Method and kit for enzyme-linked thrombin-binding assay on c1 inhibitor functional activity

FIELD: medicine.

SUBSTANCE: there are presented a method and a kit for enzyme-linked thrombin-binding assay on C1 inhibitor functional activity. The method implies thrombin sorption in microplate wells, introduction of test samples containing the functionally active C1 inhibitor to be analysed, incubation that involves C1 inhibitor binding to thrombin, and measurement of the C1 inhibitor-bound amount by an enzyme conjugate with antibodies and a substrate of said enzyme. The kit comprises a flat-bottomed microplate with the sorbed preparation of thrombin, the enzyme conjugate with the human C1 inhibitor antibodies, a substrate buffer and a reference for measuring the active C1 inhibitor.

EFFECT: group of inventions provides the novel method and kit for enzyme-linked thrombin-binding assay on C1 inhibitor functional activity.

2 cl, 2 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.

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, XIIa, XIIf and plasmin (fibrinolizina) [1]. In [2] it was shown that the ability of individual purified preparations of thrombin and C1 inhibitor to form a complex. However, it was further shown that in the blood plasma is not detected the formation of the corresponding complex. This is probably why the use of such interaction to determine the functional activity of C1 inhibitor in the blood was considered to be problematic.

The need to determine the functional activity of C1 inhibitor in the blood of patients is dictated by the fact that the deficiency of this protein leads to periodic edema, mainly affecting the limbs, face, throat and mucous membrane of the gastrointestinal tract [1, 3].

The most promising of modern methods for the determination of proteins savoro the key blood method is an enzyme immunoassay due to its sensitivity, selectivity and the possibility of automation of the analytical process. In U.S. patent [4] used the modified method of determining the activity of C1-inhibitor, based on the test system production Cycotech (San Diego, CA, USA). The method includes the following steps: 1 - on microarrays absorb avidin, 2 - obtain highly purified enzyme active drug subcomponent C1s, 3 - synthesize biotinylated drug C1s, 4 - samples of samples containing defined functionally active C1-inhibitor, and incubated with biotinylated drug C1s, 5 - formed complex of functionally active C1-inhibitor with C1s absorb in the hole microarrays, covered with Avidya, 6 - the amount of bound peroxidase in the holes C1-inhibitor determined using conjugate goat antibodies against C1-inhibitor with horseradish peroxidase and a chromogenic substrate for peroxidase. The disadvantages of the method are its multi-stage (use additional stages of interaction of the Biotin-avidin, and the need to obtain biotinylated C1s and difficulties in the acquisition of enzymatic active C1s (present in the serum in preferments form in the amount of 30 mg/l, not commercially available).

Previously developed method of determining the functional activity of C1 inhibitor [5], which provided for the: sorption in the hole microarrays fibrinolizina (pharmacy drug plasmin), samples samples containing defined functionally active C1 inhibitor, contribute to the wells microarrays and spend incubation, during which covalent binding of C1 inhibitor with fibrinolizina, the amount of bound peroxidase in the holes C1 inhibitor determined using conjugate antibodies against C1 inhibitor with horseradish peroxidase and a chromogenic substrate for peroxidase. The method is based on the ability of functionally active C1 inhibitor covalently contact fibrinolizina. The disadvantages of the method include the high proteolytic activity of plasmin and its tendency to autolysis, resulting in low stability of plasmin during storage and the possibility of unwanted hydrolysis inhibitor.

Such deficiencies deprived of thrombin, which has a more limited specificity, stability during storage, commercially available, the more that you can use the product of animal origin: thrombin bull.

Objective of the claimed invention is to reduce the cost of making the determination of the functional activity of C1 inhibitor of the human complement-based immunoassay method, improving the reliability of the determination and the use of more stable commercially available drugs.

This object is achieved by developing a method immunopharmacological functional activity of C1 inhibitor in the ability to communicate with thrombin, which provides: sorption in the hole microarrays thrombin, introduction into the wells microarrays samples containing defined functionally active C1 inhibitor, conducting incubation, during which covalent binding of C1 inhibitor with thrombin, the amount of bound peroxidase in the holes C1 inhibitor using conjugate antibodies against C1 inhibitor with the enzyme and a chromogenic substrate for this enzyme. The method is based on the ability of functionally active C1 inhibitor covalently contact with thrombin.

The technical result of the claimed invention to provide a simplified method and the kit for immunoassay determination of the functional activity of C1 inhibitor of the complement of the person using available and stable preparations.

Example 1. Immunoassay determination of the functional activity of the preparation of C1 inhibitor in the ability to communicate with thrombin. Dissolve the drug thrombin bull in 0.05 M sodium carbonate buffer, pH of 9.5, with the final concentration of 15-50 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. Three times washed tablet veronalum buffer solution, pH of 7.4, containing 0.15 M NaCl, 0.15 mm Ca2+and 0.5 mm Mg2+(VBS2+), 20 µl in each well, the tablet then dried by shaking out the remaining liquid. To each well of the tablet make 100 ál of VBS2+. In wells of each row make 100 μl of a solution containing the designated C1 inhibitor in VBS2+in the form of a progressive two-fold dilutions. 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 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 amount of component C1 inhibitor is calculated by the standard curve (figure 1).

Figure 1 shows the determination of the activity of C1 inhibitor enzyme-linked immunosorbent assay.

Example 2. The kit for immunoassay determination of the functional activity of C1 inhibitor on the ability of the tie is to thrombin. The set contains a flat-bottomed to something called a microarray adsorbed drug thrombin, 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=0.99, that allows to reliably determine the functional activity of C1 inhibitor claimed process in concentrations from 1 ng/ml by using the described set.

In addition, to verify the adequacy of the method were compared with the activities of C1 inhibitor in the blood sera of 5 patients of the claimed method and a similar method using sorbed on microarrays natural enzyme for the inhibitor: subcomponent C1s. Figure 2 presents a graphical comparison of the results of determination of the functional activity of C1 inhibitor in five patients ' sera using ELISA method associated with C1s and with the associated thrombin. You can see a good match these definitions with the correlation coefficient R2=0.99, that allows to speak about the adequacy of the proposed method.

LITERATURE

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

2. Cugno M., I. Bos, Lubbers Y., C.E. Hack, Agostoni A. In vitro interaction of C1-inhibitor with thrombin. Blood Coagul. Fibrinolysis. 2001. V.12. P. 253-260.

3. 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.

4. Pilatte Y.M., C.H. Hammer, M.M. Frank, Fries, L.F. Process for the purification of C1-inhibitor. Patent USA US5030578. July 9, 1991.

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. Method immunoassay determination of the functional activity of C1 inhibitor in the ability to communicate with thrombin, which is characterized by binding to adsorbed in the wells of microarrays by protease C1 inhibitor-defined sample of unknown activity, determining the amount of bound peroxidase C1 inhibitor with the enzyme conjugate with antibodies against C1 inhibitor and substrate of this enzyme and subsequent calculation of inhibiting thrombin activity of C1 inhibitor on the amount of the formed product of the enzymatic reaction, characterized in that the holes microarrays for enzyme immunoassay as ligand binding absorb commercial preparation of thrombin.

2. Set for immuno is armentero determine the functional activity of C1 inhibitor in the ability to communicate with thrombin, characterized in that it contains a flat-bottomed to something called a microarray with sorbed preparation of thrombin, the enzyme conjugate with antibodies to C1 the inhibitor man, substrate buffer and the standard for calculation of activity of C1 inhibitor.



 

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