CN102288756A - Preparation method and detection method of goat pox reverse indirect hemagglutination diagnostic reagent - Google Patents
Preparation method and detection method of goat pox reverse indirect hemagglutination diagnostic reagent Download PDFInfo
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- CN102288756A CN102288756A CN2011101889245A CN201110188924A CN102288756A CN 102288756 A CN102288756 A CN 102288756A CN 2011101889245 A CN2011101889245 A CN 2011101889245A CN 201110188924 A CN201110188924 A CN 201110188924A CN 102288756 A CN102288756 A CN 102288756A
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Abstract
The invention discloses a preparation method and a detection method for a goat pox reverse indirect hemagglutination diagnostic reagent capable of detecting a goat pox antigen. The preparation method comprises the following the steps of: preparing a red blood cell suspension by mixing glutaraldehyde with sheep red blood cells; mixing the red blood cell suspension with an equal amount of purified goat pox IgG (Immunoglobulin G); stirring, centrifuging and washing; and preparing 1 percent sensitized red cells from PBS (Phosphate Buffered Saline) containing 1 percent of neonatal calf serum to obtain the goat pox reverse indirect hemagglutination diagnostic reagent. Optimized preparation conditions are that: red cells are fixed with a glutaraldehyde mono-hydroformylation method, the concentration of the sensitized red cells is 0.8-1.0 percent, and the IgG content is controlled at 1.5-1.55 mg.ml<-1>, so that a good marking effect can be achieved; and the reagent is used for detecting a goat pox virus antigen with the conventional reverse indirect hemagglutination testing method, the reaction temperature is 20-25 DEG C, and the reaction time is 50 minutes. The method has high specificity, high sensitivity and high repeatability, and can be applied to quick clinical detection of goat pox antigens by a veterinarian.
Description
Technical field
The present invention relates to a kind of preparation method and detection method of goatpox reverse indirect hemagglutination diagnostic reagent.
Background technology
Goatpox is that (goat poxvirus, what GTPV) cause a kind ofly is endemy height contagious disease by the Capripoxvirus goat capripoxvirus.All can take place throughout the year, spring and autumn is common, regardless of sex, age, kind.OIE (OIE) classifies goatpox as the category-A serious infectious diseases, and No. 96 bulletin of The Ministry of Agriculture of the People's Republic of China, MOA in 1999 classified goatpox as a national class animal epidemic.In recent years, it is popular that more than 20 provinces and cities of China report the goatpox generation in succession, and the goat aquaculture that is surging forward to China has caused very big economic loss, has brought severe threat.
Chang Yong agar gel diffusion test detection method had shortcomings such as susceptibility is low, detection speed slow, specificity is not strong in the past.
For this reason, need set up a kind of sensitivity, quick, accurate, easy method detects goat capripoxvirus antigen, to be used for goatpox fast detecting, diagnosis.
Summary of the invention
The technical problem to be solved in the present invention is, a kind of preparation method and detection method of goatpox reverse indirect hemagglutination diagnostic reagent are provided, this goatpox reverse indirect hemagglutination diagnostic reagent can accurate detection goatpox antigen, reverse indirect hemagglutination assay susceptibility is higher, detection speed is faster, be used for goat capripoxvirus antigen fast detecting on the veterinary clinic, for the clinical diagnosis and the prevention and control of goatpox provide reference frame, to overcome deficiencies such as the susceptibility that prior art exists is low, detection speed slow, specificity is not strong.
