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

FIELD: medicine.

SUBSTANCE: pharmaceutical preparation derinate representing sodium deoxyribonucleate is sorbed in microplate wells. Then, an analysed sample containing a component C3 of unknown activity and a solution of reagent R3 (component C3 activity deficient human blood serum) is added in the wells. It is followed with incubation, and after washing and drying of the dish, a conjugate of enzyme with component C3 antibodies and a substratum of this enzyme are added into the wells. The component C3 activity is evaluated by the amount of the prepared product of enzymatic reaction. A kit comprises a flat-bottomed microplate with sorbed derinate, the conjugate of enzyme with human complement component C3 antibodies, the reagent R3 (component C3 activity deficient human blood serum) and the substrate buffer.

EFFECT: method allows the reliable and high sensitive component C3 test.

1 dwg, 3 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. There is a method of determining the functional activity of the component C3 [1], which provides for sorption in the wells of the microplate immunoglobulin G, then the inclusion of a microplate well 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.

The disadvantage of this method is the difficulty in the doctrine of immunochemical pure preparations of immunoglobulin G man and poor preservation of its activating ability during storage, as well as reducing the sensitivity of the method due to present heterologous component C3 Guinea pigs.

Objective of the claimed invention to provide an method and set-based immunoassay method 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 an activator of the classical pathway of complement and substitute complement Guinea pigs homologous reagent R3 - human serum devoid of activity C3.

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 entering into the wells of the microplate reagent R3 (human serum devoid of activity component C3 with preservation activities of the other components of complement, we get, for example, processing for 18 h at 4°C with a solution of KBr [2]) and the sample containing the component C3 with unknown activity, and incubation for activation of complement that involves covalent binding of C3 to the molecules of Corby is consistent Derinat. The amount of covalently bound peroxidase in the activation of the component C3 is determined by the product of the enzymatic reaction using antibodies to C3 - fraction of immunoglobulin G conjugated with an enzyme. 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 with sorbed Derinat, the enzyme conjugate with antibodies to the component C3 of the complement of man, the R3 reagent, 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 C3 with high sensitivity and without using a difficult and poorly allocated retaining its activating ability of the drug immunoglobulins.

Example 1. Determination of the functional activity of the drug component C3. Dissolve pharmacy drug Derinat in veronella buffer solution, pH 7.4, containing 0.15 M NaCl, 0.15 mm CA2+and 0.5 mm Mg2+(VBS2+), in concentrations of 10-150 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 tablet is eating the same buffer, then dry by shaking out the remaining liquid. In wells contribute 100 μl of a solution VBS2+containing 10 μl of the reagent R3 (human serum devoid of activity component C3), and analyzed the sample containing the component C3 with unknown activity. After incubation 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, (SR-T) and drainage tablet in every hole making 100 μl of peroxidase conjugate with antibodies against component C3 of the person in the same buffer at selected breeding. After incubation in an incubator for 1 h at 37°C, 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 S2 calculated by the standard curve (see drawing).

Example 2. Preparation Of R3. To 10 ml and cooled to 4°With fresh human serum at continuous stirring slowly poured 10 ml saturated at 4°C solution of KBr. The mixture is incubated for 18 h at 4°C, zatemnealsea 18 h against isotonic phosphate buffer, pH of 7.2, poured into test tubes and stored at -50°C. as a reagent R3 drug use, diluted veronalum buffer in the ratio 2:3.

Example 3. Kit for determination of the functional activity of component C3 of the human complement contains a flat-bottomed to something called a microarray with sorbed Derinat, peroxidase conjugate with antibodies to the component C3 of the complement of man, the R3 reagent, substrate buffer and a standard of known activity component C3.

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 C2 with correlation coefficient R2=0,992 that allows you to reliably determine the functional activity of the component C3 in the described manner by using the described set. The proposed method allows to determine the activity of the C3 concentration of 7 ng/ml to 1000 ng/ml, which distinguishes it from the sensitivity of the prototype, allowing to determine the activity of the C3, starting with a concentration of 50 ng/ml

Sources of information

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 2251397. Bulletin no.13. 10.05.2005.

2. Kozlov, L.V., Krylov SCI, Sneeze VP, N. Molchanov. Modified methods for determining the functional and the performance factors of the complement C2, C3, C4 and C5. Bioorganic chemistry. 1982. T.8. No. 5. S-659.

1. The method of determining the functional activity of the component C3 of the complement of the person providing the sorption in the hole microarrays activator of the classical pathway of complement, introduction into the wells microarrays analyzed samples containing component C3 complement person with unknown activity, and the solution containing the components C1, C2 and C4, incubation, pouring the contents of the hole, making the enzyme conjugate with antibodies against component C3 of the person, and then, after incubation and removal of the incubation solution, the introduction of substrate buffer, settlement activity component C3 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 and as a solution containing the components C1, C2 and C4, the R3 reagent, which is a serum of a person devoid of activity C3.

2. Kit for determination of the functional activity of component C3 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 the component C3 of the complement of man, the R3 reagent, which is a serum of a person devoid of activity C3, and substrate buffer.



 

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