The method of obtaining diagnostic agglutinins vaccine against brucella in an l-shape
The invention relates to the field of veterinary medicine and can be used for diagnosis of the causative agent of brucellosis in an L-shape. The essence of the invention lies in the fact that the serum obtained from the blood of rabbits after conducting dvuhtokovoe hyperimmunization antigen from subculture C. abortus And 206 in an L-shape. The technical result is to obtain highly active and highly specific diagnostic serum against Brucella in an L-shape. 1 PL.
The invention relates to the field of medicine and veterinary medicine, namely to Microbiology and immunology, and can be used for obtaining immune serum used for diagnosis of the causative agent of brucellosis in an L-shape.
It is known that the activity and specificity of immune serum depends on the quality of diagnostic products. Brucellosis polyvalent serum is not agglutinate brutally in a stable L-form and therefore cannot be used for their diagnosis. Therefore, obtaining high-affinity and high-level diagnostic agglutinins vaccine against Brucella in an L-shape is an important task.
A method of obtaining syware is (105M. K. - 3·109M. K.) only after 60-90 days marked mild antibody production (1:10 in RA, 1:40 in RAC and RDSC) [ishchanova R. J., Sementsova C. M., E.E. C. M., Khasanov N. X. // Veterinary medicine, 1986, No. 9, - n-30-32].
It is known that L-forms of Brucella have less pronounced compared to the original bacterial form agglutinability properties, they interacted in agglutination reactions (RA) own L-anticorodal in a dilution of 1:640, while the original culture was agglutinability with S-anticorodal up to a titer of 1:1280. However, anticavity to L-forms were agglutinative and initial bacterial forms in a dilution of 1:320 [Bazikov I. A. fabrication and characterization of L-forms of Brucella and their revertants. Abstract. Diss.... Kida. the honey. of Sciences, Saratov, 1990, - n-16]. The above messages indicate receipt of the sera against Brucella in an L-shape, but with low activity of the antibodies (AT) (titers at 1:10 and 1:640) and specificity, as they react with Brucella in the S-form in the credits AT (1:320), lower than only one breeding than with Brucella in L-form.
Closest to the present invention is a method of obtaining a vaccine against Brucella in an L-shape, including use as an antigen (AG) immunization incdpass is performed using the antigen, prepared from Brucella in an L-shape, inactivated acetone (acetone powders-up). If hyperimmunization rabbits up of Brucella in the L-form are obtained serum, in which the titles at RA was with homologous AG 1:800-1:1600, with a standard Brucella diagnosticum 1:400-1:800 [Dranovsky E. A., Tolmachev, T. A. Rostovtsev N. A. // Ukr. microbiol., 1981. No. 7. - C. 33]. However, this method does not allow to obtain serum against Brucella in an L-shape with high activity and specificity. Shown in prototype serum against Brucella in an L-shape has a low reactivity with Brucella in L-form (titres AT 1:800-1:1600) and specificity, as it reacts with the standard Brucella diagnosticum (S-form) in the credits AT less than only one dilution (1:400-1:800), than with L-antigen.
The aim of the invention is to obtain highly active and highly specific diagnostic agglutinins vaccine against Brucella in L-form.
This objective is achieved in that exercise dvuhlitrovuyu hyperimmunization rabbits AG from subculture C. abortus And 206 in an L-shape on the original scheme, to keep the blood from which it is prepared serum.
Comparative analysis of the prototype showed that the proposed technical R is of diagnostic serum used dvuhlitrovuyu scheme hyperimmunization inaktivirovannye AG, made from subculture C. abortus And-206 to L-form.
In the first cycle before immunization of rabbits in the pads of the hind paw injected with 0.5 to 0.6 ml complete adjuvant's adjuvant (PAF), then after 6-7 days injected AG combined in the popliteal lymph nodes and intramuscularly (suspension of AG with PAF). After another 3-4 days repeat injections, but in the front paws and intramuscularly. On 29-31 days from the beginning of immunization, before the second cycle, prepare the complex antigen-antibody (AG-AB). After 30-32 days to have a second cycle, which rabbits subjected to immunization in combination: a complex of antigen-antibody injected, and the suspension of AG with PAF - intramuscularly. After 3-4 days of injections of a complex of AG-AB and AG repeat. After 7-9 days after the last immunization, the animals will Krasouskaya and receive diagnostic agglutinating serum against Brucella in L-form.
