CN102253197A - Goat pox reverse indirect blood coagulation diagnostic reagent, and preparation method, and detection method thereof - Google Patents
Goat pox reverse indirect blood coagulation diagnostic reagent, and preparation method, and detection method thereof Download PDFInfo
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- CN102253197A CN102253197A CN2011101669813A CN201110166981A CN102253197A CN 102253197 A CN102253197 A CN 102253197A CN 2011101669813 A CN2011101669813 A CN 2011101669813A CN 201110166981 A CN201110166981 A CN 201110166981A CN 102253197 A CN102253197 A CN 102253197A
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Abstract
The invention discloses a goat pox reverse indirect blood coagulation diagnostic reagent and a preparation method thereof. The content of sensitized erythrocyte in the blood coagulation reagent is 0.8 to 1.0 percent; and the purified goat pox IgG content is 1.549mg/ml. Compared with the conventional common agar diffusion experiment, the preparation method has the advantages that: the goat pox reverse indirect blood coagulation diagnostic reagent is prepared by sensitizing hydroformylation sheep red blood cells by using the purified goat pox IgG, so that a goat poxvirus antibody can be detected quickly, accurately and simply; and the goat pox reverse indirect blood coagulation diagnostic reagent has higher specificity, sensitivity and repeatability and can be widely applied to quick veterinary clinical detection of the goat poxvirus antibody and provide a basis for disease prevention, diagnosis and control.
Description
Technical field
The present invention relates to reverse blood clotting diagnostic reagent technical field, particularly relate to a kind of goatpox reverse indirect hemagglutination diagnostic reagent and preparation method thereof and detection method.
Background technology
Goatpox be by the Capripoxvirus goat capripoxvirus (goat poxvirus, what GTPV) cause a kind ofly is endemy height contagious disease, 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 great economic loss, has brought severe threat.The method of traditional detection goatpox can adopt agar diffusion method, but the sensitivity of this method is not high, detection speed is slower.
Summary of the invention
Technical matters to be solved by this invention is to overcome the existing defective that sensitivity is not high, detection speed is slower that detects the method for goatpox disease, provide a kind of goatpox reverse indirect hemagglutination diagnostic reagent and preparation method thereof, the detection goat capripoxvirus antigen that this reagent can be responsive, quick, accurate, easy.
In order to solve the problems of the technologies described above, the present invention adopts following technical scheme:
In the goatpox reverse indirect hemagglutination diagnostic reagent of the present invention goatpox IgG to cause the content of sensitized erythrocyte be 0.8～1%(volume), purifying goatpox IgG content is 1.549mg/ml.
Sensitized erythrocyte in the above-mentioned goatpox reverse indirect hemagglutination diagnostic reagent is the hydroformylation sheep red blood cell (SRBC) with purifying goatpox IgG sensitization.
The present invention also provides a kind of method for preparing aforementioned goatpox reverse indirect hemagglutination diagnostic reagent: gather healthy sheep venous blood, be prepared into red cell suspension, carry out hydroformylation with glutaraldehyde and tannic acid, in acetate buffer, use purifying goatpox IgG sensitization, centrifuging, get the precipitation red blood cell and be mixed with red cell suspension, through blood clotting detect qualified after promptly.
Further, said method is: gather healthy sheep venous blood, extract red blood cell with the PBS centrifuging, be prepared into red cell suspension with phosphate buffer again, with being mixed with red cell suspension with phosphate buffer again after the glutaraldehyde hydroformylation, and then use the tannic acid hydroformylation, centrifuging gets the hydroformylation red blood cell, the hydroformylation red blood cell is used purifying goatpox IgG sensitization in acetate buffer, centrifuging is got the precipitation red blood cell with containing NaN
3With the washing of the phosphate buffer of newborn calf serum, be mixed with red cell suspension with this damping fluid again, after the blood clotting detection is qualified promptly.
