The way to obtain serum for the identification of the causative agent of anthrax |
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IPC classes for russian patent The way to obtain serum for the identification of the causative agent of anthrax (RU 2191603):
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(57) Abstract: The invention relates to medical Microbiology and the receipt of anthrax serum, allowing to distinguish Bacillus anthracis from closely related microorganisms. Cell-free culture filtrate of B. anthracis STI concentrate on ultrafilter HLP-10 and PM-10 150 times, until the protein concentration in the filtrate 8-10 mg/ml, then the selection of antigenic components carry gelchromotography on ultrathin sephacryl S-300, balanced 0.3 M NaCl solution with 0.025 M Tris-HCL buffer pH of 8.4, check out fractions at = 280 nm, to obtain a highly specific serum using antigen from M. M. more than 150 kDa, which corresponds to the first peak of the output fractions. Obtained according to the method of the serum to the antigen reacts in the reaction, immunodiffusion in gel only with antigens of different variants of the causative agent of anthrax, which allows to identify the causative agent of anthrax from closely related on the antigenic properties of the bacilli. 2 Il. The invention relates to medical Microbiology and comes to obtain serum, which allows to distinguish Bacillus anthracis from closely related microorganisms. Known antigenic with yet obtaining species-specific drugs for the diagnosis of anthrax. Currently suitable for identification of diagnostic drugs get in most cases by repeated adsorption anthrax serum closely related microorganisms (copyright certificate (19) SU (II) 1637325 A1. R. N. Nizamov, D. W. Akhmerov, R. H. Shiganova and I. N. Sadykov. A method of obtaining a fluorescent monoclonal antibodies for the diagnosis of anthrax; Afanas'ev, E. N. Scientific-methodical aspects of rapid diagnosis of zoonotic pathogens of especially dangerous infections. Doctoral dissertation, Stavropol, 2000), which adds cost and complexity to the development of diagnostic products. The closest analogue of the proposed method is the "Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens" A. P. Phillips, I. W. Ezzel; I. of Applied Bacteriology, 1989, 66, 419-432. To obtain serum, these authors used easily extracted from cells of B. anthracis protein antigens. However, serum against these antigens was not possible to fully differentiate the strains of anthrax from closely related microorganisms, even with the use of monoclonal antibodies. The disadvantage of the method of allocation of these antigens is the use of detergents, heat therapy drug that Oslo is retene is to obtain serum using native anthrax antigen, allowing you to differentiate anthrax from closely related microorganisms. This objective is achieved in that the antigen are gelchromotography on ultrathin sephacryl S-300 (Upsala, Sweden) culture filtrate of B. anthracis STI-I, which is grown in a synthetic nutrient medium at 37oWith settlerowned. Cells of B. anthracis is separated by filtration, the components are sterile culture filtrate concentrate on ultrafilter L-10 and PM-10 (Amicon company, Sweden) 150 times, until the protein concentration of 8-10 mg/ml To obtain serum of rabbits subjected to immunization with antigen from M. M. more than 150 kDa (1 peak), which eluted in the free volume with registration at =280 nm. Animals intradermally along the spine and intramuscularly groin administered increasing doses of antigen from 250 μg to 750 μg with aluminum hydroxide. The activity of serum appreciate in response immunodiffusion in gel culture filtrate (dilution 1: 4). Animals bleed when the titer of the serum of 1:8-1:16. The specificity of the test sera in the reaction, immunodiffusion in gel grown for 18 hours in a nutrient agar agents of anthrax and closely stipitata, formed between the grown culture and well with whey. Examples of specific performance, confirming the implementation of the proposed method. Example 1. Obtaining cell-free culture filtrate. B. anthracis STI-I was grown in 500 ml flasks with 200 ml of liquid nutrient medium for 21 hours, with settlerowned 72 rpm Sterilized culture medium after growing it B. anthracis by filtration through a filter, ETC-045 (firm "Amicon"). Checked for sterility by inoculation in liquid and on solid medium, then concentrated on the installation of the DS-2 with fiber filtrate HLP-10 and PM-10 150 times. The protein content in the concentrated culture filtrate was usually 6-10 mg/ml protein. Example 2. Selection gelchromotography antigenic components of B. anthracis STI. Fractionation of the components of culture filtrate Century anthrais STI was carried out in the column h cm ultrafine sephacryl S-300, which was balanced by 0.3 M solution Nl with 0.025 M Tris-Hcl buffer pH of 8.4, while the rate of elution of 25 ml/h Output fractions was detected at 280 nm with a spectrophotometer "Uvicord SII (USING, Sweden). The output of the fractions was recorded in four separate peaks (Fig.1)Example 3. Obtaining serum antigens to the individual fractions. Rabbits breed "Chinchilla" weighing 2.5-3 kg were immunized increasing doses of fractions from 250 to 750 μg of protein per animal, which was introduced with hydrocity aluminium (Alu Gel firm "Serwa") after 2 weeks, intradermally along the spine and intramuscularly in both inguinal areas. The activity of the sera was tested in the reaction, immunodiffusion in gel culture filtrate. Animals were bled at titer sera 1:8-1:16. Example 4. Determination of the specificity of the sera obtained to individual antigens Century anthracis STI. Strains Century anthracis and closely related microorganisms were seeded lawns on nutrient agar. After 18-hour incubation at 37oWith against lawns grown cultures in agar punched holes (6 mm diameter), which was made of the test serum, the Cup was left at room temperature. After 18-24 hours between grown cultures and wells with serum was formed zones of precipitates. After the formation of precipitates culture obezzarajivate pairs of formalin, washed, cups photographed (Fig. 2). In Fig. 2 (a, b, C) in the center - serum, respectively, the fractions 1, 2 and common+cap-)2 - B. anthracis 81/1 (tox+cap-) 3 - B. anthracis 81/1 (tox-cap-) 4 - B. anthracis 81/1 (tox+cap+) 5 - B. anthracis 619/42 (t+SAR+) 6 - B. anthracis 591/2 (tox+cap+) 7 - B. anthracis Davies (tox-cap+) 8 - B. cereus NP 569 9 - B. cereus was ATSS 6464 10 - B. B. megaterium 216 11 - B. thuringiensis thuringiensis V. "Pasteur" 12 - B. thuringiensis V. tburingiensis "Galleria" 13 - B. subtilis 407 14 - B. subtilis BD 430 It is evident from Fig. 2 shows that serum to the antigen fraction 1 anthrax culture filtrate forms a zone of precipitate only with antigens Century anthracis as containing plasmid virulence (px01, px02), and antigens besplatnih anthrax strains. Unlike serum fraction 1 serum fraction 2 (and fractions 3 and 4 - data not shown) and commercial anthrax globulin form a zone of precipitates as antigens anthrax strains and antigens closely related microorganisms. Thus, whey to high molecular weight antigenic complex of B. anthracis in contrast to sera to other extracellular antigens of B. anthracis and commercial anthrax globulin is highly specific and can be S="ptx2"> The way to obtain serum for the identification of the causative agent of anthrax, characterized in that the animals are subjected to immunization increasing doses of antigen from M. M. more than 150 kDa, contained in the first peak obtained by gelchromotography on ultrathin sephacryl S-300, balanced 0.3 M NaCl solution with 0.025 M Tris-HCL buffer pH of 8.4, check out fractions at = 280 nm at the division of cell-free culture filtrate of B. anthracis STI focused on ultrafilter HLP 10 and PM 10 to protein content of 8-10 mg/ml
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