Method to produce erythrocytic antigen for reaction of indirect hemagglutination in case of brucellosis. RU patent 2484481.
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Method for preparing species-specific antigen chlamydia trachomatis for enzyme immunoassay / 2244932 Invention relates to a method for preparing species-specific antigen C. trachomatis used for enzyme immunoassay. Chlamydia bodies are treated with amino acid chelate before treatment with detergent N-lauryl sarcosine and treatment with monosaccharide simple ether is carried out before treatment with detergent dodecyl sulfate sodium. The advantage of invention involves enhancement of species-specific activity of antigen. Invention can be used for serological diagnosis of urogenital chlamydiosis. |
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Method for express-diagnosis of whooping cough / 2247388 Study is carried out in brush-bioptate taken from pharynx posterior wall mucosa using reaction of indirect immunofluorescence and the presence of whooping cough bacillus antigen and specific secretory immunoglobulin A (Ig A) are determined. Whooping cough is diagnosed in simultaneous detection of whooping cough bacillus antigen and specific secretory Ig A in analyzed material. Method provides enhancing precision of diagnosis and reducing time for carrying out the diagnosis. Invention can be used for carrying out the early etiological diagnosis. |
 Preparation, method for its obtaining and method for quick test diagnostics for antigen of tick-borne encephalitis virus / 2247991The suggested preparation is being a 2%-coagglutinating reagent obtained due to adding equal amount of 0.1%-highly purified and high-avidity immunoglobulin against tick-borne encephalitis to 10%-staphylococcal reagent dissolved in distilled deionized water followed by sensitization for 1-1.5 h at 25-30 C, resuspending the residue in 0.15 M sodium chloride solution at pH 5.4-5.5 to obtain coagglutinating reagent up to 2%-content value. Method for quick test diagnostics deals with applying 15-20 mcl tested material onto a slide, adding equal amount of a certain preparation necessary for express diagnostics followed by thorough mixing by shaking a slide to visually determine the presence of antigen to tick-borne encephalitis virus in 2-7 min due to coagglutination phenomenon. |
 Method for diagnosing clamidiosis cases / 2251112Method involves mixing heparinized blood with chlamydia antigen diagnosticum in test glass tube containing physiologic saline in control test tube. Leukocyte number is determined and the leukocytes are incubated and repeatedly counted 2 h later and leukocyte fracture index is calculated. The value being greater than 0.2, clamidiosis is diagnosed. |
 Recombinant proteins containing diagnosis significant coronavirus protein antigen epitope (sars-cov), assosiated with grievous acute respiratory syndrome, and sequences of synthetic genes encoding sars-cov / 2253870Invention 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. |
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Method for titration of antirabic virus-neutralizing antibodies / 2254575 Claimed method includes interaction of vaccine rhabdovirus of specific strain with target sera; incubation of obtained mixture containing virus and serum for 60 min at 37°C; daily intact culture of saiga kidney is introduced, mixture is fed into CO2-incubator and is hold for 5-7 days. Then virus-neutralizing antibodies is determined based on limit serum dilution wherein 50 % of virus cytopathicitic action on cell culture is suppressed. |
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Method for predicting the threat of abortion in the first trimester of pregnancy / 2255339 One should detect ratio coefficients for the content of lactobacilli to conditionally pathogenic flora in scraped off samples obtained after mucus removal from the surface of posterior vaginal arch and cervical canal, and at their 2-fold decrease against the norm it is possible to conclude upon the risk of abortion. The method enables to perform efficient prediction before manifestation of clinical signs that makes it possible to carry out due therapy. |
