RU2144676C1 - Method of assay of functional activity of human c1q subcomponent of complement - Google Patents

Abstract

FIELD: medicine, medicinal immunology. SUBSTANCE: invention relates to methods of assay of functional activity of complement components in human serum blood in diagnosis of some diseases and in biological preparations. Micropanel is sensibilized with nonspecific human or animal immunoglobulins G and M. Then solution containing subcomponent C1q, conjugate of immunoglobulins with enzyme and substrate for this enzyme are added into micropanel wells successively and C1q is determined quantitatively by the enzymatic reaction end product. In part, conjugate with nonspecific human or animal immunoglobulins G and M is used as conjugate of immunoglobulins with enzyme, namely the conjugate with immunoglobulins - antibodies raised to C1q. Method ensures to determine functional activity of the first component of complement system based on immunoenzyme analysis. EFFECT: simplified method, decreased cost. 3 cl, 3 ex

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. However, the methods available today for enzyme immunoassay of complement proteins allow only their content to be determined as antigen. Nevertheless, the most informative is the assessment of the functional activity of these proteins, which characterizes the development of the pathological process. As for the determination of the functional activity of complement components, the hemolytic method [1], which is not distinguished by a high degree of reproducibility and is reluctantly used by clinicians due to the difficulties associated with the preparation of a hemolytic system based on sheep erythrocytes, is practically the only one.

 We have developed a method that allows us to determine the functional activity of the Clq subcomponent of the first component of the human complement system based on the enzyme immunoassay.

 The method is as follows: sequential sorption of the immunoglobulins of classes G or M in the micropanel wells is carried out first, then determined by C1q from solutions containing it. The amount of sorbed subcomponent C1q is determined by the product of the enzymatic reaction using immunoglobulins covalently linked to the enzyme. The method is based on the ability of a functionally active subcomponent C1q to specifically bind to aggregated immunoglobulins of classes G and M of human or animal serum.

 Example 1. Determination of the functional activity of the drug C1q.

Immunochemically pure IgG was dissolved in 0.05 M sodium carbonate buffer, pH 9.5, at a protein concentration of 10 μg / ml and 100 μl of the solution was added 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 buffered saline with detergent (tween), then the tablet is dried by shaking the remaining liquid. 100 μl of a solution containing C1q in buffered saline with detergent, in the form of progressive two-fold dilutions, is added to the wells of a row tablet. After incubation in a thermostat for 1 h at 37 ^° C, three times washing with detergent and draining the plate, 100 μl of peroxidase conjugate with immunochemically pure IgG in selected dilution of buffered saline with detergent are added to each well. After incubation in a thermostat for 1 h at 37 ^° C and three times washing with detergent and draining the plate, 100 μl of substrate buffer (10 mg of orthophenylenediamine in 25 si 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 reaction results are taken into account using a spectrophotometer with a vertical beam measuring light absorption at 492 nm. The functional activity of C1q is calculated using a standard curve.

 Example 2. The determination of Clq activity in two samples of human serum was carried out according to the method described in example 1 and the standard hemolytic method [1]. The following results were obtained: C1q activity in serum N1 - according to the claimed method - 32 ± 10 heme.ed / ml, according to the hemolytic method - 32 ± 16 heme.ed / ml; C1q activity in serum N2 - according to the claimed method - 224 ± 64 heme.ed / ml, by hemolysis - 256 ± 128 heme.ed / ml.

 Example 3. The determination of the functional activity of C1q in blood serum is carried out analogously to example 1, only immunochemically pure IgM is used to sensitize the tablet, and the conjugate is a conjugate of specific anti-C1q antibodies with peroxidase.

Literature
1. Kolb WR, Kolb LM, Podack ER Clq: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay. J. Immunol. 1979. V. 122. N 5. P. 2103 - 2111.

Claims (3)

 1. A method for determining the functional activity of the human complement subcomponent C1q, characterized in that the micropanels are sensitized by non-specific human or animal immunoglobulins G and M, sequentially a solution containing the C1q subcomponent, the immunoglobulin conjugate with the enzyme and the substrate of this enzyme are sequentially added to the micropanel wells, after which quantitative determination of C1q from the product of the enzymatic reaction.  2. The method according to claim 1, characterized in that use is made of a conjugate with non-specific immunoglobulins G and M of a human or animal.  3. The method according to claim 1, characterized in that they use a conjugate with immunoglobulins - antibodies to C1q.