Method and set for elisa determination of functional activity of human complement component c1q

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SUBSTANCE: sorption of pharmacy drug derinate, representing sodium deoxyribonucleate in micropanel holes is carried out. After that analysed sample, which contains component C1q with unknown activity, is introduced into holes. Incubation is carried out and after washing and drying of panel into holes introduced are conjugate of enzyme with antibodies against component C1q and substrate of said enzyme. Activity of component C1q is calculated by amount of formed product of enzymatic reaction. Set contains flat-bottom micropanel with sorbed derinate, conjugate of enzyme with antibodies to human complement component C1q, substrate buffer and standard with known C1q activity.

EFFECT: method application makes it possible to increase reliability of determination C1q component with application as activator of available and stable preparation derinate.

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The invention relates to medical immunology, and in particular to methods for determining the functional activity of complement components in the serum of human blood in the diagnosis of several diseases and biological agents.

The most promising of modern methods for the determination of serum proteins is an enzyme immunoassay due to its sensitivity, selectivity and the possibility of automation of the analytical process. There is a method of determining the functional activity of the component C1q, which provides for sorption in the wells of the microplate immunoglobulin G or M [1]. The amount of covalently bound peroxidase in the activation component C1q is determined by the product of the enzymatic reaction using antibodies to C1q, conjugated with an enzyme.

The disadvantages of this method are the difficulty of obtaining immunochemical pure preparations of immunoglobulin G or M man and poor preservation of activating ability when stored.

Objective of the claimed invention to provide an method and set-based immunoassay method to determine the functional activity of the component C1q classic ways of the human complement system using accessible and well preserved in normal conditions commercial prep the ATA for use as an activator of the classical pathway of complement.

This object is achieved by developing methods to determine and set. The detection method involves sorption in the hole microarrays pharmacy drug Derinat, representing deoxyribonuclease sodium, well preserved in normal conditions, then the entry in the wells microarrays analyzed sample containing the component C1q complement person and the conduct of incubation, during which C1q binds to the sorbed activator in amounts that depend on the concentration of functionally active defined component C1q, pouring the contents of the hole, and then adding the enzyme conjugate with antibodies against a component C1q person and substrate of this enzyme and settlement activity component C1q on the amount of the formed product of the enzymatic reaction.

The set contains a flat-bottomed to something called a microarray with sorbed Derinat, the enzyme conjugate with antibodies to a component C1q of the complement of man, substrate buffer and donor serum as a standard.

The technical result of the claimed invention is to develop a method and kit for determining the functional activity of the component C1q, with good reproducibility and without using a difficult and poorly allocated preserving, strengthening the existing capacity of the preparation of immunoglobulins.

Example 1. Determination of the functional activity of the drug component C1q. Dissolve pharmacy Derinat in phosphate buffer, pH 7.4, containing 0.15 M NaCl, in concentrations of 10-100 μg/ml and contribute 100 ál of the solution into each well of flat-bottomed polystyrene 96-well microarrays. Close the lid and leave overnight at 4°C. pour the Contents of the wells, washed three times tablet SFR-T (phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.05% tween-20), then dried by shaking out the remaining liquid. In wells contribute 100 ál of the sample containing C1q in SFR-So After incubation in an incubator for 1 h at 37°C, three times washing SFR-T and drainage tablet in every hole making 100 μl of peroxidase conjugate with antibodies against a component C1q person in the same buffer at selected breeding. After incubation in an incubator for 1 h at 37°C, three times washing SFR-T and drainage tablet in every hole making 100 μl of substrate buffer with TMB (3,3',5,5'-tetramethylbenzidine in 15 ml of citrate-phosphate buffer, pH 5.0, and 50 μl of 3% hydrogen peroxide). After 5-10 min incubation in the dark, the reaction is stopped by the introduction to each well 50 μl of 14% sulfuric acid. The results of the reaction consider using a spectrophotometer with a vertical beam of measuring light absorption at 450 nm. The functional activity of the drug C1q is schityvat from the standard curve (see the drawing).

Example 2. The kit for immunoassay determination of the functional activity of the component C1q complement person contains a flat-bottomed to something called a microarray with sorbed Derinat, peroxidase conjugate with antibodies to a component C1q of the complement of man, substrate buffer and a standard of known activity component C1q.

From what is shown on the drawing results, it follows that the measured optical density is linearly dependent on the concentration of the active component C1q with correlation coefficient R2=0,996 that allows you to reliably determine the functional activity of the component C1q in the described manner by using the described set.

LITERATURE

1. Kozlov, L.V., Dyakov V.L., Batalova T.N., Husova VA, Lyssakov SV Method of determining the functional activity of subcomponent C1q of the complement of man. 2000. RF patent 2144676. // Bul. No. 2. 20.01.2000.

1. The method of determining the functional activity of the component C1q complement of the person providing the sorption in the hole microarrays activator of the classical pathway of complement, introduction into the wells of the microarrays of the sample containing the component C1q complement person with unknown activity, incubation, pouring the contents of the hole, making the enzyme conjugate with antibodies against a component C1q person and then after incubation and removal Inc is belonoha solution of making the substrate of this enzyme, settlement activity component C1q on the amount of the formed product of the enzymatic reaction, characterized in that as an activator of the classical pathway of complement use Derinat.

2. Kit for determination of the functional activity of the component C1q of the complement of man, characterized in that it contains a flat-bottomed to something called a microarray with sorbed Derinat, the enzyme conjugate with antibodies to a component C1q of the complement of man, substrate buffer and a standard of known activity component C1q.



 

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