Method of serologic diagnosis of viral gastrointestinal infections in cattle by method of enzyme-immunoassay
FIELD: veterinary medicine.
SUBSTANCE: method includes the interaction of antigens with antibodies, with anti-species antibodies labeled with horseradish peroxidase, adding the substrate mixture and recording the results of the reaction on the colour intensity of the complex formed. In the reaction the plates are used with antigens of rotavirus, coronavirus, the virus of diarrhea preliminary sorbed on them, and after applying the test samples of serum 0.10-0.12 ml each. The plates were incubated for 1.5-2 h at 36-37°C and washed. Then total anti-species immunosorbent conjugate is applied of 0.10-0.12 ml consisting of enzyme-labeled antibodies against globulins of blood serum of cattle and the results of the reaction are recorded.
EFFECT: invention provides simultaneous diagnosis of rotavirus, coronavirus enteritis, viral diarrhea in cattle, sensitivity, specificity, and ease of the assay, and the ability to automate the process of research.
The present invention relates to the field of veterinary Virology, in particular to the development of a method for detection of antibodies against the viruses that cause gastrointestinal infections in cattle enzyme-linked immunosorbent assay.
A leading role in the etiology of diarrhea in young farm animals play Rota-coronavirus, virus diarrhea-diseases of the mucous membranes, to a lesser extent other infectious agents. These diseases are widely spread in the Russian Federation and abroad and put livestock farms tangible economic damage resulting from the loss from death, especially at an early age, funds spent on treatment of patients and loss of production from the animals recover. Every year half of the losses of juveniles accounted for gastrointestinal disease at an early age.
In view of the above problem diagnosis and control of these infections is important. With great efficiency to solve the problem of diagnostics allows a highly sensitive enzyme immunoassay.
There is a method of study of blood sera for antibodies to the causative agent of viral diarrhea by ELISA in epidemiological survey of livestock farms .
There is also known a method for serodiagnosis of transmissible gastroenteritis with the frost by ELISA, why in the wells of polystyrene tablet make specific antigen in 0.1 ml and adsorb on the surface of the hole. The next step in the wells microarrays make the control subjects and serum 0.1 ml and incubated for 1.5-2 h at 37°C. Unbound antibodies are removed by repeated washing, and then added to each well individuai enzyme conjugate in a volume of 0.1 ml working dilution. In positive cases, the conjugate binds fixed on the sensitized surface antibodies, forming a complex of the antigen-antibody-conjugate. The excess conjugate is removed by washing and contribute to all wells of the substrate indicator solution, which changes color according to the principle of redox reaction, is proportional to the number of conjugated with antibodies to the enzyme. The amount of enzyme is proportional to the amount of antibody bound peroxidase fixed on the surface of the wells with antigen . (Prototype).
The objective of our research was to develop a sensitive, effective way serodiagnosis of Rota-and coronavirus enteritis, viral diarrhea, bovine enzyme-linked immunosorbent assay and significantly reduce the time to diagnosis due to the simultaneous study of serum antibodies to these pathogens. Literature the data on the use of ELISA test for the study of blood sera for antibodies to Rota-, coronavirus, bovine missing.
The essence of the proposed method consists in the following. To the wells of one plate with pre-adsorbed rotavirus, coronavirus, diarrhoeal antigens automatic pipette make a test sample of blood plasma 0,10-0,12 ml in the wells with different antigens is incubated for 1.5-2 hours at a temperature of 37-38°C, washed tablet, contribute 0,10-0,12 ml individuai enzyme conjugate, consisting of enzyme labeled antibodies to globulin in the blood serum of cattle, incubated under the same conditions, the tablet is washed and after adding substrate mixture undergo simultaneous analysis of responses to the company, coronavirus enteritis, viral diarrhea in cattle.