Goatpox reverse indirect hemagglutination diagnostic reagent of the present invention, its preparation method may further comprise the steps:
The first, the healthy sheep venous blood of aseptic collection adds 10 times of amount PBS, 2 000 rev/mins centrifugal 15 minutes, wash 5 times, be made into 2.5% red cell suspension with the phosphate buffer of 0.1M pH 7.4～7.8;
The second, the red cell suspension that step 1 is made mixes with the 9:1 volume ratio with 1% glutaraldehyde, slowly stirs 1h under room temperature, 2000 rev/mins centrifugal 15 minutes, wash 5 times with phosphate buffer, make 2.5% hydroformylation red blood cell suspension again;
The 3rd, the hydroformylation red blood cell suspension that step 2 is made mixes with equivalent 1/,200 000 fresh tan-ooze, takes out after placing 37 ℃ water-bath l hour, with phosphate buffer washing 3 times, 2000 rev/mins centrifugal 15 minutes, make the precipitation red blood cell of hydroformylation, tanning;
The 4th, the precipitation red blood cell that step 3 is made adds in the acetate buffer of 10 times of amount 0.1M pH 3～4, mixes with equivalent purifying goatpox IgG dilution again, places 37 ℃ of 2h, every 15min pressure-vaccum mixing 1 time, make 0.8%～1% sensitized erythrocyte suspension therebetween; Employed purifying goatpox IgG content is controlled at 1.5～1.55mg/mL;
The 5th, centrifugal 15 minutes of 2000 rev/mins of sensitized erythrocytes that step 4 is made are got the precipitation red blood cell with containing 0.1%NaN
3, 1% newborn calf serum phosphate buffer washing 5 times, be made into concentration 0.8%～1% sensitized erythrocyte suspension diagnostic reagent at last;
The 6th, the qualified reverse indirect hemagglutination diagnostic reagent that promptly gets of analyzing and testing.
Goatpox reverse indirect hemagglutination diagnostic reagent detects the method for goat capripoxvirus antigen, the goatpox reverse indirect hemagglutination diagnostic reagent of preparation method's preparation of goatpox reverse indirect hemagglutination diagnostic reagent as described above, detection step reverse indirect hemagglutination assay method is routinely carried out, the steps include: in 96 hole V shaped slabs, to carry out, each sample is one group, every group 1～12 hole adds PBS 50 μ L, add sample 50 μ L to be checked then in first hole, be diluted to the 11st hole with liquid-transfering gun by the hole, abandon 50 μ L, the 12nd hole is the PBS blank.Last every hole adds goatpox reverse indirect hemagglutination diagnostic reagent 50 μ L, and vibration is put room temperature effect 50min, result of determination after mixing.All red blood cells are deposited at the bottom of the hole, concentrate the not aggegation that is that is 1 round dot; As red cell agglutination, then be distributed at the bottom of the hole around.Judging the power of positive reaction according to the degree of red cell agglutination, serves as tiring of detection antibody with the high dilution of serum that 50% above agglutination phenomenon (++) occurs.
The detection reaction condition of described reagent is: 20～25 ℃ of temperature of reaction, reaction time 50min.
Beneficial effect of the present invention: diagnostic reagent of the present invention and preparation method can be used for goatpox antigen on the veterinary clinic fast, a large amount of detection, for the clinical diagnosis of goatpox provides foundation.Compare with agar gel diffusion test commonly used, reverse indirect hemagglutination assay has the advantage that susceptibility is higher, detection speed is faster, specificity is stronger.
Description of drawings
Fig. 1 is the preparation flow figure of goatpox reverse indirect hemagglutination diagnostic reagent of the present invention.