Thus, the proposed solution meets the criteria of the invention of “novelty.”
The analysis of patent and scientific literature showed that the proposed method differs not only from the prototype, but also on other technical solutions in this and related fields. So, the authors have not found a way poluchenno, the proposed modes allow you to achieve this goal is to obtain a highly active and highly specific diagnostic agglutinating serum against Brucella in an L-shape. This method does not require special equipment and reagents and can be used in the production of this serum in the laboratory.
The proposed diagnostic agglutinating serum can be used in medicine and veterinary medicine for the diagnosis of modified forms of Brucella, in particular L-forms. Thus, this method meets the criteria of “inventive step” and “industrial applicability”.
The method is as follows:
Take inactivated subculture C. abortus And 206 in an L-shape and are hyperimmunization rabbits under the scheme, including two cycles.
First cycle: the rabbits in the pads of the hind paw injected with 0.5 to 0.6 ml of PAF. After 6-7 days the animals are subjected to immunization in the popliteal lymph nodes of the rear paws of 0.3-0.5 ml of a suspension of AG in buffered saline solution pH 7,2-7,4 (SFR) at a dose of 4·108-5·108microbial cells (M. K.) and intramuscularly at a dose of 4·108-5·108M. K. in a volume of 1.0 ml containing 0.5 ml of suspension of AG and 0.5 ml·109M. K. and intramuscularly 109-2·109M. K. in a volume of 1.0 ml (suspension of AG-PAF 0.5 ml).
On 29-30 days from the beginning of immunization prepare the immune complex of antigen-antibody, for what animals are immune serum, which is poured in two bottles of 1.0 ml In each tube add 1 ml of suspension of AG in the corresponding immunizing dose.
The second cycle of immunization is carried out through 30-32 days. The complex of antigen-antibody administered intravenously at the rate 2.5·109-3·109M. K., and intramuscular injection of a suspension of AG based on 2·109-3·109M. K. in the amount of 0.25-0.5 ml 0.5 ml PAF. After 3-4 days, make repeated intravenous injections of the complex AG-AB 3·109-4·109M. K. and intramuscular injection of a suspension of AG - 2·109-3·109M. K. in 0.25-0.5 ml SPR with 0.5 ml of PAF.
After 7-9 days after the last immunization, the animals spend bloodletting and get the serum in a standard way.
Take an inactivated antigen of subcultures C. abortus And 206 in an L-shape and are hyperimmunization according to the following scheme.
First cycle: the rabbits in the pads of the hind paw injected with 0.6 ml of PAF. After 7 days the animals are subjected to immunization in the popliteal lymph nodes rear l is Adam 0.5 ml of a suspension of AG and 0.5 ml of PAF. After 4 days of injection of AG carried out in pads front paws in a dose of 2·109M. K. in a volume of 0.6 ml SPR and intramuscularly 2·109M. K. in a volume of 1.0 ml (suspension of AG-PAF 0.5 ml).
On the 31st day from the beginning of the immunization prepare the immune complex of antigen-antibody, for what animals are immune serum, which is poured in two bottles of 1.0 ml In the first test tube add 1.0 ml of a suspension of AG at a dose of 3·109M. K., second 1.0 ml suspension of AG at a dose of 4·109M. K.
The second cycle immunization spend 32 days. The complex of antigen-antibody administered intravenously at the rate of 3·109M. K., and intramuscularly injected with 0.5 ml of a suspension of AG at a dose of 3·109M. K. with 0.5 ml of PAF. After 4 days, make repeated intravenous injections of the complex of antigen-antibody containing 4·109M. K., and intramuscularly injected with 0.5 ml of a suspension of AG (3·109M. K.) with 0.5 ml of PAF.
After 9 days after the last immunization, the animals will Krasouskaya and the standard method of preparing diagnostic agglutinating serum against Brucella in an L-shape. The resulting serum is a specific title AT 1:1600-1:3200.
Take an inactivated antigen of subcultures C. abortus And 206 in an L-shape and are hyperimmunization of sleh subjected to immunization in the popliteal lymph nodes of the rear paws of 0.3 ml of a suspension of AG at a dose of 4·108microbial cells (M. K.) and intramuscularly at a dose of 4·108M. K. in a volume of 1.0 ml containing 0.5 ml of antigen suspension and 0.5 ml of PAF. After 3 days, the injection of suspensions of AG carried out in pads front paws in a volume of 0.5 ml at a dose of 109M. K. and intramuscularly 109M. K. in a volume of 1.0 ml (suspension of AG-PAF 0.5 ml).
