RU2149406C1 - Method for determining functional activity of human c4 complement component - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves applying immunoenzyme analysis method. Sorption is carried out in micropanel cells of immunoglobulins of G and M class. Then, solution containing the C4 component to be determined is sorbed in the presence of Guinea pig complement. The quantity of the sorbed C4 component is determined using an enzyme reaction product with specific antibodies against human C4 bound to the enzyme with covalent bonds. Guinea pig whole serum or serum having no active C4 component is used as the Guinea pig serum. EFFECT: simplified and reliable method. 2 cl, 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. 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 component C4 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 first, native or aggregated by preliminary heating, is carried out in the micropanel wells, then determined by C4 from solutions containing it in the presence of guinea pig serum deficient in C4 or whole. The amount of component C4 covalently bound as a result of activation is determined by the product of the enzymatic reaction using antibodies to C4, a fraction of immunoglobulins G covalently linked to the enzyme. The method is based on the ability of the functional active component C4 to covalently bind to aggregated immunoglobulins of classes G and M of human or animal blood serum as a result of a cascade of complement activation reactions of C1 from guinea pig serum to aggregated IgG or IgM bound on a support and C4 using activated C1 associated with aggregated immunoglobulins.

Example 1. Determination of the functional activity of the drug C4. Immunochemically pure IgG or IgM is dissolved in 0.05 M sodium carbonate buffer, pH 9.5, at protein concentrations of 10-100 μg / ml and 100 μl of solution are added to each well of a flat-bottomed polystyrene 96-well plate. Close the lid and leave overnight at 4 ^o C. Two 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 ⁺⁺ ), 150 μl to each well, then the plate is dried by shaking out the remaining liquid. 100 μl of a solution containing C4 in VBS ⁺⁺ solution is added to the wells of a row tablet in the form of progressive two-fold dilutions. 10 μl of guinea pig serum containing no active component C4 is added to all wells of the plate. After incubation in a thermostat 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 draining the plate, 100 μl of peroxidase conjugate are added to each well with antibodies against human C4 component in the same buffer in a selected dilution. After incubating in a thermostat for 1 h at 37 ^° C and washing twice 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 drug C4 is calculated according to a standard curve.

This method determined the activity of C4 in 6 samples of human serum and the standard hemolytic method [1]. The results are obtained (in the values of effective molecules in 1 ml per 10 ¹² ), given in the table.

Example 2. Determination of the functional activity of the drug C4. Dissolve IgG or IgM aggregates obtained by preheating at 63 ^° C in 0.05 M sodium carbonate buffer, pH 9.5, at protein concentrations of 10-100 μg / ml and add 100 μl of solution to each well of flat-bottomed polystyrene 96 well tablet. Close the lid and leave overnight at 4 ^o C. Two 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 ⁺⁺ ), 150 μl to each well, then the plate is dried by shaking out the remaining liquid. 100 μl of a solution containing C4 in VBS ⁺⁺ solution is added to the wells of a row tablet in the form of progressive two-fold dilutions. 10 μl of whole guinea pig serum are added to all wells of the plate. After incubation in a thermostat for 30 min at 37 ^° C, washing twice 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 human C4 component in the same buffer in a selected dilution. After incubating in a thermostat for 1 h at 37 ^° C and washing twice 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 drug C4 is calculated according to a standard curve.

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.Vol. 8. N 5.P. 652-659.

Claims (2)

 1. A method for determining the functional activity of component C4 of a human complement, characterized in that non-specific immunoglobulins G or M of humans or animals are sorbed in the wells of the micropanels, a solution containing component C4 is added to the wells of the micropanels, and guinea pig serum that does not contain the active component C4 is carried out incubation, and then the conjugate of the enzyme with antibodies against the human component C4 and the substrate of this enzyme are introduced, after which the C4 activity is determined by the product of the enzymatic reaction.  2. A method for determining the functional activity of the complement component C4 of a human complement, characterized in that non-specific immunoglobulins G or M of humans or animals are sorbed in the micropanel wells, a solution containing component C4 and whole guinea pig serum are added to the micropanel wells, incubated, and then introduced the conjugate of the enzyme with antibodies against the component C4 of the person and the substrate of this enzyme, after which the C4 activity is determined by the product of the enzymatic reaction.

Images