Method and kit for immune-enzyme assay of functional activity of human complement component c3

FIELD: measuring equipment.

SUBSTANCE: presented group of inventions refers to medicine. There are presented method and kit for the immune-enzyme assay of functional activity of human complement component C3. The method implies thrombin sorption in microplate wells, introduction of a test sample containing the human complement component C3 with unknown activity, incubation in the presence of ethylene diamine tetraacetate (EDTA), pouring out of the well contents, introduction of an enzyme conjugate with human complement component C3 antibodies and a substrate of the above enzyme. The component C3 activity is evaluated by the amount of the prepared product of enzymatic reaction. The kit comprises the flat-bottomed microplate with sorbed thrombin, conjugated enzyme and human complement component C3 antibodies, substrate buffer and donor blood serum with known activity C3 as a reference.

EFFECT: presented group of inventions enables evaluating the functional activity of human complement component C3 without the need of activation of the whole complement system, and possesses a good result reproducibility.

2 cl, 1 dwg, 2 ex

 

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.

The known method immunoassay determination of the functional activity of component C3 [1], which provides for sorption in the hole microarrays immunoglobulin G, then make holes microarrays complement the Guinea pig, which serves as a source of components C1, C2 and C4 (present component C3 of the Guinea pig is not detected by specific antibodies against human C3) and the sample containing the component C3 of the person with unknown activity, and incubation for activation of complement, which is the binding of C3 molecules of activator - adsorbed immunoglobulin. The amount of covalently bound peroxidase in the activation component C3 of the person determined by the product of the enzymatic reaction using antibodies to C3, conjugated with an enzyme.

Also known method [2], allow the Commissioner to determine the functional activity of the component C3 of the complement of the person using accessible and well preserved in normal conditions of commercial drug Derinat for use as activator the classical pathway of complement and substitute complement Guinea pigs homologous reagent R3 - human serum devoid of activity C3.

Finally, to activate the component C3 required for binding microarrays, followed by determining the amount of bound peroxidase by the same methods, in the method [3] used an alternative C3 of convertase formed as a result of activation of the alternative pathway using sorbed on microarrays lysozyme - activator of the alternative pathway, and R3 reagent as a source of factors b and D person, necessary for the formation of the C3 convertase alternative pathway of complement.

The disadvantage of all these methods is involved in the activation process component C3, the C3 convertase generated from components of the complement of the Guinea pig in the first case, any of the components or factors of the complement R3 reagent in the second and third cases, because the activity determination component C3 in the serum inevitably creates conditions activation of complement by the classical or alternative pathways that affect other components of complement and can lead to non-specific adsorption on the surface of the hole microarrays.

Objective of the claimed invention is to develop a method and kit based on ELISA method, azuolausio to determine the functional activity of the component C3 of the complement of the person using accessible and well preserved in normal conditions, a commercial preparation of thrombin for use as activator component C3 without activation of complement and without the use of sources other components or factors of the complement.

This object is achieved by developing methods to determine and set. The detection method involves sorption in the hole microarrays pharmacy drug thrombin, well preserved in normal conditions, then the entry in the wells microarrays solution containing the component C3 complement person with unknown activity, carrying out the incubation in the presence of ethylenediaminetetraacetate sodium (EDTA) to block all pathways of activation of complement, which is the activation of functionally active C3 and covalent binding of last sorbed on the thrombin, pouring the contents of the hole, and then adding the enzyme conjugate with antibodies against component C3 human laundering unbound conjugate, the introduction of the substrate conjugio-cell enzyme and settlement activity component C3 on the number formed product of the enzymatic reaction. This therefore allows the determination of the functional activity of C3 by ELISA suitable for standardization.

The set contains a flat-bottomed to something called a microarray adsorbed thrombin, the enzyme conjugate with antibodies to a component of C3 complement person substrate Boo the EP and donor serum with a known activity of C3 as standard.

The technical result of the claimed invention is to develop a method and kit for immunoassay determination of the functional activity of component C3 of the complement of man, with good reproducibility and without the need to activate all of the complement system.

Example 1. Immunoassay determination of the functional activity of component C3 of the human complement. Dissolve the drug thrombin bull in 0.05 M sodium carbonate buffer, pH of 9.5, with a final concentration of 100 μg/ml and contribute 100 ál of the solution into each well of flat-bottomed polystyrene 96-well plate. Close the lid and leave overnight at 4°C. washed Three times to something called a microarray 0.2 M sodium phosphate buffer solution, pH of 7.4, containing 0.15 M NaCl and 50 mm EDTA (these-indianinternet sodium), filling in 150 ál into each well, and then to something called a microarray dry by shaking out the remaining liquid. To each well of the tablet make 100 μl of the same buffer solution containing EDTA. In wells microarrays make 100 μl of a solution containing the component to be determined C3 in the same buffer. After incubation in an incubator for 1 h at 37°C, three times washing with phosphate buffer, pH of 7.4, containing 0.15 M NaCl and 0.05% tween-20, and drying the tablet into each hole making 100 μl of peroxidase conjugate with antibodies against C3 persons the century in the same buffer at selected breeding. After incubation in an incubator for 1 h at 37°C, five times washing with detergent and drying the tablet into each hole making 100 μl of substrate buffer (3,3',5,5'-tetramethylbenzidine in 15 ml of citrate-phosphate buffer, pH 5.0, and 50 μl of 3% hydrogen peroxide). After 15-30 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 number of active component C3 is calculated by the standard curve (figure 1).

Figure 1 shows the definition of the functional activity of component C3 complement human enzyme-linked immunosorbent assay.

Example 2. The kit for immunoassay determination of the functional activity of the component Sz. The set contains a flat-bottomed to something called a microarray adsorbed thrombin, peroxidase conjugate with antibodies to a component Sz of the human complement, substrate buffer and donor serum with a known concentration of active component C3 as standard. This set is used in accordance with example 1.

From the above results it follows that the measured optical density is linearly dependent on the concentration of the active component C3 with correlation coefficient R=0.9998, which allows to reliably determine the functioning of the social activity component C3 in concentrations from 0.6 µg/ml in the described manner by using the described set.

LITERATURE

1. Kozlov, L.V., Romanov S.V., Dyakov V.L. Method of determining the functional activity of the component C3 of the human complement by the classical pathway. 2005. RF patent 2251697. Bull. No. 13. 10.05.2005.

2. Kozlov, L.V., MT, NV, Besucher A.M. the method and the kit for immunoassay determination of the functional activity of component C3 of the human complement. RF patent 2417375. Bull. No. 12. 27.04.2011.

3. Kozlov, L.V., Mountain, NV Method and the kit for immunoassay determination of the functional activity of component C3 of the human complement via the alternative pathway activation. Patent 2380707. 27.01.2010.

1. Method immunoassay determination of the functional activity of component C3 of the complement of man, characterized in that the holes microarrays absorb thrombin, then in wells microarrays contribute analyzed a sample containing the component C3 complement person with unknown activity, carried out the incubation in the presence of sodium ethylenediaminetetraacetate, pour the contents of the wells, and then make the enzyme conjugate with antibodies against component C3 of the person and the substrate of this enzyme, followed by the calculation of activity component C3 on the amount of the formed product of the enzymatic reaction.

2. The kit for immunoassay determination of the functional activity of component C3 of the complement of man, characterized by t the m it contains a flat-bottomed to something called a microarray adsorbed thrombin, the enzyme conjugate with antibodies to the component C3 of the complement of man, substrate buffer and donor serum with a known activity of C3 as standard.



 

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