Conjugate polysaccharide antigen with polyethylenimine as immunosorbent assay for the detection of streptococcal and pneumococcal infections
(57) Abstract:Usage area: immunology, biotechnology. The inventive synthetic immunosorbent obtained by processing the polysaccharide antigen by periodate sodium and polyethylenimine, allows to determine pneumococcal and streptococcal infections. Immunosorbent has a mol.m. about 300 million daltons, which allows its use in agglutination test in the detection of pneumococcal and streptococcal infections. table 2. The invention relates to immunobiotechnology, namely the conjugates and immunosorbents.Immunosorbent can be used for diagnosis of infectious and inflammatory diseases.Known activated matrix for immobilization of proteins (Senyk A. A. and co-authors. Activated matrix for immobilization of proteins, ed.St. USSR N 1280834, CL G 01 N 33/50) formula
P-O-CH2--CH2-NH-(CH2)nwhere P is the remainder of agarose, Sephadex or cellulose; n=2 or 3 for immobilization of proteins.Use polysaccharide matrix, which will receive in three stages.1. Activation of the polysaccharide carrier epichlorohydrin.2. The disclosure epoxy cycle of aminokislot proteins, containing a primary amino group, for example, transcortin, sexstoriesanime globulin immune globulins.However, the unsuitable matrix for immobilization of polysaccharide antigens, as the latter is unable to communicate with the connecting element.Known conjugate, in which the active group of the protein antigen associated sulfonium or alkylating or allermuir agents (agglutination reagent and the method of its production, the application of EPO N 280561, CL G 01 N 33/546).However, binding agents suitable only for protein antigens, which are often not sufficiently specific and, therefore, does not allow to identify the disease, which reduces the accuracy of diagnosis.Known also immunosorbent , which is selected as a prototype. Is a conjugate antigen is a polysaccharide-protein. Polysaccharide and protein covalently linked through
-CH2- protein. Low agglutinating properties do not allow it to be used in the solution. Small adhesive properties allow its use only with the activated solid phase carriers. In particular, the conjugate can not be used with inactivated solid phase carriers, for example, latausha good agglutinating properties, that will allow you to use it in the solution, and sufficient adhesive properties that allow its use with non-activated solid phase. This significantly extends the use of the conjugate.The technical result is that the aldehyde group of the polysaccharide is associated polyethylenimine.The essence of the invention lies in the fact that the conjugate has the formula
< / BR>where n = 103-106< / BR>From the work described in the introductory part of the description, it is known that polysaccharide is used as the antigen and activated media. Using polyethylenimine as the link is not known. Known only use it as a polycation to bind sulfo-, carboxy - and other acidic groups (Haars, A., Zonnez S. Hutterma A. Quantitative determination of lignosulfomates from sulfite spend liguors using presipitation with polyethyleneimine. Holzforsch 35(2); 59-65. 81).Therefore, there is an inventive step.The conjugate was prepared as follows.Polysaccharide antigen, for example the C-polysaccharide of pneumococcus (SPSH) or A-polysaccharide of Streptococcus gr. And( APSH) is treated with periodate sodium, then add a solution of polyethylenimine. Full reaction scheme UB>2+4O2+2Na++2H+< / BR>2.(C6H6O3(HO)2)n+ (C4H9N)n(NH2) where n = 103-106< / BR>P R I m m e R. Dosage of the ingredients are listed per 1 mg of the polysaccharide. 1.1 mg SPCH - pneumococcus antigen dissolved in 0.1 ml of 0.1 M bicarbonate buffer pH 8. Add 0.1 ml of periodate sodium (2 mg/ml). Incubated for 1 h in the dark. Then add a drop of glycerin to remove free peerdata sodium) and by vigorously shaking pour of 0.45 ml of polyethylenimine, 2 mg/ml (mol.m. 30-40 kDa) in 0.1 M bicarbonate buffer pH 8. Incubated for 2 h2. 