In the first stage reaction in all wells automatic pipette contribute to 0.1 ml of 0,01M phosphate buffer solution. In the first wells of rows coated with antigens of rotavirus, coronavirus, virus diarrhoea contribute to 0.1 ml of test serum, pipeinput, and 0.1 ml is transferred into the next pit, etc. To control reactions in parallel contribute to 0.1 ml of specific homologous sera and also titrated them as a sample. Likewise contribute and titrate with negative serum. The tablet is incubated in the incubator for 1.5 hours at 36°C, then washed it is from unbound antibodies. In the washed wells contribute to 0.1 ml individuai enzyme conjugate, consisting of globulin in the blood serum of cattle, labeled with horseradish peroxidase. Tablet incubated in an incubator at 36°C for 1.5 h and again washed three times with phosphate-buffer solution with tween-20. Then in the wells contribute to 0.1 ml of substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide. The result of the reaction into account in 30 minutes by a colour change of the formed reaction product from brown to light brown:
- intense brown staining, brown staining - positive result;
- light brown staining - equivocal result;
- staining is absent or is at the level of the negative control is a negative result.
In the study of 97 blood serum samples of cattle from disadvantaged by gastrointestinal diseases farms antibodies to rotavirus was detected in 54 of animals, for coronavirus antibodies were detected in 47, and to the virus diarrhoea - 21 animal, which accounted for 55.6 per cent to 48.4 per cent, 21.6 per cent, respectively.
The reaction is carried out analogously to example 1, but the serum, conjugate and substrate mixture contribute to the volume of 0.12 ml and tablets incubated at 37°C for 2 hours. Obtained similar results as in example 1.
The proposed method has no domestic analogues. Allows for diagnostics company, coronavirus enteritis, viral diarrhea in cattle by ELISA significantly reduce the time for setting-up the reaction and to obtain more reliable results when taking into account the reaction, as the research on these infections are carried out simultaneously on the same tablet with pre-adsorbed antigens Rota-and coronaviruses, virus diarrhoea in cattle using a General-enzyme conjugate under the same conditions. In addition, there are no published data about the use of enzyme or antibody-based test diagnostics when the Rota-, coronavirus enteritis of cattle.
The proposed method for serological diagnosis of Rota-and coronavirus enteritis, viral diarrhea cattle enzyme-linked immunosorbent assay tested by a Commission in September 2009 and has been tested in several veterinary laboratories with a positive result (FGI Central scientific-methodical vetebrate 2010, GU Kursk regional vetebrate 2010, Tverskaya interregional vetebrate 2011)
On the basis of the obtained test results, the Commission concluded that the method is sensitive and can detect these specific antic the La in the tested sera in much shorter time.
The proposed method will be applied for the inspection of cattle affected with gastrointestinal infections farms for the purpose of diagnosis Rota-, coronavirus enteritis, viral diarrhea cattle. High sensitivity, specificity, ease of setting reaction and the possibility of automation of the processes of research allows us to quickly carry out mass screening animals to determine the spread of these infections. Reducing the time to diagnosis is very important for disadvantaged by these infections farms, because Express diagnosis makes it possible to begin timely treatment and preventive measures and, thereby, improve and maintain livestock.
This test system can be used in the evaluation of the immune status of the population, to control the antigenic activity of the respective components of mono - and polyvalent vaccines.
Sources of information
1. Veterinary medicine, 4, 2004, p.20
2. Veterinary medicine, 5, 2005, p.17-20.
Method for the serological diagnosis of viral gastrointestinal infections bovine enzyme-linked immunosorbent assay, mainly Rota-, coronavirus enteritis, viral diarrhea in cattle, involving the interaction of antigens with antibodies, with antiv the annual antibodies, labeled with horseradish peroxidase, the addition of the substrate mixture and records the results on the intensity of the color formed complex, characterized in that the reaction using tablets with pre-adsorbed on them antigens Rota-coronavirus, virus diarrhea, and after application of the tested serum samples for 0,10-0,12 ml tablets incubated for 1.5-2 h at 36-37°C, washed, then contribute 0,10-0,12 ml total individualo enzyme conjugate, consisting of enzyme labeled antibodies against globulin in the blood serum of cattle and conduct analysis of reaction.
