RU2715561C1 - Method for controlling the specificity of lactoglobulin against opportunistic bacteria and salmonella by antibody content by enzyme immunoassay - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used to control specificity of lactoglobulin against opportunistic bacteria and salmonella by antibody content by enzyme immunoassay. For this purpose specificity of lactoglobulin against S. thyphimurium, S. enteritidis, S. dublin, P. mirabilis, P. vulgaris, K. pneumoniae, P. aeruginosa. Further, using a multi-scanning device, a wavelength of measurement of 450 nm, in lactoglobulin simultaneously required to assess its specificity content of antibodies to Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella thyphimurium, Salmonilla enteritidis, Salmonella dublin by optical density indicators, which must be within chromaticity of at least 0.500–0.600 standard units with such control parameters as conjugate monitoring and positive control of antibodies equal to at least 0.500–0.600 standard units.

EFFECT: invention enables selecting a more objective method of controlling lactoglobulin specificity against opportunistic bacteria and salmonellae.

1 cl, 1 dwg, 3 ex

Description

The invention relates to the field of medical biotechnology.

An analogue of the proposed method can be considered "SET FOR QUANTITATIVE DETERMINATION OF AVERMECTINS BY THE METHOD OF ONE-STAGE COMPETITIVE IMMUNO-ENZYME ANALYSIS" described in RF patent No. 2416094. The reagent kit for the quantitative determination of avermectins contains avermectin conjugate with bovine serum albumin immobilized on a polystyrene plate, a peroxidase conjugate of highly specific murine monoclonal antibodies to ivermectin, calibration samples of avermectin (with concentrations from 0 to 100 ng / ml). The monoclonal antibodies (MKAT) included in the kit are produced by a strain of hybrid cultured Mus cells. Musculus L. deposited in the Collection of transplantable somatic vertebrate cells of the Research Institute of Virology named after DI. Ivanovsky under No. 05/09. Of the non-specific components, the kit contains a buffer for diluting the MKAT conjugate with horseradish peroxidase, a buffer for diluting the analyzed samples and washing the plates, reagents for detecting peroxidase activity, a substrate solution, hydrogen peroxide and tetramethylbenzidine, a stop solution (1 M sulfuric acid). The kit according to the invention is intended to detect residual amounts of avermectins in tissues and biological fluids to control chemical contamination of animal products. (see the patent of Russia No. 2416094, class IPC G01N 33/02; publ. 04/10/2011.)

This method has several significant disadvantages. The test substance is an antigen and is fixed on the tablet. While with our method, well-known antigens are fixed on the tablet - bacteria, the antibodies to which are planned to be detected in lactoglobulin, which allows you to control in the intermediate stages of production and in the finished product. In both cases, a protein A conjugate with horseradish peroxidase was used. However, in the analogue, the conjugate is labeled with the antigen by which the wells of the tablet are sensitized, in our method for assessing the specificity of lactoglobulin, the conjugate is used at the stage of accounting for the reaction of antigen and antibody. Thus, the analogue used the method of one-stage competitive enzyme-linked immunosorbent assay. While to determine the specificity of lactoglobulin, we used the direct non-competitive 2-stage method.

Lactoglobulin against opportunistic bacteria and salmonella (LH-UPBS) - a unique domestic immunobiological preparation - is a purified fraction of bovine immune colostrum globulins and contains antibodies to S. thyphimurium, S. enteritidis, S. dublin, P. mirabilis, P. vulgaris , K. pneumoniae, P. aeraginosa. LH-UPBS is intended for the treatment of acute intestinal infections (ACI) and the correction of intestinal microbiota disorders after antibiotic therapy. The manufacturer of LG-UPBS is FBUN Rostov Research Institute of Microbiology and Parasitology, (see appendix). In 2010, the production of LG was temporarily suspended due to physical and moral aging of production. The resumption of LH production is relevant in connection with the growth of antibiotic-resistant UPB as an alternative non-addictive drug. One of the important points in the modernization of obtaining LH-UPBS is the control of the specificity of the drug. In accordance with the production regulations, the presence of antibodies in LH-UPBS is monitored at several stages of production: in the feedstock (immune colostrum); in lactoserum (intermediate) and in the finished product.

