SU1125549A1 - Immunosorbent producing method - Google Patents

Abstract

METHOD OF PREPARING IMMUNESORBENT, including adding insoluble carrier antisera to the insoluble carrier, incubating the insoluble carrier with antiserum, administering an agent that blocks the free groups of the insoluble carrier, subsequent incubation and washing the product in saline, which is simple, and in order to simplify and get riddled and washed the product in a saline solution, and in order to simplify and get riddled, and the product was washed in a saline solution, and in order to simplify the product, it is easy to remove and dissolve the product. insoluble carrier use 4-6% suspension of formalin-fixed and killed by heating microorganisms of Staphylococcus aureus carrying on and protein A, and as the blocking agent, the free groups of the insoluble carrier ispol'uet (L form a preimmune serum.

Description

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CD. The invention relates to biochemistry and immunology, in particular, to methods for producing immobilized antibodies used in affinity separation of proteins, and to obtain immunosorbents used in analytical -biochemical studies and medical diagnostics for the isolation and quantitative determination of Malge quantities of proteins, for example, immunoglobulins of various classes synthesized in human lymphocyte culture. A method for producing immunosorbent is known, based on the immobilization of protein A, derived from gold of staph Sepharose 4 lj However, the method is complicated, as it includes the protein separation stage of the microorganisms, cyanogen bromide activated Sepharose and protein immobilization. The method is multistage, and the immobilized protein A is not used directly for the antibody purification procedure. The closest to the invention to the technical essence and the achieved effect is a method of obtaining immunosorbent, including adding to the insoluble carrier (Sepharose 4 V, activated by cyanogen bromide) antisera, 24-hour incubation of insoluble carrier with antiserum, the introduction of an agent that blocks free groups of non-soluble carrier ( 1M glycine), after-incubation for 4 h and washing the product in saline 2. The disadvantages of this method are the duration (over 28 hours) and the complexity due to the multistage process. In addition, Sepharose 4B used as a carrier is a scarce and expensive compound, and its activation requires working with an extremely toxic compound, cyan bromine. The purpose of the invention is to simplify and speed up the process of obtaining immuno sorbent. The goal is achieved by the fact that according to the method of obtaining an immunosorbent, including adding an antisymer to an insoluble carrier, incubating the insoluble carrier with antiserum, introducing an agent for blocking free groups of an insoluble carrier, subsequent incubation, and washing the product in saline as an insoluble carrier, an insoluble carrier can be used as insoluble carrier. 4-6% suspension of formalin-fixed and killed by heating microorganisms of Staphylococcus aureus carrying protein A on the surface, and as TBE blocking agent, the free groups of the insoluble carriers is used, preimmune serum. The essence of the method consists in using as insoluble carrier a 4-6% suspension of killed staphylococcal microorganisms, which makes it possible to significantly shorten the incubation time with both antiserum (up to 30 min) and blocking agent (also up to 30 min), since the reaction the attachment of antibodies, the content of xc in the antiserum, and the blocking agents (proteins) contained in the non-immune serum are non-chemical and extremely fast. These reactions proceed well at physiological pH at room temperature, i.e. the implementation of the technological process proceeds in the same temperature mode (20 ° C) and using a single buffer solution with a pH of 7.6. Simplification of the method is also associated with the elimination of the need for special equipment (refrigerating chamber, electric scooper, etc.). In addition, staphylococcal microorganisms used as feedstock have a low cost and are capable of rapidly increasing their mass without any additional costs. The need to activate the insoluble carrier with cyan bromide is no longer necessary, since the antibodies contained in the antiserum are firmly attached to the staphylococcal microorganisms spontaneously. Example 1. Getting immunosorbent. A suspension of formalin-fixed and heat-killed killed Staphylococcus aureus Cowan 1 is prepared according to the Kessler method. The microorganisms are incubated in m-septon broth for 24 hours with stirring using a magnetic stirrer. The resulting microbial mass is washed twice in a physiological solution containing 0.02 M phosphate buffer pH 7.6 and 0.05% merthiolate by centrifugation.

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at 5000 rpm for 20 minutes in a TsLS-3 centrifuge. Then M1 crocodile is shaken for 1.5 h in the indicated buffer solution containing 1.5% formalin, and after washing it is heated at 3-6 min for 5 min. After additional washing, the concentration of microorganisms is adjusted to 5%. Suspension can be stored for at least 4 months. in saline containing 0.02 M phosphate buffer with a pH of 7.6 and 0.05% merthiolate. In centrifuge tubes containing 0.05 ml of a 5% suspension fixed by formalin and killed by the warming up of the microorganisms of Kovan 1 aureus, add 0.05 ml of rabbit antiserum against human i-chain immunoglobulins and incubate for 30 minutes at room temperature ( 20 C) temperature. Then, 6 MP of non-immune rabbit peripheral blood serum is added to the tubes and incubated for another 30 minutes at room temperature, after which the microorganisms are washed three times with physiological saline containing 0.02 M phosphate buffer with pH 7.6 by centrifugation at 3000 rpm within 5 min.

