Method and set for immunoenzymometric determination of functional activity of c1 inhibitor on alternative path

FIELD: chemistry.

SUBSTANCE: pharmaceutical preparation CIP (complex immunoglobulin preparation, consisting of IgG, IgA and IgM) is bound in cavities of a micropanel, said preparation containing a C3b complement component, samples containing the determined functionally active human C1 inhibitor are incubated in the cavities, and the bound C1 inhibitor is determined using a conjugate of an antibody against the human C1 inhibitor with an enzyme and a substrate for that enzyme based on the amount of the formed product of the enzymatic reaction. The set contains flat-bottomed micropanel with the sorbed CIP, the enzyme conjugate with antibodies against the human C1 inhibitor, a substrate buffer of that enzyme and a standard for the active C1 inhibitor.

EFFECT: use of the method enables to determine activity of the C1 inhibitor which regulates the complement alternative path.

2 cl, 1 dwg, 1 tbl, 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.

C1 inhibitor of the complement system as an inhibitor of serine proteases involved in the regulation of many enzymes in the blood plasma, such as C1r, C1s components of the classical pathway of complement, kallikrein kallickrein-kinin system, as well as factors of coagulation and anticoagulation systems - XIa, Hu, XIIf and plasmin (fibrinolizina) [1]. It was also discovered its ability to inhibit serine proteases pectinophora way of complement [2]. However, recently opened a new function C1 inhibitor is its ability to regulate the alternative pathway of complement by interacting with a component 3b complement and inhibiting the binding factor In complement with 3b [3]. This function is chemically different from the already known at the time of its implementation of the C1 inhibitor does not block covalently bound to the active center serine proteases, as it was in all the above cases, and is associated ecovalence component 3b, inhibiting the formation or destabilizing the final factor In [3].

The deficiency of this inhibitor in the patient's blood leads to periodic edema, setrag the living mainly the limbs, the face, throat and mucous membrane of the gastrointestinal tract.

Inherited deficiency of C1 inhibitor can be of two types. Type I is caused by the low concentrations of C1 inhibitor. When type II is produced by C1 inhibitor with reduced or absent functional activity, but normal or elevated concentrations [4]. Because type II is caused by mutations of the gene C1 inhibitor, is currently not known, are affected by these mutations on both the functional activity of C1 inhibitor: inhibition of the classical pathway and regulation of the alternative pathway of complement. Immunnofermentye methods for determining the activity of C1 inhibitor regulating the alternative pathway of complement, is not known.

Objective of the claimed invention is a method and kit for immunoassay determination of the functional activity of C1 inhibitor on an alternative path.

This object is achieved by developing a method for determining the functional activity of C1 inhibitor, which provides for binding in the hole microarrays component 3b, incubation in the wells samples containing defined functionally active C1 inhibitor man, and determination of bound peroxidase C1 inhibitor using conjugate antibodies against C1 inhibitor of the human complement with the enzyme and substra the ohms for this enzyme. For fixing in the hole microarrays 3b spend sorption pharmacy drug KIP (complex immunoglobulin preparation, consisting of immunoglobulins G, a and M people). It has been shown (see table 1)that in the samples of the commercial product KIP contains component C3 of the human complement in trace amounts that were sufficient for specific binding of C1 inhibitor.

Table 1
The content of component C3 in various series commercial complex immunoglobulin preparation preparation
SampleProtein, mg/mlC3, µg/mg protein
156,63,83
263,42,52
335,12,27
454,30,09
526,06,10
661,6The set contains a flat-bottomed to something called a microarray with an associated drug KIP, the enzyme conjugate with antibodies to C1 the inhibitor man, substrate buffer and the standard for active C1 inhibitor.

The technical result of the claimed invention is to develop a method and kit for determining the functional activity of C1 inhibitor of the human complement by the alternative pathway of complement by using as source component 3b complement pharmacy drug KIP.