SUBSTANCE: for the purpose of prediction of developing atopic dermatitis in newborns, umbilical blood plasma is examined for a level of interleukin-18. If the related level is 34 pg/ml or lower, developing atopic dermatitis in the newborn is predicted.
EFFECT: use of the given method enables well-timed evaluation of a risk of developing atopic dermatitis in newborns and early preventive measures for prevention thereof.
SUBSTANCE: method under the invention provides that the complex immunoglobulin preparation containing a component of C3b complement is sorbed in microplate wells for the purpose of immunoassay. Then the microplate wells are added with a solution of an analysed sample containing human complement C1 inhibitor with the unknown activity. It is followed by incubation, drying and washing of the dish; the wells are added with a conjugate of enzyme with serine proteinase in the form of preparation of fibrinolysin and a substrate of this enzyme. The amount of the prepared enzymatic reaction product is used to derive the content of active C1 inhibitor. A kit for implementing the method comprises a flat-bottomed microplate with bound C3b, the conjugate of enzyme with serine proteinase, the substrate buffer of this enzyme and a reference for active C1 inhibitor.
EFFECT: use of the method under the invention enables determining activity of C1 inhibitor, simultaneously bound both with serine proteinase inhibition, and with binding inhibition of complement factor B and C3b complement component.
2 cl, 1 tbl, 1 dwg
SUBSTANCE: method under the invention provides that the preparation pyrogenal is sorbed in microplate wells for the purpose of immunoassay; then the microplate wells are added with a solution of an analysed samples containing complement human C1 complement inhibitor with the unknown concentration. Then the plates are incubated, and after washing and drying, a conjugate of enzyme with C1 inhibitor antibodies and a substratum of this enzyme are added into the wells. The amount of the prepared enzymatic reaction product enables calculating the content of the C1 inhibitor in the analysed sample. A kit for implementing the method comprises a flat-bottomed microplate with the sorbed preparation of pyrogenal, the conjugate of the enzyme with the human C1 inhibitor antibodies, a substrate buffer of this enzyme and a reference for the C1 inhibitor.
EFFECT: use of the method enables implementing quantitation of the C1 inhibitor using an ability of the latter to bind to lipopolysaccharide.
2 cl, 1 dwg, 2 ex
SUBSTANCE: method under the invention provides that the preparation heparin is sorbed in microplate wells for the purpose of immunoassay. Then the microplate wells are added with a solution of an analysed samples containing complement C1 inhibitor with the unknown concentration. It is followed by incubation, and after washing and drying of the dish, a conjugate of enzyme with C1 inhibitor antibodies and a substratum of this enzyme are added into the wells. The quantitation is enabled by the amount of the prepared enzymatic reaction product. A kit for implementing the method comprises a flat-bottomed microplate with the sorbed preparation heparin, the conjugate of the enzyme with the human C1 inhibitor antibodies, a substrate buffer of this enzyme and a reference for the C1 inhibitor.
EFFECT: use of an ability of the C1 inhibitor to bind to heparin to determine its content in the analysed samples.
2 cl, 2 dwg, 2 ex
SUBSTANCE: pharmaceutical preparation CIP (complex immunoglobulin preparation, consisting of IgG, IgA and IgM) is bound in cavities of a micropanel, said preparation containing a C3b complement component, samples containing the determined functionally active human C1 inhibitor are incubated in the cavities, and the bound C1 inhibitor is determined using a conjugate of an antibody against the human C1 inhibitor with an enzyme and a substrate for that enzyme based on the amount of the formed product of the enzymatic reaction. The set contains flat-bottomed micropanel with the sorbed CIP, the enzyme conjugate with antibodies against the human C1 inhibitor, a substrate buffer of that enzyme and a standard for the active C1 inhibitor.
EFFECT: use of the method enables to determine activity of the C1 inhibitor which regulates the complement alternative path.