The prototype of the proposed method is a reaction of passive hemagglutination of agglutination (RPHA) with the original erythrocyte diagnostics obtained at one time in the production of lactoglobulins based on lipopolysaccharides S. thyphimurium, S. enteritidis, S. dublin, P. mirabilis, P. vulgaris, K. pneumoniae, P. aeruginosa isolated by Westphal water-phenolic extraction, adsorbed on sheep erythrocytes. Recently, RPGA has produced false positive results due to the instability of the controls - mainly related to sheep erythrocytes. In addition, the reaction was evaluated subjectively using a 4-cross system with visual recognition of the sediment in the holes in the form of an “umbrella” (loose sediment at the bottom of the hole) and “buttons” (the sediment in the hole is concentrated in the center), (see Appendix)

In this regard, the aim of the work is to select an objective way to control the specificity of lactoglobulin against opportunistic bacteria and salmonella based on the ELISA method, which gives results comparable to the previously used RPGA.

The advantage of the invention is that the ELISA method is objective (i.e., the determination is carried out using a multiscan device, and with RPHA it is subjective by the presence of a characteristic precipitate). According to the conditions of production, we can change the method of specificity control to a new one, when it is comparable with the previous one.

SOP (standard production sample) is lactoglobulin against opportunistic bacteria and bovine salmonella with ELISA activity of not more than 62.5 μg of the drug in a volume of 10 μl of PBS.

The proposed method is as follows.

1. Preparatory phase

1) Preparation of lactoglobulin samples.

500 mg of dry LH-UPBS was dissolved in 10 ml of purified water, and an initial concentration of 50 mg / ml was obtained. Two serial dilutions in phosphate-buffered saline (PBS) were prepared in test tubes, starting from 1: 2 to 1: 128, receiving, respectively, from 500 mg to 3.9 mg in 1 ml.

2) Tablet sensitization.

Bacterial cultures (Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, Salmonitta enteritidis, Salmonella dublin) were plated on beveled microbiological agar. Cultivated at 37 degrees from 18-24 hours. Then washed away the culture of the FSBR. The microbial suspension was standardized to 1 billion mt / ml. The resulting suspension was boiled in a water bath for 15 minutes. It was cooled to 20 degrees C. A solution of 1: 10000 merthiolate was added. The resulting antigens are stored in a refrigerator at 8 ± 2 degrees. C for 3 months.

3) Preparation of controls.

For ELISA controls, a multicomponent antigen was prepared. Received antigens using a dispenser, 1 ml of each item is taken and transferred to a test tube. Thoroughly mixed (The mixture is stored for 1 month in the refrigerator in a sealed state at 8 ± 2 degrees C).

4) Preparation of ELISA components.

Preparation of the conjugate is carried out in accordance with the instructions. Staphylococcus aureus A protein conjugated with horseradish peroxidase is used as the conjugate. The solvent used is PBS. The conjugate is prepared ex temporae. The substrate mixture consists of tetramethylbenzidine (TMB) and hydrogen peroxide. The substrate solution is prepared ex temporae.

The invention is illustrated in the figure, which shows a diagram of the introduction of components for performing ELISA. (see Fig. 1)

2. Statement of ELISA (Fig. In Fig. 1).

1) Sensitization of polystyrene tablet

Sensitization was carried out using a dispenser of 100 μl of antigens in each well with columns from A to N. Each number from 1 to 7 corresponds to different antigens of UPB and Salmonella. 100 μl of the multicomponent antigen is added to wells 12-G and 12-H (antibody control is positive and negative). After the introduction of antigens, the tablet is covered with a lid and left at 8 ± 2 degrees. With at 18-24 hours.

2) The introduction of lactoglobulin.

Lactoglobulin prepared in dilutions was added to each well of the plate 100 μl. Distribute accordingly: A - 500 mg; B - 250 mg; C - 125 mg; D - 62.5 mg; E - 31.25 mg; F - 15.6 mg; G - 7.8 mg; H - 3.9 mg. The concentration of the drug decreases from top to bottom (from A to H). After the introduction of lactoglobulin, the plate is incubated at 37 degrees. C for an hour.

3) Making controls

Positive control (antibodies - SOP) and negative control of antibodies - non-immune serum is added in quantities of 100 μl to the wells of G-12 and H-12 plate). Incubation of controls takes place in the same mode as lactoglobulin.

