RU2798124C1 - Method of obtaining brucellosis polystyrene latex diagnosticum - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of obtaining a brucellosis polystyrene latex diagnosticum is proposed, including mixing in equal volumes of a 2% solution of polystyrene latex and an antigen of a brucellosis microbe from a strain of Brucella (B.) abortus 19 BA at a concentration of 2×10⁹ mc/ml in 12% sodium chloride solution, stained with brilliant green — 0.004%, gentian violet — 0.002%; incubation 2 h at 37°C and 12–16 hours in the refrigerator at 4°C; then washed twice from unbound antigen with 0.1 M phosphate-buffered saline pH 7.2–7.4 by centrifugation at 5000 rpm for 10 min; the precipitate is resuspended in 0.1% gelatin solution to 0.2% concentration with formalin to 0.1%.

EFFECT: invention provides an extension of the arsenal of methods of obtaining a specific brucellosis latex diagnosticum for the detection of antibodies in the reaction of latex agglutination.

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Description

The invention relates to biotechnology, microbiology and, in particular, to a method for producing latex diagnosticums for latex agglutination reaction.

Polymeric microspheres (latex particles) with adsorbed molecules of antibodies or antigens react and agglutinate with the corresponding antigens of microorganisms or antibodies. Polymer microspheres are a convenient and flexible system for the development of diagnostic preparations used in the latex agglutination test (RAL). When choosing polymer microspheres, one usually takes into account the diameter and size distribution of particles, composition, chemical properties of the surface (reaction groups, functionality, charge), and other more specific properties, such as the presence of chromophore fillers (dyes), nanoparticles of metals and their oxides. For latex agglutination, microspheres (colored, unpainted) with a size of 0.2-1.0 microns, activated (for covalent crosslinking) and non-activated (for physical sorption) are more often used. An important parameter is the composition of polymeric microspheres. Typically, polymer microspheres consist of polyacrolein, polystyrene, polymethyl methacrylate. These materials have different physical and optical properties, which may show advantages or impose some restrictions on their use [1].

To detect antibodies against the causative agent of brucellosis, various serological methods are used (indirect hemagglutination reaction - RIGA; enzyme immunoassay - ELISA; latex agglutination reaction - RAL, etc.), which have their own advantages and disadvantages.

A known method of obtaining R-brucella erythrocyte diagnosticum for RIGA [2], which consists in the preparation of formalized sheep erythrocytes, followed by their sensitization with sensitin obtained by growing the bacterial mass of B. abortus 16/4, its washing, inactivation, extraction, separation of sensitin by centrifugation, exposure on bacterial cells with sodium dodecyl sulfate.

The disadvantage of this method is the use of biological raw materials - sheep erythrocytes, difficult to standardize, whose parameters depend on factors such as sex, age of the animal, time of blood sampling. In addition, to obtain high-quality erythrocyte diagnosticums, formalized erythrocytes must be kept for a year for stabilization.

A known method of obtaining enzyme immunoassay test systems based on monoclonal antibodies against S-LPS (S-lipopolysaccharide) and R-LPS (R-lipopolysaccharide), labeled with horseradish peroxidase, for the differential diagnosis of brucellosis [3].

The disadvantage of this method is the relative complexity of obtaining a test system and analysis, the possibility of false positive results, the need to use chemically clean glassware. In addition, a prerequisite is the availability of a photometer in the laboratory to record the results.

Closest to the claimed method is a method of obtaining immunodiagnosticum based on polystyrene latex [4]. To obtain an immunodiagnosticum in accordance with the invention, an equal volume of α-casein solution (10 mg/ml) in buffer prepared as follows: α-casein is dissolved in the calculated amount of buffer, heated for 30 minutes at 37°C, the solution is clarified by centrifugation for 15 minutes at 5000 rpm. The optimal concentration of α-casein solution to saturate free binding sites is 0.4 mg/ml. The mixture of latex suspension with α-casein solution is thoroughly shaken and left at room temperature for 30 min, after which it is placed in a refrigerator (4°С) for 24 h. polymer, however, the presence of casein in the composition of the drug significantly reduces its shelf life due to the possibility of bacterial growth. The disadvantage of this method is that the diagnosticums obtained by the described method are not sensitive enough: the maximum serum dilution that this drug can detect is 1:320.