 Strain of mammalian hybrid cell c3/s-3e5 of mus musculus l. producing monoclonal antibody to bernet's coxiellas / 2257414Invention represents a new strain of mammalian hybrid cells C3/S-3E5 of Mus musculus L. producing monoclonal antibodies (MCAb) to Bernet's coxiellas (strain "Grita") in cell cultures and abdominal cavity of syngenic animals. Hybridoma C3/S-3E5 producing MCAb to this pathogen is obtained by fusion of murine myeloma of strain Sp-2/0 and murine splenocytes of strain BALB/c immunized with the concentrated and purified Bernet's coxiella preparation (strain "Grita) inactivated with formalin using polyethylene glycol of molecular mass 1000 Da as a fusing agent and the following cloning by method of maximal dilutions. Specificity of prepared MCAb: absence of cross-reactions in IFA with Provacheck's rickettsia antigen and with the non-infected accumulation substrate. Using prepared MCAb it is possible to carry out specific detection of Bernet's coxiellas by method IFA (direct and indirect variants). IFA sensitivity based on these MCAb is 2.0 x 103 ID50 x cm-3 for white rats. Applying the present invention allows detecting and identifying pathogens of rickettsial etiology. |
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Method for diagnosing irritated intestine syndrome / 2259165 Method involves applying chromato-mass-spetrometric techniques for determining small and large intestine bioptate fatty acids content. The number of microbial cells is determined from chromato-mass-spetrogram peak areas. The calculated proportions are interpreted for diagnosing irritated intestine syndrome. |
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Differential diagnosis method for predicting intestine dysbacteriosis severity degree in chronic bronchitis cases / 2260808 Method involves carrying out bacteriological examination of feces with following intestine microflora composition being determined. Mean degree of fat, protein and d-xylose excretion in 5 g version is to be determined. Mean fat excretion degree being equal to 2.71±0.04, mean J131 albumin excretion degree being equal to 2.66±0.03, mean d-xylose excretion degree being equal to 1.77±0.04, dysbacteriosis of mild severity degree is to be predicted. Mean fat excretion degree being equal to 4.37±0.28, mean J131 albumin excretion degree being equal to 3.57±0.14, mean d-xylose excretion degree being equal to 1.45±0.14, dysbacteriosis of moderate severity degree is to be predicted. Mean fat excretion degree being equal to 5.22±0.18, mean J131 albumin excretion degree being equal to 4.26±0.12, mean d-xylose excretion degree being equal to 1.11±0.04, severe dysbacteriosis is to be predicted. |
FIELD: veterinary science.
SUBSTANCE: method to produce erythocytic antigen for reaction of indirect hemagglutination in case of brucellosis includes preparation of formalinised erythrocytes of sheep and their sensibilisation with sensitin produced by growth of bacterial brucella mass, its washout with hypertonic solution of sodium chloride, inactivation, extraction and separation of sensitin by centrifugation. At the same time formalinised erythrocytes of sheep are sensibilised with antigen made by exposure of the inactivated bacterial mass of the strain B.abortus 19 in the dose of 40-50 billion microbial cells per 1 ml of sodium dodecyl sulfate in 1% concentration at 70-72°C for 45 minutes, with further loading of erythrocytes with sensitin in terms of 0.2-0.4 ml of antigen per 1 ml of 5% suspension of erythrocytes.
EFFECT: method improvement.
4 tbl
The invention relates to the field of veterinary medicine, in particular to the manufacturing of diagnostic products, and can be used for serological diagnosis of brucellosis in animals.
A method of obtaining erythrocytic diagnosticum by the disintegration of Brucella ultrasound with subsequent release of the antigen and the sensitization of the erythrocytes, the disintegration is carried out at pH 8.0 and 9.0, sensitization of erythrocytes is carried out at 70-72º within 5-10 min [A.S. №784879, 1980]. The disadvantage of this method is that in the manufacture of diagnosticum necessary to carry out erythrocytes, which complicates the production process, and when you use certain parties tannin antigen can be , when examining Ihar blood serum of healthy animals, in addition, diagnosticum has underactive.
The closest analogy is the way of getting erythrocite antigen reaction indirect haemagglutination in brucellosis, including the preparation of erythrocytes with their subsequent sensitization received the cultivation of bacterial mass bruzell, enveloping her gipertoniceski solution of sodium chloride, inactivation, extraction and separation of by centrifugation, before processing , red blood cells pretreated sodium 1%concentration at 50-60oC for 30 minutes [EN 2283498 C1, 22.02.2005,]. However, this method can't get antigen of high activity, in connection with which the diagnosis of brucellosis in disadvantaged farms Ihar using a well-known diagnosticum part of patients having animals in blood serum specific in diagnostic titers. Besides, production of erythrocytic diagnosticum in this way is expensive because it requires a large number of original material - bacterial mass to extract and load them erythrocytes.