2 cl, 1 dwg, 1 tbl, 2 ex
SUBSTANCE: complex antigen of measles virus used as a component of immunoenzymometric test system for diagnostics of antibodies to measles virus was produced from culture liquid containing measles virus stain Leningrad 16 with titer no less than 105.0 50% tissue cytopathic dose/ml, Edmonston - no less than 106.0 50% tissue cytopathic dose/ml, NovO/96 no less than 108.0 50% tissue cytopathic dose/ml by inactivation of its infectious activity by detergent and separation of protein from cellular lysate by chromatographic purification in amount no less than 2 mcg/ml with the purity no less than 70%. Culture virus-containing liquid was processed using separate cultivation of measles virus stains Leningrad 16, Edmonston and NovO/96 on monolayer of Vero cells culture with subsequent mixing of culture virus containing liquid in proportion 1:1:1 v/v.
EFFECT: Usage of invention provides for increased sensitivity and specificity of a complex antigen possessing the property to detect antibodies in blood serum.
2 cl, 4 tbl, 6 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention concerns medical virology and microbiology. Strain is deposited in culture collection of Federal State Research Institution State Research Centre of Virology and Microbiology "Vektor" of Rospotrebnadzor, under registration No VB-05. Strain features higher productivity. More sensitive immunoenzyme test system for hepatitis virus antibody diagnostics is created on the basis of this strain. Invention can be applied in virology.
EFFECT: production of more sensitive immunoenzyme test system for hepatitis virus antibody diagnostics.
2 cl, 1 dwg, 1 tbl, 4 ex
FIELD: medicine; ophthalmology.
SUBSTANCE: invention is intended for simultaneous verification of uveal melanoma and forecast of metastasis development. Immunohistochemical analysis of protein S-100 and melanin-A expression within tumour cell is accompanied with simultaneous estimation of S-100-positive cells number within visual field. Uveal melanoma is verified if reaction of S-100 and melanin-A is positive with forecasted high possibility of tumour dissemination in case number of S-100-positive cells number is less than 50.
EFFECT: simplified examination associated with high specificity and sensibility of simultaneously verified uveal melanoma and forecasted metastasis.
1 tbl, 1 dwg, 2 ex
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
SUBSTANCE: method involves determining total IgA quantity using immunoenzyme method based on polyclonal IgA antibodies as binding antibodies on the micropanel and the same antibodies in conjugate with peroxidase for detecting bound IgA in sample before and after treatment with specific IgA1-protease. Total IgA1 and IgA2 content is determined before being treated with enzyme. IgA2 content only is determined after being treated with enzyme. IgA1 is not determined after being disintegrated with IgA1-protease. Kit has microplate containing absorbed polyclonal IgA antibodies, conjugate of the same antibodies with peroxidase, reference sample having given IgA concentration, IgA1-protease and substrate buffer.
EFFECT: simplified method and kit for determining IgA1 and IgA2; improved results reproducibility; no specific monoclonal antibodies being used.
2 cl, 1 tbl
FIELD: medicine, ophthalmology.
SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.
EFFECT: higher accuracy of prediction.
FIELD: medicine, ophthalmology.
SUBSTANCE: one should detect the content of tumor necrosis factor alpha in acute period, moreover, the above-mentioned factor should be determined in lacrimal liquid on the 11th - 15th d against the onset of herpetic ophthalmoinspection, at its content ranged 176-250 pcg/ml prediction is considered to be favorable, and from the value of 300 pcg/ml and higher - as unfavorable.
EFFECT: higher accuracy of prediction.
FIELD: medicine, surgery.
SUBSTANCE: one should carry out virological testing patient's blood serum and hepatic bioptates. At detecting TTVDNA and HGVRNA it is necessary to perform ultrasound survey, and at availability of biliary sludge one should conclude upon early stage of cholelithiasis.
EFFECT: higher accuracy of diagnostics.
FIELD: medicine, cardiology.
SUBSTANCE: one should detect the level of activity of IgM and IgG immunoglobulins to cytomegalovirus on the 5th and 15th d of large-focus myocardial infarction. At increased diagnostically valuable result of specific immunoglobulins of type M by 0.10-0.20 times and for type G by 0.73-2.09 times it is possible to predict favorable clinical flow of large-focus myocardial infarction without exudative pericarditis. At the value of specific immunoglobulins exceeding diagnostically valuable result for type M - by 0.86-1.67 times and for type G by 2.42-3.01 times one should predict early clinical flow of post-infarction pericarditis. The method enables to carry out prophylactic measures in due time.