The purpose of the invention is to develop a method for producing an active and specific brucellosis polystyrene latex diagnosticum for the detection of antibodies in the reaction of latex agglutination.

The technical result of the invention lies in the fact that the polymer suspension is centrifuged, adjusted with 0.9% sodium chloride solution to a concentration of 2% by dry residue of the polymer and mixed in equal volumes with the antigen of the brucellosis microbe from the strain Brucella (B.) abortus 19 destroyed on an ultrasonic disintegrator VA at a concentration of 2×10 ⁹ mc/ml in 12% sodium chloride solution, stained with brilliant green (0.004%) and gentian violet (0.002%); incubated at 37°C for 2 hours and 12-16 hours in a refrigerator at 4°C; twice washed from unbound antigen with 0.1 M phosphate-buffered saline, pH 7.2-7.4 by centrifugation at 5000 rpm for 10 min; the precipitate is resuspended in 0.1% gelatin solution to 0.2% concentration and stabilized with formalin to 0.1%.

To obtain a brucellosis latex diagnosticum for the detection of antibodies in RAL, latex for agglutination (OOO Diaem, Moscow) with a particle size of 0.8 μm, a dry residue content of polystyrene of 10% and sodium dodecyl sulfate of 0.01% was used. A suspension of the brucellosis microbe B. abortus 19 VA grown on Albimi agar at a temperature of (37±1)°C for (48±1) hours was used as a ligand. The backmass was washed out with 12% sodium chloride solution. Next, the backmass was heated at 100°С for 60 min. After controlling the general and specific sterility of the back mass, the suspension was diluted to a concentration of 2×10 ⁹ mc/ml with 12% sodium chloride solution with 0.5% phenol. Aniline dyes of the triphenylmethane series were added: brilliant green up to 0.004% concentration and gentian violet up to 0.002% concentration. The antigen was destroyed on an ultrasonic homogenizer (Sonicator Q125, Qsonica LLC) for 2 min (frequency 20 Hz).

Control of sensitivity and specificity of the developed experimental series of the drug was carried out with homologous and heterologous sera. RAL was placed in U-shaped (round-bottom) microplates.

To control the sensitivity of RAL, brucellosis sera were used: immune rabbit serum obtained at the FKUZ Stavropol Anti-Plague Institute of Rospotrebnadzor (experimental series) and diagnostic polyvalent brucellosis dry serum for the agglutination test (FKUZ Irkutsk Research Anti-Plague Institute of Rospotrebnadzor).

To control the specificity, the following sera were used: diagnostic cholera O1 adsorbed dry for RA (FKUN Russian Research Anti-Plague Institute Microb); diagnostic Salmonella adsorbed O-polyvalent for RA (CJSC "Ecolab"); diagnostic dry tularemia for RA (FKUZ Irkutsk Research Anti-Plague Institute of Rospotrebnadzor).

The RAL setting (micromethod) was carried out as follows: 25 µl of dilution liquid Tween-80 at a dilution of 1:25 thousand was added to all wells (8 rows) of the plate. Further, in the first wells of the rows, 25 µl of homologous and heterologous sera at dilution 1: 100, titrated in steps of 2, getting dilutions in the wells from 1:200 to 1:12800, the eighth rows are negative controls (serum was not added to them). Then, 25 µl of the developed brucellosis latex diagnosticum was dripped into all wells. The sensitivity of RAL was 1:800-1:1600, which corresponds to the sensitivity of the brucellosis erythrocyte diagnosticum. In the control of specificity, it was found that there was no cross-reaction with salmonella, cholera sera, and with tularemia, a cross-reaction up to ¼ titer of diagnosticum was observed, which meets the requirements of regulatory documents for these types of diagnosticums. The possibility of practical application of the invention is illustrated by examples of its specific implementation using the totality of the claimed features.

