RU2376606C1 - Method and set for immune-enzyme detection of functional activity of component c4 of human complement - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is related to the field of medicine and to method and set for immune-enzyme detection of functional activity of component C4 of human complement. Substance of invention includes introduction of complement-derivative as activator of classical route into micropanel holes, then reagent of R4 and analysed sample, incubation, removal of holes content, introduction of ferment conjugate with antibodies against component C3 of complement, incubation, removal of incubation solution, introduction of ferment substrate and performance of C4 component calculation by amount of product generated as a result of fermentative reaction. Invention also includes set for detection of functional activity of component C4 of human complement.

EFFECT: increased sensitivity of method.

2 cl, 2 ex, 1 dwg

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. A known method for determining the functional activity of component C4, mainly its isotype C4B [1], which provides for the sorption in the wells of a microplate of a lipopolysaccharide, then determined by C4 from solutions containing it in the presence of R4 reagent, i.e. human serum lacking C4 activity. The amount of covalently bound component C3 depends on the concentration of the functionally active detectable component C4. The amount of C3 component 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 only drawback of this method is the difficulty in sorption of lipopolysaccharide on polystyrene micropanels commonly used in enzyme-linked immunosorbent assay, 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 the component C4 of the classical pathway of the human complement system 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 into the micropanel wells a solution containing R4 reagent (human serum lacking the activity of component C4) and an analyzed sample containing component C4 the complement of a person with unknown activity, and the incubation, during which the formation of C3-convertase (C4b2a) occurs on the sorbed activator in quantities, depending they depend on the concentration of the functionally active determined component C4, which results in the activation of component C3 by this convertase and covalent binding of the latter on the sorbed activator, pouring out the contents of the wells, and then introducing the enzyme conjugate with antibodies against the human component C3 and the substrate of this enzyme and calculating the component activity C3 by the amount of the resulting enzymatic reaction product.

The kit contains a flat-bottomed micropanel with adsorbed derinate, R4 reagent (human serum lacking the activity of component C4), an enzyme conjugate with antibodies to human complement component C3, a substrate buffer and donor blood serum as a standard.

The technical result of the claimed invention is the development of a method and kit for determining the functional activity of the component C4, namely its C4B isotype, having good reproducibility of results and the absence of the need for the use of a lipopolysaccharide preparation that is difficult to absorb and does not preserve its activating ability.

Example 1. Determination of the functional activity of the drug component C4

The derinate pharmacy preparation is dissolved in phosphate buffer, pH 7.4, containing 0.15 M NaCl, at concentrations of 10-100 μg / ml and 100 μl of the solution is added to each well of a flat-bottomed polystyrene 96-well micropanel. Cover and leave overnight at 4 ° C. The micropanel is washed three times with Veronal buffer solution, pH 7.4, containing 0.15 M NaCl, 0.15 mM Ca ²⁺ and 0.5 mM Mg ²⁺ . 100 μl of the same buffer containing 10 μl of R4 reagent (R4 is human serum lacking the activity of component C4 [2]) and an assay sample containing component C4 with unknown activity are added to the wells of a row of micropanels. After incubating in an incubator 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 the 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 (3.3 ', 5.5'-tetramethylbenzidine in 15 ml of citrate-phosphate buffer, pH, are added to each well) 5.0, and 50 μl of 3% hydrogen peroxide). After 15-30 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 450 nm. The functional activity of the preparation of component C4 is calculated according to a standard curve.

Example 2. A kit for determining the functional activity of component C4

The kit contains a flat-bottomed micropanel with sorbed derinate, R4 reagent (human serum lacking the activity of component C4), a peroxidase conjugate with antibodies to human complement component C3, a substrate buffer and donor blood serum as a standard. This kit is used in accordance with example 1.

From the results shown in the drawing, it follows that the measured optical density linearly depends on the concentration of the active component C4 with a correlation coefficient of R ² = 0.998, which allows you to reliably determine the functional activity of component C4 in the described manner using the described set.

LITERATURE

1. Kozlov L.V., Lakhtin M.V., Lakhtin V.M., Dyakov V.L. A method for determining the functional activity of the complement component C4 of a person. RF patent 2253117. Bull. No. 15. 05/27/2005.

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.V.8. No. 5. S.652-659.

Claims (2)

1. A method for determining the functional activity of the complement component C4 of a person, comprising sorbing the complement pathway activator in the micropanel of the classic complement pathway, introducing the R4 reagent micropanel into the wells of an assay sample containing a human complement component C4 with unknown activity, incubating, pouring out the contents of the wells, introducing the enzyme conjugate with antibodies against human component C3, and then after incubation and removal of the incubation solution, making the substrate of this enzyme, after which even the activity of component C4 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 C4, characterized in that it contains a flat-bottomed micropanel with sorbed derinate, R4 reagent, an enzyme conjugate with antibodies to the human complement component C3, a substrate buffer and a standard with known activity of the C4 component.

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