On the 29th day from the beginning of the immunization prepare the immune complex of antigen-antibody, for what animals are immune serum, which is poured in two bottles of 1.0 ml In the first test tube add 1.0 ml of a suspension of AG in the dose of 2.5·109M. K., second 1.0 ml suspension of AG at a dose of 3·109M. K.
The second cycle immunization carried out in 30 days. The complex of antigen-antibody administered intravenously at the rate 2.5·109M. K., and intramuscularly suspension of AG 2·109M. K. 0.25 ml with 0.5 ml of PAF. After 3 days, make repeated intravenous injections of the complex of antigen-antibody at a dose of 3·109M. K. and intramuscular suspension of AG at a dose of 2·109M. K. in a volume of 0.25 ml with 0.5 ml of PAF.
Through 7 days after the last immunization, the animals will Krasouskaya and the standard method of preparing diagnostic agglutinating serum against Brucella in an L-shape. The resulting serum is a specific title AT 1:1600-1:3200.
In addition, the use of serum, obtained by the proposed method allows to detect soluble antigens of Brucella in the L-form in the reaction, immunodiffusion in gel formation of one or two bands of precipitation and does not form any with antigens of Brucella S-form. Brucellosis polyvalent serum in the reaction, immunodiffusion in gel with Brucella antigens in S-shape forms a 2-3 or more bands of precipitation.
The proposed method for the rabbit hyperimmune serum against Brucella in the L-form is more efficient because it involves the immunization of animals inaktivirovannye antigen that does not require special measures for the protection of operating personnel.
Compared with the prototype of this method is, first and foremost those that apply dvuhlitrovuyu hyperimmunization on the original scheme, allowing you to get more specific and more active Syv what about this, compared with the prototype, where the antibody titers with standard Brucella diagnosticum (S-form) was 1:400-1:800, RA proposed L-serum is not registered positive reactions with Brucella in the S - and R - forms, indicating the absence of the necessary adsorption for removal of antibodies against S - and R-antigens of Brucella. The high specificity indicates the absence of interaction diagnostic serum against Brucella in an L-shape with some microorganisms, with several common antigens.
The method of obtaining diagnostic agglutinins vaccine against Brucella in L-form, including immunization of animals with the antigen of subcultures C. abortus in an L-shape, bleeding the animals and obtaining serum, characterized in that hyperimmunization spend antigen obtained from subcultures C. abortus And 206, two cycles as follows: in the first cycle, the rabbits in the pads of the hind paw injected with 0.5 to 0.6 ml complete adjuvant's adjuvant, after 6-7 days enter suspension of antigen in combination: in the popliteal lymph nodes of the rear legs at a dose of 4-10 - 5-10 M. K., and intramuscularly 4·108- 5·108M. K. with complete adjuvant's adjuvant, after 3-4 days of injection powa then through 29-31 days from the beginning of the immunization, the animals spend bloodletting and get the immune serum, to obtain a complex of the antigen - antibody type antigen in a 1:1 ratio in immunizing doses of 2.5·109- 3·109M. K., and 3·109- 4·109M. K. through 30-32 days to have a second cycle of immunization, which includes the introduction of complex antigen - antibody intravenously at a dose of 2.5·109- 3·109M. K., and suspension of the antigen with complete adjuvant's adjuvant intramuscularly at a dose of 2·109- 3·109M. K. after 3-4 days of injections of a complex of antigen - antibody and the antigen suspension is repeated similarly in doses of 3·109- 4·109M. K., and 2·109- 3·109M. K. accordingly, to obtain the serum of the animals will Krasouskaya in 7-9 days.
FIELD: microbiology and immunology, in particular immunodiagnosis.
SUBSTANCE: atypical strain of melioidose Burkholderia pseudomallei-111-6-1 with altered phenotype defected with respect to synthesis of 8 antigen and acting as immunosuppressor is used as antigen for animal immunization. Immune serum is obtained after 2 immunization cycles of animal-producer with titer in gel immunodiffusion reaction not less than 1:128.
EFFECT: immune serum with increased specific activity.