Best, aforementioned preparation method is: gather healthy sheep venous blood, extract red blood cell with the PBS centrifuging, use 0.1M again, it is 2.5% red cell suspension that the phosphate buffer of pH7.4 is prepared into percent by volume, with using 0.1M after the glutaraldehyde hydroformylation again, it is 2.5% red cell suspension that the phosphate buffer of pH7.4 is mixed with percent by volume, and then with fresh tannic acid in 37 ℃ water-bath hydroformylation l hour, use 0.1M, the washing of the phosphate buffer of pH7.4, centrifuging gets the hydroformylation red blood cell, the hydroformylation red blood cell is added the 0.1M that 10 volumes are doubly measured, in the acetate buffer of pH 3～4, mix with equal volume amounts purifying goatpox IgG dilution again, place 37 ℃ of senses to make 2h sensitization, centrifuging is got the precipitation red blood cell with containing 0.1%(weight) NaN
3With the 1%(volume) 0.1M of newborn calf serum, the washing of pH7.4 phosphate buffer, be mixed with red cell suspension with this damping fluid again, after the blood clotting detection is qualified promptly.
Adopt aforementioned diagnostic reagent to detect the method for goatpox: during detection, the pH value is controlled at 7.4～7.8,20～25 ℃ of temperature of reaction, reaction time 50min.
Compare with existing agar diffusion experiment commonly used, the present invention has prepared goatpox reverse indirect hemagglutination diagnostic reagent with purifying goatpox IgG sensitization hydroformylation sheep red blood cell (SRBC), detection goat capripoxvirus antigen that can be quick, accurate, easy, have specificity, susceptibility and repeatability preferably, the fast detecting that can be widely used in goat capripoxvirus antigen on the veterinary clinic is for prevention from suffering from the diseases, diagnosis and prevention and control provide foundation.
Embodiment
Goatpox reverse indirect hemagglutination diagnostic reagent is by following proportioning and method preparation
1, preparation hydroformylation red blood cell
The healthy sheep venous blood of aseptic collection, add 10 volumes times PBS, 2000 rev/mins centrifugal 15 minutes, wash 5 times with PBS, with phosphate buffer (0.1M, pH 7.4) be made into the 2.5%(percent by volume) red cell suspension, then with this red cell suspension and 1%(percent by volume) glutaraldehyde water solution mixes with the volume ratio of 9:1, under room temperature, slowly stir 1h, 2000 rev/mins centrifugal 15 minutes, wash 5 times with phosphate buffer, again make 2.5% suspending liquid, then with this suspending liquid and equal volume amounts 1/200000(volume ratio) fresh tan-ooze mixes, and put in 37 ℃ the water-bath and take out after l hour, with phosphate buffer washing 3 times, 2000 rev/mins centrifugal 15 minutes hydroformylation precipitation red blood cell.
2, sensitization: hydroformylation is precipitated red blood cell add 10 volumes and doubly measure acetate buffer (0.1M, pH 3～4) in, mix with equal volume amounts purifying goatpox IgG dilution (1.549mg/ml) again, place 37 ℃ of senses to make 2h, therebetween every 15min pressure-vaccum mixing 1 time, 2000 rev/mins centrifugal 15 minutes, must precipitate sensitized erythrocyte.
3, get the precipitation sensitized erythrocyte with containing 0.1%(weight) NaN
3, the 1%(volume) phosphate buffer (0.1M, the pH 7.4) washing 5 times of newborn calf serum, be made into sensitized erythrocyte concentration 1%(volume then), the red cell suspension of IgG content 1.549mg/ml.
4, the reverse indirect hemagglutination assay methods analyst detects and promptly to make the reverse indirect hemagglutination diagnostic reagent after qualified routinely.
During detection, the pH value is controlled at 7.4～7.8,20～25 ℃ of temperature of reaction, and reaction time 50min is an optimum reaction condition.
Claims (6)
1. goatpox reverse indirect hemagglutination diagnostic reagent, it is characterized in that: the content of goatpox IgG sensitized erythrocyte is 0.8～1% in the hemagg lutination diagnostic reagent, purifying goatpox IgG content is 1.549mg/ml.
2. according to the described goatpox reverse indirect hemagglutination of claim 1 diagnostic reagent, it is characterized in that: described sensitized erythrocyte is the hydroformylation sheep red blood cell (SRBC) with purifying goatpox IgG sensitization.