Example 1. Determination of the functional activity of C1 inhibitor in alternative ways. Dissolve Pharmacopeia drug KIP at a concentration of 40 µg/ml in 0.05 M sodium carbonate buffer, pH of 9.5, 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. washed Three times to something called a microarray veronalum buffer solution, pH 7.4, containing 0.15 M NaCl and 50 mm EDTA (ethylenediaminetetraacetate sodium), filling in 150 ál into each well, and then to something called a microarray dry by shaking out the remaining liquid. In wells microarrays make the sample containing C1 inhibitor 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, and drying Microban is whether in each well contribute 100 μl of peroxidase conjugate with antibodies against C1 inhibitor man 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 functional activity of the component C1 inhibitor is calculated by the standard curve (figure 1).

Example 2. Kit for determination of the functional activity of C1 inhibitor in alternative ways. The set contains a flat-bottomed to something called a microarray adsorbed drug KIP, peroxidase conjugate with antibodies to C1 the inhibitor man, substrate buffer and a standard with a known activity of C1 inhibitor man. This set is used in accordance with example 1.

From the above figure results, it follows that the measured optical density is linearly dependent on the concentration of active C1 inhibitor with correlation coefficient R2=0,999, which allows to reliably determine the functional activity of C1 inhibitor in alternative ways in concentrations from 1 ng/ml in the described manner by using the described set.

Cu is IU, carried out a determination of the functional activity of C1 inhibitor in the serum of a patient with hereditary angioneurotic edema type II, i.e. with a functional deficiency of this protein. The methods developed previously [4, 5], it was found that the content of C1 inhibitor in the serum of this patient was 204 mg/ml, and the activity of the classical pathway inhibition corresponded to 93 µg/ml active. Determination of the functional activity of C1 inhibitor on alternative ways of the blood of this patient gave a value of 95 mg/ml active. This implies that the proposed method represents the activity of C1 inhibitor, and does not quantify as protein, and that the activity shown by C1 inhibitor of the alternative pathway of complement, in this particular case corresponds to its activity in the classical pathway.

LITERATURE

1. Davis AE 3rd. CI inhibitor and hereditary angioneurotic edema. Ann. Rev. Immunol. 1988. V.66. P.595-628.

2. Matsushita M., Thiel, S., Jensenius J.C., Terai L, Fujita T. Proteolytic activities of two types of mannosebinding lectin-associated serine protease. J. Immunol. 2000. V.165. P.2637-2642.

3. Jiang H., Wagner, E., Zhang H., Frank, M.M. Complement 1 inhibitor is a regulator of the alternative complement pathway. J Exp Med. 2001. V.194. P.1609-1616.

4. Andina C.C., Kozlov, L.V., Dyakov V.L. Determination of functional activity, the number of C1 inhibitor and autoantibodies to it as a tool for differential diagnosis of edema. Biomedical chemistry 2004. T. No. 1. Pp.86-91.

5. Kozlov, L.V., Andina S., Husova VA, Dyakov V.L., Batalova so-CALLED. The method of determining the functional activity of C1-inhibitor of the human complement. RF patent №2195662. Bull. No. 36. 27.12.2002.

1. The method of determining the functional activity of C1-inhibitor of the human complement via the alternative pathway, characterized by the fact that the holes microarrays for enzyme immunoassay absorb pharmacy drug KIP, then in wells microarrays make a solution of the sample containing C1-inhibitor of the human complement of unknown activity, carry out incubation and after drying the tablet and washing the wells, making the enzyme conjugate with antibodies against C1-inhibitor and substrate of this enzyme, followed by calculation of the content of active C1-inhibitor on the amount of the formed product of the enzymatic reaction.

2. Kit for determination of the functional activity of C1-inhibitor on the alternative path, characterized in that it contains a flat-bottomed to something called a microarray adsorbed drug KIP, the enzyme conjugate with antibodies to C1-inhibitor man, substrate buffer and the standard for active C1-inhibitor.



 

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