2 tbl, 2 ex
FIELD: molecular biology, biotechnology, gene engineering, in particular diagnosis of atypical pneumonias.
SUBSTANCE: invention relates to DNA based on identified protein antigen epitope SARS-CoV. Particularly disclosed are nucleotide sequences of synthetic genes encoding recombinant protein fragments containing coronavirus protein antigen determinants SARS-CoV, represented in SEQ ID NO:1 - SEQ ID NO:8; as well as SARS-CoV virus protein fragments encoded by DNA sequences with amino acid sequences represented in SEQ ID NO:10 - SEQ ID NO:17. Said fragments are isolated by using preliminary obtained fusion protein by attachment to its N-terminal end GST-S-transferase protein with sequence represented in SEQ ID NO:9. Fusion proteins are useful in manufacturing of preparations for diagnosis of acute respiratory virus SARS-CoV.
EFFECT: new diagnostic agents for diagnosis of atypical pneumonias.
FIELD: medicine, radiation biology.
SUBSTANCE: invention proposes a method for preparing allergenic preparation used for diagnosis of body radiation injures. Method involves irradiation of potato tubers by the dose 350-400 Gr, the following extraction of quinoid radiotoxin by extraction with ethanol followed by removing extractant in rotary evaporator and additional extraction with ethyl acetate. The final prepared fraction is subjected for chromatography and separated in the system: (a) 2% acetic acid, and (b) mixture benzene - acetic acid - water in the ratio = 2:4:1, respectively. The prepared allergenic fraction is eluted with 2% acetic acid solution and standardized by dry matter as measured for 1 mg/cm3. Also, invention proposes a method for diagnosis of body radiation injures involving intracutaneous administration of specific allergen and detection of autosensitization symptom. Diagnosis of radiation diseases is proved by the allergy index. Proposed methods provide preparing the specific radiation allergen for detection of radiosensitization of body and to carry out diagnosis of radiation disease. Invention can be used in radiation biology.
EFFECT: improved preparing method, improved method for diagnosis.
1 tbl, 3 ex
FIELD: biotechnology, medicine, immunology.
SUBSTANCE: method involves preparing control samples by preparing solution of heterologous chimeric antibodies consisting of whole molecules or fragments of immunoglobulin isolated from immunized animal serum and associated with whole molecules or fragments of human immunoglobulin in phosphate-saline buffer, pH 6.0 followed by preparing different dilutions in indicated buffer, their control in IFA and selection of dilutions at optical density values 1.0, not less. Invention provides preparing control samples comprising specific antibodies that elicit high specificity and capacity for detection in combination with their infectious safety, standard indices and availability with respect to economy aspects.
EFFECT: improved preparing method.
FIELD: veterinary microbiology.
SUBSTANCE: claimed method includes providing of stable culture from B.abortus 19 strain in L-form followed by rabbit immunization. Culture is obtained in dense broth containing additionally 10-15 % of normal hoarse serum wherein broth is singly exposed to streptomycin action in dose of 2.5-5.0 U/ml of medium. Then slurry is prepared from obtained culture, inactivated at 85-90°C for 160 min and triply intravenously administrated to rabbits in increasing doses with 72 h intervals.
EFFECT: method for more exact estimation of brucelliasis epizootic situation on basis of typical, dissociated and deep-altered brucella forms.
6 tbl, 4 ex
FIELD: veterinary virology, in particular production of latex diagnosticum for diagnosis of horned cattle and poultry infections.
SUBSTANCE: claimed method includes centrifugation of polymeric suspension and adjusting of polymeric suspension with distilled water up to 0.5 % (calculated as polymer dry residue)/ Then equal volumes of viral antigen in infective titer of 6.5 lg TCD/50 ml (tissue cytopatic doses) and 0.5 %-1.0 % polymeric suspension are mixed and mixture is hold in thermostatic regulator at 36-38°C for 4-6 h. Further 0.07-0.15 % aqueous solution of human serum albumin is added into total suspension volume, mixture is incubated .at 4-8°C for 11-13 h; suspension is washed, and diagnosticum concentration is adjusted with phosphate buffered saline with pH 7.2-7.4 up to 0.1-0.3 %. Prepared diagnosticum is stored at 4°C not more than 1 year.
EFFECT: diagnosticum for before-the-fact detection of different infective diseases, prophylaxis and treatment.
7 ex, 7 tbl
FIELD: medical engineering.
