RU2232992C1 - Method for assay of functional activity of human complement component c3 by alternative pathway of activation - Google Patents

Abstract

FIELD: medicinal immunology.

SUBSTANCE: invention relates to methods for assay of functional activity of complement factors in human serum blood and can be used in diagnosis of some diseases and in biological preparations. Method involves sorption of activator of the complement alternative pathway in microplate wells and then solution containing reagent R3 and analyzed sample containing component C3 of human complement with unknown activity are added. Incubation is carried out in the presence of Mg²⁺ ions and in the absence of Ca²⁺ and then conjugate of enzyme with antibodies raised against the human component C3 and substrate for the same enzyme are added. The calculation of C3 activity is carried out by the amount of formed product of enzymatic reaction. Invention provides the development of method allowing the assay of functional activity of component C3 in human complement system in alternative pathway of activation.

EFFECT: improved assay method.

1 tbl, 5 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 determining serum proteins is enzyme immunoassay due to its sensitivity, selectivity and the ability to automate the analytical process. However, currently available methods of 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 the complement component C3, the hemolytic method [1] for determining the activity of C3, which manifests itself in the activation of the system in the classical way, is not distinguished by a high degree of reproducibility and is reluctantly used by clinicians because of the difficulties associated with the preparation of the hemolytic system for erythrocyte-based sheep. Component C3 is key in activating an alternative path. Methods for determining the activity exerted by the human complement component C3 in an alternative path are apparently not described.

The objective of the claimed invention is to develop a method that allows to determine the functional activity of component C3 of the human complement system in an alternative activation pathway based on the enzyme immunoassay.

The task is achieved by developing a determination method that provides for the sorption of an alternative complement pathway activator selected from the series: immunoglobulin A, G or a mixture of immunoglobulins A, G, M, component C3, lipopolysaccharide in the wells of the microplate, then adding a solution containing the reagent to the wells of the microplate R3 (human serum lacking the activity of component C3), and an analyzed sample containing component C3 of a complement of a person with unknown activity, incubation in the presence of an ion to Mg ²⁺ and in the absence of Ca ²⁺ ions (which occurs during formation of the C3 convertase (C3bBb) adsorbed on the alternative pathway activator in amounts dependent on the concentration of functionally active detecting component C3, resulting in the activation of the C3 convertase and covalent binding by the latter on a sorbed activator), pouring out the contents of the wells, and then introducing the conjugate of the enzyme with antibodies against component C3 of the person and the substrate of this enzyme and calculating the activity of component C3 p the amount of the resulting enzymatic reaction product.

The technical result of the claimed invention is the development of a simplified method for determining the functional activity of component C3 of a human complement, which has good reproducibility of the results and the absence of the need to use such poorly stored and difficult to standardize components, such as red blood cells.

Example 1. Determination of the functional activity of the drug C3. The activator of the alternative complement pathway, immunochemically pure IgA, was dissolved in 0.05 M sodium carbonate buffer, pH 9.5, at concentrations of 10-100 μg / ml and 100 μl of solution was added into each well of a flat-bottomed polystyrene 96-well plate. Close the lid and leave overnight at 4 ° C. The tablet is washed three times with Veronal buffer solution, pH 7.4, containing 0.15 M NaCl, 10 mM ethylene glycol bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetic acid and 5 mM MgCl ₂ ( Mg-EGTA), pouring 150 μl into each well, then the plate is dried by shaking the remaining liquid. 100 μl of Mg-EGTA solution containing 10 μl of R3 reagent (R3 is human serum treated with a semi-saturated KBr solution for 18 hours in the cold [1]) and an assay sample containing component C3 with unknown activity are added to the wells of a row tablet. After incubation in a thermostat for 1 h at 37 ° C, twice washing with phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.05% tween-20, and draining the plate, 100 μl of peroxidase conjugate are added to each well with antibodies against component C3 person in the same buffer in a selected dilution. After incubating in a thermostat for 1 h at 37 ° C, washing twice with detergent and draining the plate, 100 μl of substrate buffer (10 mg of orthophenylenediamine in 25 ml of nitrate-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 drug C3 is calculated according to a standard curve. This method determined the activity of C3 in samples with a known content of active C3. The results are shown in the table.

Example 2. The determination of the functional activity of the drug C3 is carried out analogously to example 1, but immunoglobulin G is used as an alternative pathway activator. The results are shown in the table.

Example 3. Determination of the functional activity of the drug C3 is carried out analogously to example 1, but as an activator of the alternative path, KIP is used - a complex immunoglobulin preparation containing immunoglobulins G, A and M. The results are shown in the table.

Example 4. The determination of the functional activity of the drug C3 is carried out analogously to example 1, but as an activator of the alternative pathway, the drug component C3 of the guinea pig is used. The results are shown in the table.

Example 5. Determination of the functional activity of the drug C3 is carried out analogously to example 1, but as an activator of the alternative pathway, use a lipopolysaccharide (LPS - preparation of the bacterial cell wall, pyrogenal). The results are shown in the table.

LITERATURE

1. Kozlov L.V., Krylova Yu.I., Chikh V.P., Molchanova N.N. Modified methods for determining the functional activity of complement factors C2, C3, C4 and C5. Bioorganic chemistry. 1982. T. 8. No. 5. S.652-659.

Claims (1)

A method for determining the functional activity of a human complement component C3, characterized in that an alternative complement pathway activator selected from a number of immunoglobulins A, G or a mixture of immunoglobulins A, G, M, component C3, lipopolysaccharide is sorbed in the wells of the microplate, then a solution is introduced into the wells of the microplate, containing the reagent R3, and the test sample containing the component C3 of the complement of a person with unknown activity, incubate in the presence of Mg ^{2+ ions} and in the absence of Ca ^{2+ ions} , the contents of the wells are poured, and then the conjugate of the enzyme with antibodies against the human component C3 and the substrate of this enzyme are introduced, after which the C3 activity is calculated by the amount of the resulting enzymatic reaction product.