The effectiveness of the resulting immunosorbents is illustrated by the following examples.

Example 2. The preparation of immunosorbents was carried out as in Example 1, but using 1, 4, 5, and 6% cell suspension concentrations. Immunosorbents with a suspension concentration of 4, 5 and 6% sorb immunoglobulins G and M with almost the same efficiency (the specificity of the immunosorbent depends on the type of antibodies used for its floor). The sorption efficiency for immunosorbent, obtained on the basis of a 1% cell suspension, decreases in relation to immunoglobulins G and M, respectively, by 5-6 and 8-8.5 times. Thus, the effectiveness of the last sorbent is low. The use of suspended matter concentrate above 6% is impractical due to overrun of the starting material.

Example 3. Determination of the content of carbon-14 immunoglobulin G and immunoglobulin M in a mixture of labeled proteins.

Washed immunosorbents (0.05 ml) are suspended in 1 ml of the sample about 255494

the sample is a mixture of carbon-14-labeled proteins with an unknown content of radioactive immunoglobulins G and immunoglobulins M. The tubes are incubated for 40 min at room temperature. The recorded quantitative characteristic of the studied immunoglobulins G and immunoglobulins M, attached as a result of incubation, to immunosorbents carrying antibodies against the human immunoglobulin I-chains corresponding to human radioactivity is directly proportional to the amount of immunoglobulins. The specificity of anti-immunoglobulin immunosorbents is checked by blocking their binding capacity with non-radioactive standard high-purity immunoglobulins. For this, a part of immunosorbents are incubated for 40 minutes at room temperature with an excess of inactive dioactive immunoglobulin G (at the rate of 25 μg per 0.06 mp 5% suspension of the immunosorbent) or M (at the rate of 100 μg per 0.05) before incubating with the test sample. ml of 5% suspension of immunosorbent) and washed three times.

0 The final radioactivity of proteins attached to anti-immunoglobulin immunosorbents is determined using a beta-spectrometer after their suspension in physiological solution,

containing 0.02 M phosphate buffer with a pH of 7.6, and transferred to vials containing scintillation fluid. Brae The degree of specificity of immunoglobulin attachment to anti-immunQ noglobulin immunosorbents is expressed by the coefficient (K) of specificity, which is calculated according to the formula

.

SO And

8. And

W

kr,

where A is the radioactivity of proteins attached to an unblocked anti-immunoglobulin immunosorbent;

B — radioactivity of proteins attached to an anti-immunoglobulin immunosorbent blocked by a non-radioactive immunoglobulin of the appropriate class; B is the radioactivity of proteins attached to an anti-immunoglobulin immunosorbent treated with a non-radioactive immunoglobulin of the opposite class. Anti-immunoglobulin immunosorbents, characterized by an attachment specificity coefficient, have the highest specificity. The table gives the definition of the content of labeled with carbon-14 and D4-heglobulin M and immunoglobulins M in a mixture of labeled proteins. (In experiments 1 and 2, two different samples of a mixture of labeled proteins were used. As can be seen from the table, the immunosorbents obtained according to the proposed method, carrying antibodies against the Y and U chains of human immunoglobulins, are suitable for highly specific determination of the immunoglobulin M immunoglobulins M in a mixture proteins. Obtained according to the proposed method ymm rhinosubbents are suitable for use as a diagnostic tool in assessing the functional state: the humoral immunity system in patients, in In particular, to determine the humoral immune response of the studied lymphocytes to mitogenic stimulation. Thus, the use of the invention significantly simplifies (by reducing the number of stages and their complexity) and speeds up (reducing the process time to 1-2 hours instead of 28 hours according to a known method) the process of obtaining immunosorbent. At the same time, there is also saving of inaccessible phosphorus and exclusion from the process up to BfiToro reagent (cyanogen bromide). These advantages allow the proposed method to be used for routine production of immunosorbents in a wide range of research and medical diagnostic laboratories.

Anti-ImmunogloGene Bulin G

Not used Immunoglobulin G Immunoglobulin M Immunoglobulin M Immunoglobulin G

96741287 38461130

61t1 1,641

9753186 3788146

1125549

U Continuation of the table

Claims (1)

METHOD FOR PRODUCING AN IMMUNOSORBENT, including adding antiserum to an insoluble carrier, incubating an insoluble carrier with an antiserum, introducing an agent that blocks the free groups of an insoluble carrier, subsequent incubation and washing of the product in physiological saline, characterized in that, in order to simplify and simplify the process a 4-6% suspension of formalin fixed and staphylococcus aureus killed by heating microorganisms carrying white And, as a blocking agent, free _of insoluble carrier trup- nN, The use ® form a preimmune serum. SU, .., 1125549 1 1125549