Method and kit for immunoassay for c1 inhibitor activity shown in both pathways of complement activation

FIELD: medicine.

SUBSTANCE: method under the invention provides that the complex immunoglobulin preparation containing a component of C3b complement is sorbed in microplate wells for the purpose of immunoassay. Then the microplate wells are added with a solution of an analysed sample containing human complement C1 inhibitor with the unknown activity. It is followed by incubation, drying and washing of the dish; the wells are added with a conjugate of enzyme with serine proteinase in the form of preparation of fibrinolysin and a substrate of this enzyme. The amount of the prepared enzymatic reaction product is used to derive the content of active C1 inhibitor. A kit for implementing the method comprises a flat-bottomed microplate with bound C3b, the conjugate of enzyme with serine proteinase, the substrate buffer of this enzyme and a reference for active C1 inhibitor.

EFFECT: use of the method under the invention enables determining activity of C1 inhibitor, simultaneously bound both with serine proteinase inhibition, and with binding inhibition of complement factor B and C3b complement component.

2 cl, 1 tbl, 1 dwg

 

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 Proteas 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, XIIa, 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 C3b complement and inhibiting the binding factor In complement with C3b [3]. This function is chemically different from the already known at the time of its implementation of the C1 inhibitor does not block covalently-active center serine proteases, as it was in all the above cases, and is associated ecovalence component C3b, inhibiting the formation or destabilizing the final factor In [3].

The deficiency of this inhibitor in the patient's blood leads to periodic edema, tatraiva is relevant 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, such mutations on both the functional activity of C1 inhibitor can have different effects. Thus, it is now known that C1 inhibitor can bind to serine proteases covalently (for example, components of the C1s and C1r complement or fibrinolizina [5], forming the ester bond of carboxyl group of the inhibitor to the hydroxyl group of serine in the active center of the protease and inhibition of the classical pathway of activation of complement. In addition, it is known that C1 inhibitor can contact ecovalence component C3b complement, blocking its interaction with the factor and In the inhibition of alternative pathway activation of complement. Are these two sites of interaction of C1 inhibitor with complement in one molecule inhibitor or spatially separated and can interact independently in the literature is unknown. With the existence of independent sites of interaction of C1 inhibitor with to monentary complement C1s and C3b is a possible method for determining the activity of C1 inhibitor, where C1 inhibitor is simultaneously associated with both components or substances that imitate it.

Objective of the claimed invention is a method and kit for immunoassay determination of the minimum of the activities of C1 inhibitor by the classical and alternative pathways using simple and available funds without specific antibodies.

This object is achieved by developing a method immunoassay determination of C1 inhibitor, which provides for binding in the hole microarrays component C3b, incubation in the wells samples containing defined functionally active C1 inhibitor man, and determination of bound peroxidase C1 inhibitor using conjugate serine proteinase enzyme and substrate for this enzyme.

For fixing in the hole microarrays C3b 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
661,63,89

The set contains a flat-bottomed to something called a microarray with the associated C3b in the medication KIP, a conjugate of an enzyme with serine-protease, substrate buffer and the standard for active C1 inhibitor.

The technical result of the claimed invention is to develop a method and kit for immunoassay determination of C1 inhibitor possessing both complementarianism activities using as source component C3b complement pharmacy drug KIP (complex immunodeficiency is globulinemia drug consisting of immunoglobulins G, a and M people), which, as it was discovered, contains a component C3b complement person and for the determination of bound peroxidase C1 inhibitor use conjugate pharmacy drug fibrinolizina with horseradish peroxidase. Thus, when implementing this method there is no need to obtain specific antibodies can also be used pharmaceutical drugs, and the way answers the question of the existence of a deficiency in the activity of C1 inhibitor, regardless of whether this is due to the deficiency of activity of C1 inhibitor in the classical or alternative pathways of complement.

Example 1. The determination of the activity of C1 inhibitor, shown in both pathways activate the complement. 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), 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 7°C, double washing phosphate buffer (SFR), pH 7.4, containing 0.15 M NaCl and 0.05% tween-20, and drainage microarrays in each well contribute 100 μl of peroxidase conjugate with pharmacopoeial drug fibrinolizina 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 (drawing).

The drawing shows the determination of the activity of C1 inhibitor, shown in both pathways activate the complement.

Example 2. The kit immunoassay for determining the activity of C1 inhibitor, shown in both pathways activate the complement.

Set to determine the minimum of the complement inhibiting activity of C1 inhibitor. The set contains a flat-bottomed to something called a microarray with an associated component C3b, a conjugate of an enzyme with serine-protease, substrate buffer and a standard with a known asset is awn C1 inhibitor man. This set is used in accordance with example 1.

From what is shown on the drawing results, it follows that the measured optical density is linearly dependent on the concentration of active C1 inhibitor with correlation coefficient R2=0.99, that allows you to reliably determine the activity of the C1 inhibitor shown in both pathways activate the complement in concentrations of 1.5 ng/ml in the described manner by using the described set.

LITERATURE

1. Davis, A.E. 3rd. C1 inhibitor and hereditary angioneurotic edema. Ann. Rev. Immunol. 1988. V.66. P.595-628.

2. Matsushita M., Thiel, S., Jensenius J.C., Terai I., 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 S., 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 immunoassay method for determining the activity of C1 inhibitor, shown in both pathways activate the complement, characterized in that the holes microarrays for enzyme immunoassay SOR is irout 3b in the form of pharmaceutical 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 wash the wells make a conjugate of an enzyme with serine-proteinase in the form of pharmaceutical drug fibrinolizina and the substrate of this enzyme, followed by calculation of the content of active C1 inhibitor according to the amount of the formed product of the enzymatic reaction.

2. The kit immunoassay for determining the activity of C1 inhibitor, shown in both pathways activate the complement, characterized in that the kit contains flat-bottomed to something called a microarray with an associated component of complement 3b, the conjugate of an enzyme with serine-protease, substrate buffer and the standard for active C1 inhibitor.



 

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