Example 1. Development of a method for obtaining a brucellosis latex diagnosticum for the agglutination reaction with B. abortus 19 BA brucella antigen in a ratio of 1:2.

To obtain a brucellosis antigenic latex diagnosticum, polystyrene latex was used for agglutination with a particle size of 0.8 μm. As a ligand for immobilization, a suspension of B. abortus 19 VA was used at a concentration of 2 × 10 ⁹ mc/ml in 12% sodium chloride solution, inactivated by heating at 100°C for 60 min with the addition of 0.5% phenol and destroyed on an ultrasonic homogenizer for 2 min. Brilliant green - up to 0.004%, gentian violet - up to 0.002% concentration were used as antiseptics and dyes. Latex (2%) and antigen were mixed in a 1:2 volume ratio. The mixture was incubated with stirring and a temperature of 37°C for 2 hours and 12-16 hours in a refrigerator at a temperature of 4°C. Then it was centrifuged at 5000 rpm for 10 min, washed twice from unbound antigen with 0.1 M PBS, pH 7.2-7.4, and the last precipitate was resuspended in 0.1% gelatin solution to 0.2% concentration. To stabilize and store for a long time, formalin was added to a concentration of 0.1%. When setting up RAL in a microplate, the sensitivity was 1:1600, in the absence of cross-reactions with cholera and salmonella sera and up to ¼ titer of diagnosticum with tularemia, which corresponds to the regulatory documentation for these drugs and can be used in the work.

Example 2. Development of a method for obtaining a brucellosis latex diagnosticum for the agglutination reaction with B. abortus 19 BA brucella antigen in a ratio of 1:1.

Received diagnosticum analogously to example 1, with only one difference - latex was immobilized with brucellosis antigen in a ratio of 1:1. The sensitivity was 1:1600.

Thus, it is more expedient to use the preparation of the diagnosticum according to example 2, excluding unnecessary overspending of the antigen.

Used Books

1. G&T Polymer Technologies [Electronic resource]: URL http://gtpt.ru (date of access: 05/24/2021).

2. A method of manufacturing R-brucella erythrocyte antigen for the reaction of indirect hemagglutination (RIGA): US Pat. 2491553 Ros. Federation. No. 2011140108/15 / Karlova M.Yu., Degtyarenko L.V.; dec. 03.10.2011; publ. 27.08.2013, Bull. No. 24. 7 s.

3. Method for the differential diagnosis of brucellosis: Pat. 2490647 Ros. Federation. No. 2009144376/10 / Yudin V.I., Kozlov V.E., Bezgin V.M., Sklyarov O.D.; dec. 11/30/2009; publ. 20.08.2013, Bull. No. 23. 9 p.

4. Immunodiagnosticum based on polystyrene latex: Pat. 2231366 Ros. Federation. No. 2002113997/14 / Leonova V.B., Rosenfeld M.A.; dec. May 29, 2002; publ. 06/27/2004, Bull. No. 18. 8 s.

Claims (1)

A method for producing a brucellosis polystyrene latex diagnosticum, characterized in that a 2% solution of polystyrene latex is mixed in equal volumes and an antigen of a brucellosis microbe from a strain of Brucella (B.) abortus 19 BA at a concentration of 2 × 10 ⁹ m, destroyed on an ultrasonic disintegrator for 2 minutes. k./ml in 12% sodium chloride solution, stained with brilliant green - 0.004%, gentian violet - 0.002%; incubated at 37°C for 2 hours and 12-16 hours in a refrigerator at 4°C; washed twice from unbound antigen with 0.1 M phosphate-buffered saline pH 7.2-7.4 by centrifugation at 5000 rpm for 10 min; the precipitate is resuspended in 0.1% gelatin solution to 0.2% concentration with formalin to 0.1%.