SK101492A3 - Solution for stabilization of biological active substances bonded on a solid support - Google Patents

Description

Pharmaceutical industry.

BACKGROUND OF THE INVENTION

A method is currently used in which a biologically active substance binds to a solid support by passive adsorption, typically at alkaline pH, for several hours at 4-37 ° C. After washing, a blocking solution, usually containing proteins, most commonly bovine serum albumin (BSA), where the protein binds to unoccupied active sites of the polystyrene carrier, is usually applied further, thereby reducing non-specific reactions when performing the assay itself. After washing and drying, the carrier is sealed in foil and kept dry and refrigerated. It is usually stable for several months without measurable loss of activity.

Some biologically active substances (eg antibodies) isolated from hyperimmune sera, ascites fluids or other biological material may contain, despite partial purification, traces of proteolytic enzymes, while some enzymes bind to the carrier together with the antibody and cause degradation during storage, and thereby a decrease in carrier activity. Some biologically active substances are more sensitive to the presence of these enzymes, others less. The amount of proteolytic enzymes present may also vary from lot to lot, and it is not easy to determine exactly how stable the lot of carrier with the biologically active agent will be. Sometimes, bovine albumin used for blocking may also contain proteolytic enzymes rather than adenoviruses.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION It is an object of the present invention to provide a blocking solution which contains β-aminocaproic acid instead of proteins, thereby eliminating the above disadvantages. E-aminocaproic acid (EACA) is an inhibitor of proteolytic enzymes and is used to stabilize protein-containing solutions. It is also capable of passively adsorbing to a solid support (e.g. polystyrene). Thus, these two EACA properties can be utilized to stabilize biologically active substances bound to a solid support. If an EACA solution is used instead of a protein blocking solution, the EACA will bind to the yet unoccupied active sites of the polystyrene carrier. This, on the one hand, occupies undesirable free active sites (thus blocking and reducing non-specific reactions) and, on the other hand, the bound EACA by its ability to inhibit proteases prevents the degradation of the bound biologically active substance by the potentially present proteolytic enzymes.

The binding of biologically active substances (most commonly antigens or antibodies) to insoluble polymeric carriers (most often polystyrene) has a wide application in the production of diagnostic kits based on enzyme immunoassay (EIA, ELISA), radioimmunoassay (RIA) and some other methods. These kits then serve to detect or determine biologically active substances in the diagnosis and monitoring of a variety of diseases (infectious, tumor, metabolic, genetic, etc.).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

a) A monoclonal anti-HBe antibody, clone A2 (i.e. against hepatitis B virus e antigen), was bound by passive adsorption to a polystyrene microtiter plate. Binding was carried out overnight at + 4 ° C, in an environment of 0.01 mol / l bicarbonate buffer at pH = 9.6, the antibody had a concentration of 0.01 mg / ml.

b) The solution was aspirated from the wells and the blocking solution of the invention (at the optimum concentration, i.e. 0.1% EACA in water) or a conventional blocking solution containing 1% BSA was applied. Incubate for 1 h at room temperature.

c) After aspiration of the blocking solution, the plate was dried with compressed air and sealed under vacuum into a triple layer polyamide / (Al 1) polyethylene.

U prepared in this way stability was monitored for platelets % activity of antibodies bound to + 4 ° C and + dot at 37 ° C, as EL ISA test (evidence of serum HBeAg). classical blocking blocking solution solution s 1% BSA according to the invention Low: 14 ° C Deň: 32 ° C 5 ° C time ACTIVITY (%) ACTIVITY (%) 0 100 100 100 100 1 month 100 9 1 100 100 2 months 100 8 2 100 100 3 months 9 8 45 100 100 4 months 97 20 100 100 6 months 96 1 1 100 42

The results of the stability tests show that the use of the blocking solution of the invention at an optimal concentration of EACA significantly increases the stability of the anti-HBe antibodies bound to the polystyrene plate. After half a year, the activity of the plate stabilized according to the invention at 37 ° C is the same as the plates blocked in the classical manner after 3 months. The expiration of the plate prepared according to the invention is approximately twice that of the blocking solution.

Example 1 a d 2

a) A monoclonal antibody was bound to the polystyrene microtiter plate by passive adsorption (i.e. anti-hepatitis e antigen was run overnight at + 4 ° C, anti-HBe, clone A2B). Binding to an environment of 0.01 mol / l bicarbonate buffer pH-9.6, the antibody had a concentration of 0.01 mg / ml

b) The solution was aspirated from the wells and a blocking solution of the invention (at a minimum effective concentration, i.e., 0.001% EACA in water) or a conventional blocking solution containing 1% BSA was applied. Incubate for 1 h at room temperature.

c) After aspiration of the blocking solution, the plate was dried with compressed air and sealed under vacuum into a triple layer film of poly (A 1) polyethylene.

The plate thus prepared was monitored for stability at + 4 ° C and + 37 ° C, as a% of the activity of the plate-bound antibodies in an ELISA (proof of serum HBeAg).

classical blocking solution with 1% BSA b 1 eye solution according to the invention

Low: 14 ° C Deň: 32 ° C Low: 14 ° C 37 ° time' ACTIVITY (%) ACTIVITY (£) 0 100 100 100 100 1 month 100 9 1 100 100 2 months 100 82 100 90 3 months 98 45 100 60 4 months 97 20 100 38 6 months 96 1 1 100 20

The stability of the plates stabilized according to the invention with a minimum effective EACA concentration is slightly better than the plates prepared by the classical method.

Ex a <1 a d 3

a) Monoclonal anti-HBe antibody, clone A2 (i.e. against hepatitis B virus e antigen), was passively adsorbed to the polystyrene microtiter plate. Binding was carried out overnight at + 4 ° C, in an environment of 0.01 mol / l b i carbonate buffer pH = 9.6, the antibody having a concentration of 0.01 mg / ml.

b) The solution was aspirated from the wells and a blocking solution according to the invention (with the maximum possible concentration of EACA, i.e. 10% EACA in water) or a conventional blocking solution containing 1% EASA was applied. Everything was incubated for 1 h at room temperature.

c) After aspiration of the blocking solution, the plate was dried with compressed air and sealed under vacuum into a triple layer polyamide / (Al 1) for 1 yet ylene.

Plates thus prepared were monitored for stability at + 4 ° C and + 37 ° C, as% of plate-bound antibody activity in an ELISA (evidence of serum HBeAg).

classical blocking solution with 1% BSA blocking solution according to the invention

Low: 14 ° C 37 “C Low: 14 ° C Deň: 32 ° C time ACTIVITY (%) QUALITY (%) 0 100 100 100 100 1 month 100 9 1 100 100 2 months 100 82 100 100 3 months 98 45 100 98

months 97 20 100 · 70 6 months 96 11 100 44

Thus, the stability of the plates prepared according to the invention with a maximum concentration of EACA is comparable to the results obtained with an optimal concentration (Example 1).

Claims (1)

A solution for the stabilization of biologically active substances bound to a solid support, characterized in that it contains epsilon amino caproic acid at a concentration of 10 m 1 k rograms / m 1 to 100 mg / m 1.