SUBSTANCE: device has substrate having polymeric working layer on it, produced from copolymer based on methacrylic acid derivatives with biological macromolecules (probes) immobilized thereon. The substrate is manufactured from activated or not activated glass, metal or polymer material. The working layer has macroporous monolithic copolymer glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass with affine biological probes immobilized thereon. Probe-copolymer proportion is 2-10 mg/g of copolymer, for protein, 1-20 mg/g of copolymer for peptide and for oligonucleotide, nucleic acid - 0.5-3 mg/g of copolymer, pore radius of 0.4-1.5 mcm, it has thickness of 50-700 microns and is manufactured as continuous or discrete microcellular layer. The method for manufacturing biochip involves preparing substrate, producing working layer by monomer copolymerization on methacrylic acid derivatives base, immobilizing biological macromolecules - probes on forming copolymer, washing, drying the received biochip. Radical copolymerization of glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass is carried out for producing working layer with photo-or thermal initiation in poregenic solvent medium being applied. Proportion of the sum of monomer volumes to solvent volume being equal to 6:9, initiator concentration in reactionary medium being equal to 0.2-1.0% by weight, given reaction mixture is placed on substrate as continuous or discrete layer. Macroporous monolithic continuous or discrete microcellular layer is formed as a result of copolymerization on the substrate. Then, covalent immobilization of biological macromolecules is carried out in the layer pores or their direct synthesis on formed copolymer with its native or modified epoxy groups being used. Biological affine probe is produced. The probe is introduced into copolymer in quantity of 2-10 mg/g of copolymer for fiber, for peptide - 1-20 mg/g of copolymer and for oligonucleotide or nucleic acid - 0.5-3 mg/g of copolymer.
EFFECT: manufacturing reusable biochip with predetermined controllable and reproduced quality.
FIELD: medicine, veterinary.
SUBSTANCE: specific antibodies to virus of goose enteritis are determined by IEA where virus received following injection of epizootic strain "P-75" of enteritis virus, is used as an antigen, and macro porous glass with diameter not less than 700 is used for purification of virus-containing material, the goose Ig G specific immunoperoxidase conjugate is used as an anti-specific agent, and the IEA results are calculated by use of the formula: titer = antiIg[2.02(lg S/P)+3.51], where S is for optical density of tested serum; P is for optical density of the positive control.
EFFECT: method reduces the test time and economical costs.
2 tbl, 2 ex
FIELD: medicine; gastroenterology.
SUBSTANCE: cell lysate is received by double destruction of bacterial cells: first, treatment with 1% sodium desoxycholate solution at a rate of 0.5 ml solution to 0.2 ml suspension with concentration (5-7) × 1010 microbes/ml, with following stirring at magnetic agitator in thermostat at 40°С for 2 hours, second, by performing 5 per 45 sec cycles of ultrasound disintegration, after which, the obtained suspension is precipitated by centrifuging at 8000 rpm for 40 min, and the obtained cell lysate is used as a sensitin, which is immobilised at polymer carrier with the following lyophilisation. In sensitin, the protein concentration 1.5-2 mg/m with wide immunoglobulin spectrum is received. As sensitin carrier, the globular polymeric particles with diameter 1.5 mcm and containing 1.3 mmol/g aldehyde groups can be used, and the lysate-produced immobilisation of micro spheres is performed by use of 0.1 mol carbonate buffer with pH 9.2. Interlocking of free aldehyde groups is performed by adding 2.0 ml 0.5% gelatose on 0.9% sodium chloride to suspension with carrier and soluble antigen, and leave to stay at constant stirring at the agitator at 20°С fro 120 min.
EFFECT: method provides the quantitative determination of antibodies to HPylory and efficient application for controlling the eradicative therapy.
2 tbl, 2 ex, 4 cl
FIELD: medicine; veterinary science.
SUBSTANCE: produced plasma after it is separated from blood and refined from trypanosome deposition is processed with 20-25% polyethylene glycol solution taken in proportion equal to blood plasma volume. Then produced mixture is kept at room temperature for 12-15 min, recentrifuged at 6000 revolutions/min for 15-20 min, after that supernatant is removed, and produced deposition is used as trypanosome exoantigen for serological reaction. At that its activity should be 95-97%.
EFFECT: timely finding of sick animals and possibility to take emergency measures for invasion elimination.