SU1704090A1 - Method for preparing erythrocytar diagnosticum for inmmunoanalysis - Google Patents

Abstract

This invention relates to the field of immunobiotechnology. specifically, to engineering immunochemistry, and can be used in the production of immunodiagnostic test systems for the determination of antigens and antibodies. The aim of the invention is to increase the sensitivity of the immunoassay. This goal is achieved by covalent immobilization of ana-specific immunoreagents on erythrocytes activated by sodium meta-period in the presence of high molecular weight dextran. This makes it possible to increase the sensitivity of the determination of antigen and antibodies by 8-16 times in comparison with known methods. 2 tab. WITH

Description

The invention relates to the field of immunobiotechnology, namely engineering immunochemistry, and can be used in the manufacture of immunodiagnostic test systems for the determination of antigens and antibodies.

Immuno-analitic methods are known, which are based on the use of stereospecific detectable ligand of efficacy cells labeled with erythrocytes (direct and passive hemagglutination reactions — RIGA and RPHA, erythroimmuno-adsorption — HerA 1 and 2.

The covalent immobilization of immunoreagents on erythrocytes allows

receive highly active and stable erythrocyte diagnostica. The most effective are the two-stage methods of conjugation, which involve the preliminary activation of erythrocytes (stage I) with the subsequent immobilization on their surface of intact antigens, antibodies, and other immunoreactive compounds (stage II). Two-step methods guarantee the safety of the functional activity of immunoreagents labeled with erythrocytes. Activation can be accomplished by directly chemically modifying the peptide or carbohydrate groups of the viewer membrane.

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of roocytes into reactive groups, or by the introduction of exogenous reactive groups during the treatment of erythrocytes with homo- and heterobifunctional reagents. In the known methods, either peptide (often primary ainic) are selectively activated. or carbohydrate (usually hydroxyl) groups of erythrocyte membranes. Accordingly, antigens and antibodies are immobilized either on protein portions of integral and peripheral glycoproteins, or on polysaccharides of glycocalyx of the activated surface of erythrocytes. Therefore, the known methods do not allow covalently binding specific analyte immunoreagents to be 100% theoretically functionally active in relation to chemical modification of the erythrocyte membrane area, i.e. achieve the highest possible density of their covalent immobilization.

The sensitivity of immunoassay methods with erythrocyte diagnostics directly depends on the density of immobilization of specific analyte immunoreagents on the surface of erythrocytes and is theoretically maximum at the maximum density of their immobilization.

The closest to the present invention is the method of preparing spectrocytic diagnostics for immunoassay, which is as follows: 50% suspension Formalized sheep red blood cells are treated with 0.001 M mega-periodate sodium in 0.02 M acetate buffer pH 4.9 at + 4 ° C for 30 minutes . Activated erythrocytes are washed with 0.05 M phosphate buffer (FBI) pH 8.5. and used for conjugation with human immunoglobulin G. 1% immunoglobulin G (10 mg / ml) in PBS was incubated with activated erythrocytes for 2 hours at 22 ° C, after which sodium borohydride was added to a final concentration of 0.01% and incubated for another 1 hour at + 4 ° C in the dark. The prepared erythrocyte diagnosticum (C. immunoglobulin labeled with erythrocytes) is used (after washing with buffered phosphates (0.15 M sodium chloride solution pH 7.2, EGF in RPGP) to determine rheumatoid factor 3.

The known method is based on the direct chemical modification of paired hydroxyl groups of the hexose and pentose rings of erythrocyte membrane carbohydrates due to their oxidation by meta-period anions into reactive aldehyde groups. Covenant bond is formed as base.

Schiffra with the participation of aldehyde groups of activated erythrocyte polysaccharides and primary amino groups of immobilized IgG, followed by stabilization of the valylamine bond by reduction with sodium borohydride. IgG covalently binds to glycocalyx polysaccharides. but not on the activated red cell membrane proteins. As a result, IgGs are immobilized with

0 by a non-maximal density, since the selective nature of covalent binding prevents their conjugation from 100% of the theoretical area of the erythrocyte membrane.

The disadvantage of the known method is the low sensitivity of the immunoassay with the prepared diagnosticum, which is a consequence of the non-maximal density of immobilization of the analyte-specific immunoreagent (tgG) on the surface of the viewer cell mark. In addition, when red blood cells are treated with a very low concentration of sodium meta-periodate (0.001 M), incomplete activation occurs.

