed, during which covalent binding of the Cl inhibitor occurs with fibrinolysin, the amount of Cl inhibitor bound in the wells is determined using the conjugate of antibodies against the Cl inhibitor with horseradish peroxidase and a chromogenic substrate for perks idazy. The method is based on the ability of a functionally active Cl inhibitor to covalently bind to fibrinolysin.

 The technical result of the claimed invention is the development of a simplified method for determining the functional activity of a human complement complement inhibitor using available drugs.

Example. Determination of the functional activity of the drug Cl inhibitor. Dissolve the pharmacopoeial preparation of fibrinolisin in 0.05 M sodium carbonate buffer, pH 9.5, with a final activity of 1-10 units / ml and add 100 μl of solution to each well of a flat-bottomed polystyrene 96-well plate. Close the lid and leave overnight at 4 ^o C. Three times the tablet is washed with Veronal buffer solution, pH 7.4, containing 0.15 M NaCl, 0.15 mm Ca ²⁺ and 0.5 mm Mg ²⁺ (VBS ²⁺ ), 200 μl to each well, then the plate is dried by shaking out the remaining liquid. 100 μl of VBS ^{2 was} added to each well of the plate. 100 μl of a solution containing a detectable Cl inhibitor in VBS ²⁺ in the form of progressive two-fold dilutions is added to the wells of each row of tablets. After incubation in a thermostat for 1 h at 37 ^° C, washing three times with phosphate buffer, pH 7.4, containing 0.15 M MaCl and 0.05% Tween-20, and draining the plate, 100 μl of peroxidase conjugate are added to each well with antibodies against human Cl inhibitor in the same buffer in a selected dilution. After incubation in a thermostat for 1 h at 37 ^° C, five times washing with detergent and draining the plate, 100 μl of substrate buffer (10 mg of orthophenylenediamine in 25 ml of citrate-phosphate buffer, pH 5.0, and 50 μl 3 % hydrogen peroxide). After 30 minutes of incubation in the dark, the reaction is stopped by adding 50 μl of 14% sulfuric acid to each well. The results of the reaction are taken into account using a spectrophotometer with a vertical beam by measuring light absorption at 492 nm. The functional activity of the Cl inhibitor preparation is calculated from the standard curve obtained from the same experiment with the standard solution with the known activity of the Cl inhibitor.

 This method determined the activity of the Cl inhibitor in 10 samples of human serum and compared with its quantitative determination in the same sera by the standard method of radial immunodiffusion (RID). The results are shown in the table.

 From the table it follows that the results obtained by the two methods are consistent with each other, i.e. the claimed method really determines the functional activity of the complement inhibitor Cl. The small discrepancies observed in individual results are explained by the fact that the RID method is less accurate due to difficulties in measuring the diameters of the precipitation rings (the determined concentration is proportional to the square of the diameter), which causes an increase in errors.

LITERATURE
1. Pilatte YM, Hammer CH, Frank MM, Fries LF Process for the purification of Cl-inhibitor. Patent US 5030578. July 9, 1991.

Claims (1)

 A method for determining the functional activity of a human complement C1-inhibitor, characterized in that fibrinolysin is adsorbed in the micropanel wells for enzyme-linked immunosorbent assay, then a sample of the test sample containing human complement C1-inhibitor with unknown activity is added to the micropanel wells, incubated and, after the plate is dried and washed the conjugate of the enzyme with antibodies against the C1-inhibitor and the substrate of this enzyme are added to the wells, after which the content of the active C1-inhibitor is calculated by the number of razovavshegosya product of the enzymatic reaction.

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