4) Introduction of the conjugate.

The conjugate was added at a rate of 100 μl to each working well and to the F-12 well (conjugate control). The tablet after this manipulation is incubated at 37 degrees. C for an hour. The substrate mixture is added to all working wells of 100 μl. The tablet can withstand 20 minutes at 20 degrees. C in the dark place. Then add 20 μl of stop reagent.

5) Accounting for the reaction.

Reading the results was carried out on a multiscan at a wavelength of 450 nm. The resulting optical density in the negative control should be two times or more lower than the positive control. Reference values of the level of optical density should be in the range of not less than 0.500-0.600. Control of the conjugate should be positive (give a color of at least 0.500-0.600).

When selecting the optimal amounts of antigens and lactoglobulin, 3 options were used 200 μl, 100 μl and 50 μl, proposed in examples 1, 2, 3.

Example 1. Determination of the presence of antibodies in lactoglobulin during sensitization of the tablet with antigens in an amount of 200 μl in one well.

The method was carried out according to the scheme proposed in Fig. 1. Sensitization of the tablet with antigens Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, Salmonilla was carried out with a microdoser in a dose of 200 μl per 1 well. Next, a prototype lactoglobulin was added in an amount of 200 μl per 1 well. The conjugate and substrate mixture were also added according to the scheme in an amount of 200 μl per 1 well. The ELISA of the reaction was evaluated by a multiscan at a wavelength of 450 nm. Indicators of optical density in accordance with the amount of 62.5 mg / ml protein were in the range of 522-600 units. In this case, the control conjugate gave indicators of 650 units. optical density, positive antibody control 600 units. optical density.

Example 2. Determination of the presence of antibodies in lactoglobulin during sensitization of the tablet with antigens in an amount of 50 μl in one well.

The method was carried out according to the scheme proposed in FIG. 1. The tablet was sensitized with the antigens Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, and Salmonilla by a microdoser at a dose of 50 μl per 1 well. Next, a prototype lactoglobulin was added in an amount of 50 μl to 1 well. The conjugate and substrate mixture were also added according to the scheme in an amount of 50 μl per 1 well. The ELISA of the reaction was evaluated by a multiscan at a wavelength of 450 nm. Indicators of optical density in accordance with the amount of 62.5 mg / ml protein were in the range of 450-520 units. In this case, the control conjugate gave indicators of 400 units. optical density, positive antibody control 300 units. optical density.

Example 3. Determination of the presence of antibodies in lactoglobulin during sensitization of the tablet with antigens in an amount of 100 μl in one well.

The method was carried out according to the scheme proposed in FIG. 1. The tablet was sensitized with the antigens Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, Salmonilla with a microdoser at a dose of 100 μl per 1 well. Next, a prototype lactoglobulin was added in an amount of 100 μl per 1 well. The conjugate and substrate mixture were also added according to the scheme in an amount of 100 μl per 1 well. The ELISA of the reaction was evaluated by a multiscan at a wavelength of 450 nm. Indicators of optical density in accordance with the amount of 62.5 mg / ml protein were in the range of 500-600 units. In this case, the control conjugate gave indicators of 500 units. optical density, positive antibody control 600 units. optical density.

A comparison of the optical density obtained by evaluating the ELISA settings with different amounts of introduced antigens and drug samples revealed similar values at 200 μl and 100 μl of substances in the wells. The optimal amount of introduced substances is recognized as 100 μl of substances per well (example 3).

Claims (1)

A method for controlling the specificity of lactoglobulin against opportunistic bacteria and salmonella by the content of antibodies by enzyme immunoassay, including determining the specificity of lactoglobulin against S. thyphimurium, S. enteritidis, S. dublin, P. mirabilis, P. vulgaris, K. pneumoniae, P. aeruginosa and characterized in that, using a multiscanning device, measuring wavelength 450 nm, in lactoglobulin, the content of antibodies to Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella thyphimurium, Salmonilla enteritidis, Salm is simultaneously determined to assess its specificity onella dublin in terms of the level of optical density, which should be within the chromaticity range of at least 0.500-0.600 conventional units with such control indicators as conjugate control and positive antibody control equal to at least 0.500-0.600 conventional units.

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