1 mg of group-specific polysaccharide of Streptococcus group a antigen of Streptococcus dissolved in 0.1 ml of 0.1 M bicarbonate buffer pH 8. Add 0.1 ml of periodate sodium (2 mg/ml) in bicarbonate buffer and incubated for 1 h in the dark. To remove free periodate sodium pour in a drop of glycerine. Then add 0.45 ml of a solution of polyethylenimine (2 mg/ml) and incubated in the dark for 2 hThus prepared conjugates lead phosphate buffer to 40 ml and poured into wells for assay 100 ál into each well. Stand for 12 h at 4aboutC, washed on the elemental analysis.Since the polysaccharide antigen is specific for acute infectious-inflammatory diseases, immunosorbent suitable for the diagnosis of this class of diseases. Thus sorbent with SPSH - pneumococcus antigen suitable for the diagnosis of diseases caused by pneumococcus (such as acute pneumonia, with APSH - antigen of Streptococcus group A - for the diagnosis of diseases caused by Streptococcus group A (rheumatism, tonsillitis, glomerulonephritis)), with Re - glycolipid a antigen of gram-negative microorganisms for the diagnosis of diseases caused by gram - negative organisms enterobacteria (pyelonephritis, enteritis, adnexitis, etc). For diagnostic purposes in wells with conjugate make 100 ml of 0.5% solution of bovine serum albumin in phosphate buffer, incubated 1 h at 37aboutWith, and then remove the contents from the wells and make them 100 μl of the test serum in dilutions from 1:5 to 1:640. Incubated 1 h at 37aboutC, washed 4 times add to the wells, 100 μl of antibodies to human immunoglobulins conjugated to horseradish peroxidase at a dilution of 1:200 and incubated 1 h at 37aboutC, washed 5 times, make 100 ál of substrate - orthophenanthroline (1 mg/CMA. As an example, the results of studies immunosorbent SPCH five patients.1. Patient d, acute pneumonia. From sputum sown pneumococcus. The titer of antibodies to SPSH identified using the developed conjugate as immunosorbent assay, 1:320.2. Sick Yu, acute pneumonia, bronchial asthma. From sputum sown pneumococcus. The titer of antibodies to conjugate with SPSH identified using the proposed immunosorbent assay, 1:640.3. Patient C, tuberculosis of the lungs. The antibody titer of 1:40.4. Patient N., sarcoidosis. The antibody titer of 1:10.5. Patient G, chronic bronchitis, sown from sputum aureus. The titer of antibodies to conjugate with SPSH identified using the developed immunosorbent assay, 1:20.These examples show that high titers of antibodies were observed in patients in the acute phase of the disease caused by pneumococcus.In the case of use of a sorbent solution to 50 μl of the suspension of sorbent granules with media add 50 ál of test serum, diluted saline solution from 1:1 to 1:1024, incubated for at least 15 min, the reaction account for the formation of a colored precipitate.As an example, robotopia (standard) and using the proposed immunosorbent assay in patients with streptococcal infection. The results are shown in table. 1.From table. 1 shows that when using standard it is impossible to establish the degree of reliability at different activity of the process, since the data obtained with its help, do not correspond to the state of the process. Using this proposed immunosorbent assay marked a highly reliable difference between groups of patients.Parallel research was conducted in the titer of antibodies on the proposed invention and prototype. Investigated the serum of patients shown in the examples described above. The results are shown in table. 2.Analysis of the data table. 2 shows that the sorbent-prototype is not suitable due to their low molecular weight to work, as it does not have agglutinating properties. The proposed conjugate can be used as immunosorbent assay in agglutination reactions. CONJUGATE POLYSACCHARIDE ANTIGEN WITH POLYETHYLENIMINE AS IMMUNOSORBENT ASSAY FOR THE DETECTION OF STREPTOCOCCAL AND PNEUMOCOCCAL INFECTIONS.Conjugate polysaccharide antigen with polyethylenimine General formula
< / BR>where n = 103- 106,
as immunosorbent assay for the detection of streptococcal and pneumococcal infections.
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.