RU2405042C1 - Method and kit for immune-enzyme assay of functional activity of human complement component c2 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: micro-well attachment of the pharmaceutical preparation Derinate with drug substance represented with sodium deoxyribonucleate. Thereafter, an analysed sample containing component C2 of undefined activity and reagent R2 solution (human blood serum with inactivated component C2) is introduced into the wells. It is followed with incubation, and after washing and drying the tray, conjugated enzyme with component C3 antibodies, and its substratum are introduced into the wells. The component C2 activity is calculated by the amount of the enzymatic reaction product. A kit comprises a flat-bottomed micro-tray with attached Derinate, conjugated enzyme with human complement component C3 antibodies, reagent R2 (human blood serum with inactivated component C2), a substratum buffer and a standard with defined C2 activity.

EFFECT: method allows reliable component C2 detection with the use of the available and stable preparation Derinate as an activator.

2 cl, 1 dwg, 1 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 the enzyme immunoassay due to its sensitivity, selectivity and the ability to automate the analytical process. A known method for determining the functional activity of component C2 [1], which provides for the sorption of the immunoglobulin G microtiter plate in the wells, then introducing the reagent R2 (human serum devoid of component C2 while maintaining the activities of the remaining complement components, obtained, for example, by heating it for 30 min at 50 ° С [2]) and the analyzed sample containing component C2 with unknown activity, and incubation to activate complement, during which formation takes place on the sorbed ohm immunoglobulin C3 convertase (C4bC2a) in amounts depending on the concentration of functionally active detecting component C2, and the activation of the C3 convertase components and covalent binding of the latter to immunoglobulin molecules sorbed. Thus, the amount of bound C3 depends on the amount of C3 convertase, i.e. the amount of the determined component C2. The amount of component C3 covalently bound as a result of activation is determined by the product of the enzymatic reaction using anti-C3 antibodies conjugated to the enzyme. The method is based on the ability of the functionally active component C2 to form a C3 convertase, the amount of which is determined by the amount of activated C3 bound in the wells. Thereby, the determination of the functional activity of C2 is achieved by an enzyme immunoassay suitable for standardization.

The disadvantage of this method is the difficulty of obtaining immunochemically pure preparations of human immunoglobulin G and poor preservation of the activating ability during storage.

The objective of the claimed invention is to develop a method and kit based on an enzyme-linked immunosorbent assay that allows you to determine the functional activity of component C2 of a person’s complement using an affordable and well-preserved commercial preparation for use as an activator of the classical complement pathway.

The task is achieved by developing a method for determining and recruiting. The method of determination involves sorption in the micropanel wells of a pharmaceutical preparation of derinat, which is sodium deoxyribonucleate, well preserved under ordinary conditions, then introducing the reagent R2 (human serum devoid of component C2 into the microtiter wells while maintaining the activity of the remaining complement components, obtained, for example, by heating it for 30 min at 50 ° С [2]) and an analyzed sample containing component C2 with unknown activity, and incubation to activate complement, during which is formed on the adsorbed immunoglobulin C3 convertase (C4bC2a) in amounts depending on the concentration of functionally active detecting component C2, and the activation of the C3 convertase components and covalent binding of the latter to immunoglobulin molecules sorbed. Thus, the amount of bound C3 depends on the amount of C3 convertase, i.e. the amount of the determined component C2. The amount of component C3 covalently bound as a result of activation is determined by the product of the enzymatic reaction using antibodies to the C3 fraction of immunoglobulins G conjugated to the enzyme. The method is based on the ability of the functionally active component C2 to form a C3 convertase, the amount of which is determined by the amount of activated C3 bound in the wells. Thereby, the determination of the functional activity of C2 is achieved by an enzyme immunoassay suitable for standardization.

The kit contains a flat-bottomed micropanel with sorbed derinate, an enzyme conjugate with antibodies to the human complement component C3, reagent R2, substrate buffer and donor blood serum as a standard.

The technical result of the claimed invention is the development of a method and a kit for determining the functional activity of component C2, which have good reproducibility of results and the absence of the need to use an immunoglobulin preparation that is difficult to secrete and does not preserve its activating ability.

Example 1. Determination of the functional activity of the drug component C2. The pharmaceutical preparation of Derinat is dissolved in Veronal buffer solution, pH 7.4, containing 0.15 M NaCl, 0.15 mm Ca ²⁺ and 0.5 mm Mg ²⁺ (VBS ²⁺ ), in concentrations of 10-100 μg / ml and add 100 μl of the solution to each well of a flat-bottomed polystyrene 96-well micro-panel. Close the lid and leave overnight at 4 ° C. The contents of the wells are poured, the plate is washed with the same buffer, then dried by shaking out the remaining liquid. 100 μl of VBS ²⁺ solution containing 10 μl of reagent R2 (R2 is human serum heated at 50 ° C for 35 min) and an assay sample containing component C2 with unknown activity are added to the wells of a row tablet. 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, (PBS-T), and draining the plate in each well make 100 μl of peroxidase conjugate with antibodies against human component C3 in the same buffer in a selected dilution. After incubation in a thermostat for 1 h at 37 ° C, washing with PBS-T and draining the plate, 100 μl of TMB substrate buffer (3.3 ', 5.5'-tetramethylbenzidine in 15 ml of citrate-phosphate buffer are added to each well) , pH 5.0, and 50 μl of 3% hydrogen peroxide). After 5-10 min 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 450 nm. The functional activity of the drug C2 is calculated according to the standard curve (see drawing).

Example 2. A kit for determining the functional activity of component C2 of a human complement contains a flat-bottomed micropanel with adsorbed derinate, a conjugate of peroxidase with antibodies to component C3 of a human complement, reagent R2, substrate buffer and a standard with known activity of component C2.

From the results shown in the drawing, it follows that the measured optical density linearly depends on the concentration of the active component C2 with a correlation coefficient of R ² = 0.996, which allows you to reliably determine the functional activity of component C2 in the described manner using the described set.

LITERATURE

1. Kozlov L.V., Romanov S.V., Dyakov V.L., Batalova T.N., Lakhtin V.M., Guzova V.A. A method for determining the functional activity of the complement component C2 of a person. 2000. RF patent 2195670. Bull. Number 36. 12/27/2002.

2. 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 (2)

1. The method of determining the functional activity of the complement component C2 of a person’s complement, providing for the sorption of the activator of the classical complement pathway in the micropanel wells, introducing into the micropanel wells an assayed sample containing a human complement component C2 with unknown activity, and a solution containing R2 reagent, incubation, pouring out the contents of the wells, introduction of an enzyme conjugate with antibodies against human component C3, and then, after incubation and removal of the incubation solution, introduction of a substrate of this enzyme, p Calculation of the activity of component C2 by the amount of the resulting enzymatic reaction product, characterized in that Derinat is used as an activator of the classical complement pathway. 2. A kit for determining the functional activity of the human complement component C2, characterized in that it contains a flat-bottomed micropanel with adsorbed derinate, an enzyme conjugate with antibodies to the human complement component C3, reagent R2, substrate buffer and a standard with known activity of component C2.

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