The technical result of the invention is a method of receiving erythrocytic antigen reaction indirect haemagglutination in brucellosis (Ihar), which has a high activity at detection of animals with brucellosis in reducing the economic cost of producing it.
The technical result is achieved by the fact that prepare RAM erythrocytes and their received the cultivation of bacterial mass bruzell, flush it gipertoniceski solution of sodium chloride, inactivate, extracted and separated by centrifugation , RAM erythrocytes antigen made when exposed to inactivate bacterial lot of strain .abortus 19 dose 40-50 billion microbial cells in 1 ml of sodium dodecyl sulfate in 1%concentration of 70 72º within 45 minutes followed load of erythrocytes the rate of 0.2-0.4 ml antigen on 1 ml of 5%mist of red blood cells.
The proposed method is as follows.
culture Brucella strain .abortus 19 wash off with 12-percent solution of sodium chloride, filtered and subjected to inactivation by autoclaving at 1 ATM. (120oC) for 30 minutes, bring the concentration of Brucella to 40-50 billion M.K. in 1 ml of 12%solution of NaCl. The bacterial suspension add 1% of sodium dodecyl sulphate, and then it was heated in water bath at a temperature of 70-72º within 45 minutes with occasional stirring every 5 minutes.
The suspension is decanted by centrifugation at 7-8 thousand rpm for 30 minutes, liquid (antigen) is used for sensitization sheep erythrocytes.
Sensitisation erythrocytes spend the optimal dose , which is determined at the titration with standard pattern of serum .
The specificity and activity check when examining Ihar negative serum and standard serum sample .
An example of definition of optimum conditions of sensitization erythrocytes obtained when exposed to bacterial suspension different concentrations of sodium dodecylsulfate, is presented in table 1. Data of table 1 show that the optimal concentration of sodium dodecyl sulfate in removing the antigen is 1%concentration, allowing to get the active with a given activity, Ihar not lower titer of 1:3200 with a rating of 4 cross when setting the standard pattern of serum and a negative result with negative serum.
Optimal dose necessary to receive the erythrocytic antigen for Ihar, determined by its adsorption in increasing concentrations on erythrocytes and testing activity of erythrocytes sensitized in Ihar when titration with standard pattern of serum , specificity - with negative serum and physiological solution.
When titration for sensitization of erythrocytes it is added in quantities 0,1; 0,2; 0,4; 1,0; 1,5; 2,0; 3,0 ml to 1 ml of 5%mist erythrocytes.
The results of the validation activity and specificity of antigen, received at the sensitization of erythrocytes different doses , are presented in table 2. Analysis of the data of table 2 shows that antigen produced by processing erythrocytes 1%concentration of sodium dodecylsulfate sodium with their subsequent sensitization learned by autoclaving, has lower serological activity in comparison with the antigen obtained during the sensitization erythrocytes learned when exposed to a 1%concentration of sodium dodecylsulfate. The optimal number of for processing of erythrocytes is 0.2-0.4 ml to 1 ml of 5%mist of red blood cells, antigen has activity (titer 1:6400-1:12800) and specificity (negative result with negative serum and physiological saline).
When sensitization of erythrocytes, pretreated detergent (analogue), to obtain titer Ihar 1:3200 with standard pattern of serum requires 1.5 ml , i.e. 7.5 times more than the proposed antigen.
Therefore, sensitisation erythrocytes made by the proposed method, the rate of 0.2-0.4 ml it to 1 ml of 5%mist of red blood cells, allows to receive specific and more active (compared with the equivalent) brucellosis antigen for Ihar.
The results of the test specificity and activity erythrocyte antigens produced in different ways, are presented in tables 3, 4. Data in table 3 and 4 show that the proposed method allows to obtain specific and more active brucellosis antigen in comparison with the equivalent.