EFFECT: higher accuracy of prediction.
5 ex, 2 tbl
SUBSTANCE: one should test blood serumal samples in immunoenzymatic assay by applying a test system ELI-N-1, the components of which are being antigens of nervous tissue or their immunochemical analogs specifically binding antibodies with tendency to antigens of nervous tissue, and, also, idiotypical antibodies to them, or their variable parts. Prediction should be performed according to the level of idiotypical and anti-idiotypical autoantibodies to antigens of nervous tissue and, also, according to their ratio. Thus, a new test system has been elaborated that enables to predict the flow of available nervous-psychic diseases.
EFFECT: higher accuracy of prediction.
2 cl, 7 ex
FIELD: medicine, obstetrics.
SUBSTANCE: in pregnant women at pregnancy terms being after 39 wk in average portion of morning urine one should detect due to a solid-phase immunoenzymatic assay the concentration of pregnancy-associated protein-A (PAPP-A). At the level of PAPP-A being 768.2 ng/ml and more it is possible to conclude upon physiological mature pregnancy. The innovation is noninvasive and enables to increase accuracy in predicting true over-mature pregnancy.
EFFECT: higher efficiency and accuracy of diagnostics.
2 ex, 1 tbl
FIELD: medicine, biotechnology.
SUBSTANCE: the present innovation deals with elaborating diagnostic reagents for testing prionic protein in mammalian cerebral tissue due to IEA technique and refers to antibodies specifically reacting with prionic protein PrP or its fragment. The innovation includes polyclonal antibodies obtained to synthetic peptides including amino acid sequences of bovine prionic protein 143-168, 101-134 and 211-241, their conjugates with horseradish peroxidase and diagnostic reagents obtained upon their basis that enable to detect prionic protein in mammalian cerebral tissues.
EFFECT: higher specificity of detection.
15 cl, 8 ex, 8 tbl
FIELD: veterinary and medicine.
SUBSTANCE: invention, in particular, relates to production and use of biological preparations intended for differential diagnostics of brucellosis and to a method of differentially diagnosing brucellosis. Method involves serologic analysis of sera using antigenic "IFK", which is horse-radish peroxidase-labeled electrophoretically purified polypeptide fraction of virulent strain B.arborus 54. Diagnosis of brucellosis is stated when anti-brucellosis antibodies in sick animal sera diluted to 1/100 and higher are revealed at CSP reaction intensity 2.1 and higher.
EFFECT: elaborated method increasing immuno-enzymatic test specificity and allowing performing differentiation of postvaccinal immunological reactions and postinfectious reactions induced by microorganisms having antigenic affinity with brucellas, especially Yersinia enteriocolitica.
2 cl, 4 ex
FIELD: biotechnology, analytical chemistry.
SUBSTANCE: claimed method includes providing of complexes between antigen molecules and specific antibodies on carrier surface, wherein said complexes are disclosed by addition of enzyme label thereto followed detection thereof based on formation of enzyme reaction product. Enzyme label addition is carried out by two protein interaction, namely bacterial ribonucleaze barnase and barstare, which is inhibitor thereof, wherein either of the two is added to immunoreagent, and the other one is added to enzyme label. Abovementioned complexes have high affinity, specificity, and binding constant of 1014 M-1.
EFFECT: new method for antigen detection.
6 dwg, 2 ex
FIELD: veterinary virology.
SUBSTANCE: the present innovation deals with interaction of antibodies with an antigen, with antibodies labeled with horseradish peroxidase, addition of substrate mixture and registration of reaction results. Moreover, one should apply plotting boards with presorbed antibodies and general immunoenzymatic conjugate. The innovation enables to shorten terms for diagnosis and obtain more significant results of diagnostics.
EFFECT: higher efficiency.