Embodiment
Embodiments of the invention: each component of goatpox reverse indirect hemagglutination diagnostic reagent of the present invention is: phosphate buffer (0.1M, pH 7.4), acetate buffer (0.1M, pH 3～4), glutaraldehyde, tannic acid, sheep red blood cell (SRBC), purifying goatpox IgG, new-born calve serum, NaN
3
As schematically shown in Figure 1, goatpox reverse indirect hemagglutination diagnostic reagent of the present invention the preparation method, may further comprise the steps:
The first, the healthy sheep venous blood of aseptic collection adds 10 times of amount PBS, 2 000 rev/mins centrifugal 15 minutes, wash 5 times, be made into 2.5% red cell suspension with phosphate buffer (0.1M, pH 7.4);
The second, the red cell suspension that step 1 is made mixes with the 9:1 volume ratio with 1% glutaraldehyde, slowly stirs 1h under room temperature, 2000 rev/mins centrifugal 15 minutes, wash 5 times with phosphate buffer, make 2.5% suspending liquid again;
The 3rd, step 2 is made red blood cell suspension mix with equivalent 1/,200 000 fresh tan-ooze, take out after placing 37 ℃ water-bath l hour, with phosphate buffer washing 3 times, 2000 rev/mins are centrifugal 15 minutes;
The 4th, the precipitation red blood cell that step 3 is made adds in 10 times of amount acetate buffers (0.1M, pH 3～4), mixes with equivalent IgG purification dilution again, places 37 ℃ of 2h, therebetween every 15min pressure-vaccum mixing 1 time; Employed purifying goatpox IgG content is 1.549mg/mL;
The 5th, centrifugal 15 minutes of 2000 rev/mins of sensitized erythrocytes that step 4 is made are got the precipitation red blood cell with containing 0.1%NaN
3, 1% newborn calf serum phosphate buffer washing 5 times, be made into 0.8%～1.0% sensitized erythrocyte suspension diagnostic reagent at last;
The 6th, the qualified reverse indirect hemagglutination diagnostic reagent that promptly makes of analyzing and testing;
Goatpox reverse indirect hemagglutination diagnostic reagent detects the method for goat capripoxvirus antigen, detection step reverse indirect hemagglutination assay method is routinely carried out, the steps include: in 96 hole V shaped slabs, to carry out, each sample is one group, every group 1～12 hole adds PBS 50 μ L, adds sample 50 μ L to be checked then in first hole, is diluted to the 11st hole with liquid-transfering gun by the hole, abandon 50 μ L, the 12nd hole is the PBS blank.Last every hole adds goatpox reverse indirect hemagglutination diagnostic reagent 50 μ L, and vibration is put room temperature effect 50min, result of determination after mixing.All red blood cells are deposited at the bottom of the hole, concentrate the not aggegation that is that is 1 round dot; As red cell agglutination, then be distributed at the bottom of the hole around.Judging the power of positive reaction according to the degree of red cell agglutination, serves as tiring of detection antibody with the high dilution of serum that 50% above agglutination phenomenon (++) occurs.
The detection reaction condition of described reagent is: 20～25 ℃ of temperature of reaction, reaction time 50min.
Claims (2)
1. the preparation method of a goatpox reverse indirect hemagglutination diagnostic reagent is characterized in that: may further comprise the steps:
The first, the healthy sheep venous blood of aseptic collection adds 10 times of amount PBS, 2 000 rev/mins centrifugal 15 minutes, wash 5 times, be made into 2.5% red cell suspension with the phosphate buffer of 0.1M--pH 7.4～7.8;
The second, the red cell suspension that step 1 is made mixes with the 9:1 volume ratio with 1% glutaraldehyde, slowly stirs 1h under room temperature, 2000 rev/mins centrifugal 15 minutes, wash 5 times with phosphate buffer, make 2.5% hydroformylation red blood cell suspension again;
The 3rd, the hydroformylation red blood cell suspension that step 2 is made mixes with equivalent 1/,200 000 fresh tan-ooze, takes out after placing 37 ℃ water-bath l hour, with phosphate buffer washing 3 times, 2000 rev/mins centrifugal 15 minutes, make the precipitation red blood cell of hydroformylation, tanning;
The 4th, the precipitation red blood cell that step 3 is made adds in the acetate buffer of 10 times of amount 0.1M--pH 3～4, mixes with equivalent purifying goatpox IgG dilution again, places 37 ℃ of 2h, every 15min pressure-vaccum mixing 1 time, make 0.8%～1% sensitized erythrocyte suspension therebetween; Employed purifying goatpox IgG content is controlled at 1.5～1.55mg/mL;
The 5th, centrifugal 15 minutes of 2000 rev/mins of sensitized erythrocytes that step 4 is made are got the precipitation red blood cell with containing 0.1%NaN
3, 1% newborn calf serum phosphate buffer washing 5 times, be made into concentration 0.8%～1% sensitized erythrocyte suspension diagnostic reagent at last;
The 6th, the qualified reverse indirect hemagglutination diagnostic reagent that promptly gets of analyzing and testing.