Activity diagnosticum enhanced through the impact on microbial suspension chemical detergent - 1% of sodium dodecyl sulphate. The economic cost of production decreased in two times at the expense of the reduction of the concentration of microbial cells when removing and decrease of its number under load erythrocytes.
brucellosis antigen for Ihar made by the method of Б, tested at conducting epizootic control for the welfare of households on brucellosis in cattle and small cattle and rehabilitation of the affected areas with high economic and effects in the farms of the Omsk region.
Table 1The influence of concentration of sodium dodecyl sulphate on activity and specificity erythrocytic antigen
The concentration of sodium dodecyl sulfate
The amount of antigen per 1 ml to 5.0% suspension of erythrocytes
Titre with standard pattern of serum
Control with negative serum
Control with saline solution
0,25% 0,2 - - - 0,4 - - - 1 - - - 2 1:400+++ - - 3 1:800+++ - - 0,5% 0,2 - - - 0,41:200++++
- - 11:200++++
- - 21:800++++
- - 31:1600++++
- - 1,0% 0,21:6400++++
- - 0,41:12800++++
- - 11:25600+++
- - 21:25600+++
- - 31:51200+++
- - 1,5% 0,21:3200++++
- ++ 0,41:6400++++
++ ++++ 11:12800++++
++++ ++++ 21:25600++++
++++ ++++ 31:51200++++
++++ ++++ 2,0% 0,21:6400++++
++++ ++++ 0,41:25600++++
++++ ++++ 11:51200++++
++++ ++++ 21:51200++++
++++ ++++ 31:51200++++
++++ ++++ Table 2The results of titration and alive, specificity of erythrocyte antigens in Ihar
The number of on 1 ml of 5% mist
Caption at Ihar
with a standard specimen serum
negative serum
with natural solution
1:200 1:400 1:800 1:1600 1:3200 1:6400 1:12800 1:25600 1:50 1:100 1:200 1:400 1 2 3 4 5 6 7 8 9 10 11 12 13 14with the antigen Б
0,1 ++++ ++++ ++++ ++++ +++ + - - - - - - - 0,2 ++++ ++++ ++++ ++++ ++++ ++++ ++ + - - - - - 0,4 ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ - - - - - 1,0 ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ - - - - - 1,5 ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ - - - - - 2,0 ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ - - - - - 3,0 ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ - - - -with the antigen of the Caspian
0,1 - - - - - - - - - - - - - 0,2 - - - - - - - - - - - - - 0,4 ++ ++ ++ ++ - - - - - - - - - 1,0 ++++ ++++ ++++ +++ + - - - - - - - - 1,5 ++++ ++++ ++++ ++++ ++++ + - - - - - - - 2,0 ++++ ++++ ++++ ++++ ++++ ++ - - - - - - - 3,0 ++++ ++++ ++++ ++++ ++++ +++ ++ - - - - - - Table 4Results of comparative tests of activity erythrocyte antigens
Animal species
The number of samples studied
Responded positively in Ihar
with the antigen Б
with the antigen of the Caspian (analogue)
anti-lock brakes.
%the average titer
anti-lock brakes.
%the average titer
cattle troubled by brucellosis farms
78 20 25,6 1:290 15 19,2 1:213small cattle troubled by brucellosis farms
157 92 58,6 1:133 59 37,6 1:101The method of obtaining the erythrocytic antigen reaction indirect haemagglutination when brucellosis, including the preparation of sheep erythrocytes with their further sensitization received the cultivation of bacterial mass bruzell, flush it gipertoniceski solution of sodium chloride, inactivation, extraction and separation of by centrifugation wherein RAM erythrocytes antigen made when exposed to inactivate bacterial lot of strain Century abortus 19 dose 40-50 billion microbial cells in 1 ml of sodium dodecyl sulfate in 1%concentration at 70-72 OC for 45 min, follow-up load of erythrocytes the rate of 0.2-0.4 ml antigen on 1 ml of 5%mist of red blood cells.