RU2798124C9 - 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.

1 cl, 2 ex

Description

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

Polymer microspheres (latex particles) with molecules of antibodies or antigens adsorbed on them react and agglutinate with the corresponding antigens of microorganisms or antibodies. Polymer microspheres are a convenient and flexible system for the development of diagnostic drugs used in the latex agglutination reaction (LAR). When choosing polymer microspheres, the diameter and size distribution of particles, composition, chemical properties of the surface (reactive groups, functionality, charge) and other more specific properties, such as the presence of chromophoric fillers (dyes), metal nanoparticles and their oxides are usually taken into account. For latex agglutination, microspheres (colored, uncolored) with a size of 0.2-1.0 microns, activated (for covalent cross-linking) and non-activated (for physical sorption) are most often used. The composition of polymer microspheres is also an important parameter. Typically, polymer microspheres consist of polyacrolein, polystyrene, and polymethyl methacrylate. These materials have different physical and optical properties, which may show advantages or impose some limitations on their use [1].

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

There is a known method for obtaining R-brucellosis erythrocyte diagnosticum for RIGA [2], which consists in the preparation of formalinized sheep erythrocytes, followed by their sensitization with sensitin obtained by growing the bacterial mass 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, which are difficult to standardize, whose parameters depend on factors such as gender, age of the animal, and time of blood collection. In addition, to obtain high-quality erythrocyte diagnostics, formalinized erythrocytes must be kept for a year for stabilization.

There is a known method for producing enzyme-linked immunosorbent 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 performing an analysis, the possibility of false positive results, and the need to use chemically clean glassware. In addition, a prerequisite is the presence of a photometer in the laboratory to record the results.

The closest to the claimed method is a method for producing an immunodiagnosticum based on polystyrene latex [4]. To obtain an immunodiagnosticum in accordance with the present invention, an equal volume of α-casein solution (10 mg/ml) in a 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 the latex suspension with the α-casein solution is thoroughly shaken and left at room temperature for 30 minutes, after which it is placed in the refrigerator (4°C) for 24 hours. The described general approach makes it possible to obtain a wide range of diagnostics, differing only in the specific protein immobilized on the surface 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 diagnostics obtained by the described method are not sensitive enough: the maximum dilution of serum that allows the detection of this drug is 1:320.

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

The technical result of the invention is that the polymer suspension is centrifuged, adjusted with a 0.9% sodium chloride solution to a concentration of 2% based on the dry residue of the polymer and mixed in equal volumes with the brucellosis microbe antigen from the Brucella (B.) abortus 19 strain destroyed on an ultrasonic disintegrator VA at a concentration of 2×10 ⁹ mc/ml in a 12% sodium chloride solution, colored with brilliant green (0.004%) and gentian violet (0.002%); incubate at a temperature of 37°C for 2 hours and 12-16 hours in a refrigerator at a temperature of 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 minutes; the sediment is resuspended in a 0.1% gelatin solution to 0.2% concentration and stabilized with formaldehyde to 0.1%.

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

The sensitivity and specificity of the developed experimental series of the drug were monitored with homologous and heterologous sera. RAL was placed in U-shaped (round-bottomed) microplates.

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

To control specificity, the following sera were used: diagnostic cholera O1 adsorbed dry sera for RA (FKUN “Russian Research Antiplague Institute “Microbe”); diagnostic salmonella adsorbed O-polyvalent for RA (JSC "Ekolab"); diagnostic dry tularemia for RA (FKUZ Irkutsk Research Antiplague Institute of Rospotrebnadzor).

The RAL setup (micromethod) was carried out as follows: 25 μl of Tween-80 dilution liquid at a dilution of 1:25 thousand was added to all wells (8 rows) of the plate. Next, 25 μl of homologous and heterologous sera at a dilution of 1 were added to the first wells of the rows: 100, titrated in steps of 2, obtaining dilutions in wells from 1:200 to 1:12800, the eighth rows are negative controls (no serum was added to them). Then 25 μl of the developed brucellosis latex diagnosticum was dropped into all wells. The sensitivity of RAL was 1:800-1:1600, which corresponds to the sensitivity of the brucellosis erythrocyte diagnosticum. When monitoring specificity, it was found that there was no cross-reaction with salmonella and cholera sera, and with tularemia, a cross-reaction of up to ¼ titer of the 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 set of claimed features.

Example 1. Development of a method for producing brucellosis latex diagnosticum for an agglutination reaction with the brucellosis antigen B. abortus 19 BA 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 microns. A suspension of B. abortus 19 BA was used as a ligand for immobilization at a concentration of 2 × 10 ⁹ m.c./ml in a 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 minutes. 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 volume ratio of 1:2. 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. Next, it was centrifuged at 5000 rpm for 10 minutes, washed twice from unbound antigen with 0.1 M PBS, pH 7.2-7.4, and the last sediment was resuspended in a 0.1% gelatin solution to a 0.2% concentration. For stabilization and storage for a long time, formaldehyde was added to a concentration of 0.1%. When performing 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 the diagnosticum with tularemia, which corresponds to the regulatory documentation for these drugs and can be used in work.

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

The diagnosticum was prepared similarly to example 1, with only one difference - the latex was immobilized with brucellosis antigen in a 1:1 ratio. In this case, the sensitivity was 1:1600.

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

Used Books

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

2. Method of manufacturing R-brucellosis erythrocyte antigen for the indirect hemagglutination reaction (RIHA): Pat. 2491553 Ross. Federation. No. 2011140108/15 / Karlova M.Yu., Degtyarenko L.V.; application 03.10.2011; publ. 08/27/2013, Bulletin. No. 24. 7 p.

3. Method for differential diagnosis of brucellosis: Pat. 2490647 Ross. Federation. No. 2009144376/10 / Yudin V.I., Kozlov V.E., Bezgin V.M., Sklyarov O.D.; application November 30, 2009; publ. 08/20/2013, Bulletin. No. 23. 9 p.

4. Immunodiagnosticum based on polystyrene latex: patent. 2231366 Ross. Federation. No. 2002113997/14 / Leonova V.B., Rosenfeld M.A.; application 05/29/2002; publ. 06/27/2004, Bulletin. No. 18. 8 p.

Claims (1)

A method for producing brucellosis polystyrene latex diagnosticum, characterized in that equal volumes of a 2% solution of polystyrene latex are mixed and the antigen of the brucellosis microbe from the strain Brucella (B.) abortus 19 BA, destroyed on an ultrasonic disintegrator for 2 minutes, is destroyed in a concentration of 2 × 10 ⁹ m. k./ml in a 12% sodium chloride solution, colored with brilliant green - 0.004%, gentian violet - 0.002%; incubate at a temperature of 37°C for 2 hours and 12-16 hours in a refrigerator at a temperature of 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 minutes; the sediment is resuspended in a 0.1% gelatin solution to 0.2% concentration with formalin to 0.1%.