5 glycocalyx polysaccharides.

At the same time, the prototype does not allow an increase in the concentration of meta-periodate anions. since at higher concentrations it actively oxidizes heme

0 pigments, which leads to discoloration of activated red blood cells (this significantly reduces the contrast of the label). The aim of the invention is to increase the sensitivity of the immunoassay.

5 The goal is achieved by covalent immobilization of analyte-specific immunoreagents on erythrocytes activated by sodium meta-periodate in a mixture with high-molecular dextran.

0 When activated, which is carried out under alkaline conditions (pH 9.6) at room temperature (22 ° C) for 20 minutes, simultaneous modification of the erythrocyte glycocalyx erythrocyte polysaccharides takes place.

5 dextran molecules in solution. Activated high molecular weight dextran is an in situ polyacetate homo-polyfunctional compound that is formed in situ.

0 reagent (according to the nature of the reactive groups - popaldehyde). The activation pH correlates to the conjugation pH. therefore, the zldehyde dextran molecules are covalently bound to amino groups during the activation time.

5 integral and peripheral proteins of the erythrocyte membrane, exposing a large number of free reactive aldehyde groups to the solution.

Thus, a maximum (100%) of the theoretical surface area of the erythrocytes becomes activated. Covalent binding of activated dextran creates favorable spatial conditions for the further immobilization of ligand-specific immunoreagents at some distance from the erythrocyte membrane surface (spacer effect), which increases the degree of freedom of their reaction with definable ligands. In addition, the difference from the known method consists in using a 20-fold higher concentration of sodium meta-periodate (0.002 M, and 0.001 M in the known method, which provides a more complete activation of erythrocyte membrane polysaccharides, while no decrease in their contrast ratio was observed. Example 1: Erythrocyte Activation: To 1 ml of a 50% suspension of formalinized irradiated erythrocytes of the drum in a 0.05 M carbonate bicarbonate buffer (CBB) pH 9.6. Add 3 ml of 10% -. dextran t-500 o KBB and 1 ml of a 0.1 M solution of sodium meta-periodate (chemically pure). The mixture is incubated, sively with mixing by shaking for 20 minutes at 22 ° C. The activated erythrocytes were washed 5 times by centrifugation in HEC.

Immobilization of immunoreagents. 0.5 ml of a 50% suspension of activated erythrocytes is mixed with 2 mg of an immunoreagent specific to a detectable ligand in 2 ml of KBB. Incubate for 1 hour at 22 ° C and constant stirring, and then, after adding sodium borohydride (chemically pure), to a final concentration of 0.02% for another 1 hour at 4 ° C in the dark.

The suspension of the prepared erythrocytic diagnostic agent is washed in EGF with 0.05% tween-20 and stored in the same solution with 0.02% sodium azide.

The results of an experimental comparison of the known method are presented in Table 1.

EXAMPLE 2 The method is carried out analogously to example 1, but the conjugation conditions vary: concentration of dextran, periodate concentration, activation time, pH.

The sensitivity of the determination of the columnar toxoid in EIA with the diagnostics obtained is given in Table. 2

Thus, the sensitivity of immunoassays with erythrocyteric diagnostics. prepared by the proposed method, 8/16 times higher sensitivity, achievable in the known method.

In addition, the method makes it possible to shorten the preparation time of erythrocyte immunodiagnostics by 2 times — conjugation (immobilization of immunoreagents on erythrocytes) lasts 1 hour versus 2 hours in a known way. and a 10-fold decrease in the concentration of immobilized immunoreagents.

Claims (1)

Invention Formula A method for preparing an erythrocyte diagnosticum for immunoassay, including treating formalinized erythrocytes with periodate followed by incubation with an immunoreagent and restoring sodium borohydride, in order to increase immunoassay sensitivity. periodate treatment is carried out at pH 8.5-9.6 in the presence of 8-12 vol.% dextran mol. m. 40-500 kilodalton at 20-25 ° C for 10-30 minutes, while periodate is used in concentrations of 0.01-0.04 m.  Pure antibodies and F (ab) 2 - fragments of pure antibodies were obtained by affinity chromatography of horse hyperimmune antisera on antigenic immunosorbents.  Immunoglobulins G are obtained from hyperimmune antiserum by ion exchange chromatography on DEAE - Sephadex A-50.  Antibody titers were determined in unfractionated immune rabbit antisera.  Used commercial reagent affinity chromatographic protein A. table 2