2. a goatpox reverse indirect hemagglutination diagnostic reagent detects the method for goat capripoxvirus antigen, it is characterized in that: the goatpox reverse indirect hemagglutination diagnostic reagent of preparation method's preparation of goatpox reverse indirect hemagglutination diagnostic reagent as claimed in claim 1, detection step reverse indirect hemagglutination assay method is routinely carried out, the steps include: in 96 hole V shaped slabs, to carry out, each sample is one group, every group 1～12 hole adds PBS 50 μ L, add sample 50 μ L to be checked then in first hole, be diluted to the 11st hole with liquid-transfering gun by the hole, abandon 50 μ L, the 12nd hole is the PBS blank; Last every hole adds goatpox reverse indirect hemagglutination diagnostic reagent 50 μ L, and vibration is put room temperature effect 50min, result of determination after mixing; All red blood cells are deposited at the bottom of the hole, concentrate the not aggegation that is that is 1 round dot; As red cell agglutination, then be distributed at the bottom of the hole around; Judging the power of positive reaction according to the degree of red cell agglutination, serves as tiring of detection antibody with the high dilution of serum that 50% above agglutination phenomenon occurs;
The detection reaction condition of described reagent is: 20～25 ℃ of temperature of reaction, reaction time 50min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109682977A (en) * | 2018-06-21 | 2019-04-26 | 中国农业科学院兰州兽医研究所 | A kind of Hai Shi enterococcus indirect hemagglutination antibody assay kit and preparation method thereof |
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| CN1432811A (en) * | 2002-01-06 | 2003-07-30 | 安徽安科生物工程股份有限公司 | Sperm antibody testing reagent |
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| CN101712971A (en) * | 2009-11-07 | 2010-05-26 | 中国农业科学院兰州兽医研究所 | Mycoplasma capricolum subsp. pneumonia antigen of goats and preparation method thereof |
| CN102253197A (en) * | 2011-06-21 | 2011-11-23 | 贵州大学 | Goat pox reverse indirect blood coagulation diagnostic reagent, and preparation method, and detection method thereof |
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Patent Citations (5)
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|---|---|---|---|---|
| CN1432811A (en) * | 2002-01-06 | 2003-07-30 | 安徽安科生物工程股份有限公司 | Sperm antibody testing reagent |
| CN1932518A (en) * | 2005-09-16 | 2007-03-21 | 中国农业科学院兰州兽医研究所 | Toxoplasma indirect hemagg lutination diagnostic reagent and producing process thereof |
| CN101423548A (en) * | 2008-12-12 | 2009-05-06 | 陶海静 | Preparation of duck hepatitis I type virus indirect hemagglutination diagnostic antigen and kit |
| CN101712971A (en) * | 2009-11-07 | 2010-05-26 | 中国农业科学院兰州兽医研究所 | Mycoplasma capricolum subsp. pneumonia antigen of goats and preparation method thereof |
| CN102253197A (en) * | 2011-06-21 | 2011-11-23 | 贵州大学 | Goat pox reverse indirect blood coagulation diagnostic reagent, and preparation method, and detection method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109682977A (en) * | 2018-06-21 | 2019-04-26 | 中国农业科学院兰州兽医研究所 | A kind of Hai Shi enterococcus indirect hemagglutination antibody assay kit and preparation method thereof |
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Application publication date: 20111221 |