3. according to the preparation method of claim 1 or 2 described goatpox reverse indirect hemagglutination diagnostic reagents, it is characterized in that: gather healthy sheep venous blood, be prepared into red cell suspension, carry out hydroformylation with glutaraldehyde and tannic acid, in acetate buffer, use purifying goatpox IgG sensitization, centrifuging is got the precipitation red blood cell and is mixed with red cell suspension, through blood clotting detect qualified after promptly.
4. according to the preparation method of the described goatpox reverse indirect hemagglutination of claim 3 diagnostic reagent, it is characterized in that: gather healthy sheep venous blood, extract red blood cell with the PBS centrifuging, be prepared into red cell suspension with phosphate buffer again, with being mixed with red cell suspension with phosphate buffer again after the glutaraldehyde hydroformylation, and then use the tannic acid hydroformylation, centrifuging gets the hydroformylation red blood cell, the hydroformylation red blood cell is used purifying goatpox IgG sensitization in acetate buffer, centrifuging is got the precipitation red blood cell with containing NaN
3With the washing of the phosphate buffer of newborn calf serum, be mixed with red cell suspension with this damping fluid again, after the blood clotting detection is qualified promptly.
5. according to the preparation method of the described goatpox reverse indirect hemagglutination of claim 4 diagnostic reagent, it is characterized in that: gather healthy sheep venous blood, extract red blood cell with the PBS centrifuging, use 0.1M again, it is 2.5% red cell suspension that the phosphate buffer of pH7.4 is prepared into percent by volume, with using 0.1M after the glutaraldehyde hydroformylation again, it is 2.5% red cell suspension that the phosphate buffer of pH7.4 is mixed with percent by volume, and then with fresh tannic acid in 37 ℃ water-bath hydroformylation l hour, use 0.1M, the washing of the phosphate buffer of pH7.4, centrifuging gets the hydroformylation red blood cell, the hydroformylation red blood cell is added the 0.1M that 10 volumes are doubly measured, in the acetate buffer of pH 3～4, mix with equal volume amounts purifying goatpox IgG dilution again, place 37 ℃ of senses to make 2h sensitization, centrifuging is got the precipitation red blood cell with containing 0.1%(weight) NaN
3With the 1%(volume) 0.1M of newborn calf serum, the washing of pH7.4 phosphate buffer, be mixed with red cell suspension with this damping fluid again, after the blood clotting detection is qualified promptly.
6. the arbitrary described diagnostic reagent of claim 1 to 5 detects the method for goatpox, and it is characterized in that: during detection, the pH value is controlled at 7.4～7.8,20～25 ℃ of temperature of reaction, reaction time 50min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102288756A (en) * | 2011-07-07 | 2011-12-21 | 贵州大学 | Preparation method and detection method of goat pox reverse indirect hemagglutination diagnostic reagent |
| CN108957000A (en) * | 2018-06-21 | 2018-12-07 | 中国农业科学院兰州兽医研究所 | A kind of staphylococcus saprophyticus indirect hemagglutination antibody assay kit and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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2011
- 2011-06-21 CN CN2011101669813A patent/CN102253197A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
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| 周碧君 等: "山羊痘血清学诊断技术研究", 《中国预防兽医学报》, vol. 28, no. 4, 31 July 2006 (2006-07-31), pages 423 - 426 * |
| 王开功 等: "应用反向间接血凝试验检测山羊痘病毒抗原", 《山地农业生物学报》, vol. 24, no. 01, 28 February 2005 (2005-02-28), pages 29 - 32 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102288756A (en) * | 2011-07-07 | 2011-12-21 | 贵州大学 | Preparation method and detection method of goat pox reverse indirect hemagglutination diagnostic reagent |
| CN108957000A (en) * | 2018-06-21 | 2018-12-07 | 中国农业科学院兰州兽医研究所 | A kind of staphylococcus saprophyticus indirect hemagglutination antibody assay kit and preparation method thereof |
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Application publication date: 20111123 |