CN102313803A - Goat pox indirect hemagglutination diagnosis reagent as well as preparation method and detecting method thereof - Google Patents
Goat pox indirect hemagglutination diagnosis reagent as well as preparation method and detecting method thereof Download PDFInfo
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- CN102313803A CN102313803A CN201110166920A CN201110166920A CN102313803A CN 102313803 A CN102313803 A CN 102313803A CN 201110166920 A CN201110166920 A CN 201110166920A CN 201110166920 A CN201110166920 A CN 201110166920A CN 102313803 A CN102313803 A CN 102313803A
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Abstract
The invention discloses a goat pox indirect hemagglutination diagnosis reagent as well as a preparation method and a detecting method thereof. The hemagglutination diagnosis reagent comprises the following components by weight percent: 0.6-1.2% of goat pox virus antigen sensitized red blood cells, and 20-200 mu g/ml of purified goat pox virus antigen. Compared with the commonly used agar diffusion experiment, the goat pox indirect hemagglutination diagnosis reagent is prepared from purified goat pox virus antigen sensitized hydroformylated sheep red blood cells in the invention, thus, the goat pox virus antibody can be detected fast, accurately and simply, the result can be determined in 50 minutes, and the antibody with low valence can be detected; moreover, the goat pox indirect hemagglutination diagnosis reagent has good specificity, sensitivity and repeatability, and can be widely used for fast and substantive detection of goat pox virus antibodies in clinical vet, and can provide the basis for prevention, diagnosis and prevention and control of a disease.
Description
Technical field
The present invention relates to forward hemagg lutination diagnostic reagent technical field, particularly relate to a kind of goatpox forward indirect hemagg lutination 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 country one type of 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; A kind of goatpox forward indirect hemagg lutination diagnostic reagent and preparation method thereof is provided, the detection goat capripoxvirus antibody 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:
The erythrocytic content of goat capripoxvirus antigen sensibilization is 0.6～1.2% (volume) in the goatpox forward indirect hemagg lutination diagnostic reagent of the present invention, and purifying goat capripoxvirus antigenic content is 20～200 μ g/ml.
Preferably, the erythrocytic content of goat capripoxvirus antigen sensibilization is 0.8～1% in the above-mentioned hemagg lutination diagnostic reagent, and purifying goat capripoxvirus antigenic content is 100 μ g/ml.
Sensitized erythrocyte in the aforementioned goatpox forward indirect hemagg lutination diagnostic reagent is the hydroformylation sheep red blood cell (SRBC) with purifying goat capripoxvirus antigen sensibilization.
The present invention also provides a kind of method for preparing aforementioned goatpox forward indirect hemagg lutination diagnostic reagent: gather healthy sheep venous blood; Be prepared into red cell suspension; Carry out hydroformylation with glutaraldehyde, with purifying goat capripoxvirus antigen sensibilization, centrifuging; Get the deposition red blood cell and be mixed with red cell suspension, after the blood clotting detection is qualified, promptly get.
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; Use the glutaraldehyde hydroformylation, centrifuging gets the hydroformylation red blood cell, with purifying goat capripoxvirus antigen sensibilization; Centrifuging is got the deposition red blood cell with containing NaN
3Phosphate buffer washing with new-born calve serum is mixed with red cell suspension with this damping fluid again, after the blood clotting detection is qualified, promptly gets.
Best, aforementioned preparation method is: gather healthy sheep venous blood, extract red blood cell with the PBS centrifuging; Be prepared into the red cell suspension of 2.5% (volume) again with the phosphate buffer of 0.1M, pH7.4; With centrifuging after the glutaraldehyde hydroformylation, with washing of the phosphate buffer of 0.1M, pH7.4 the hydroformylation red blood cell, the hydroformylation red blood cell is dissolved in the phosphate buffer of 0.1M that 10 volumes doubly measure, pH 7.4; Again with equal volume amounts purifying goat capripoxvirus mixed antigen; Stir 2h sensitization down in room temperature condition, centrifuging is got the deposition red blood cell with containing 0.1% (quality) NaN
30.1M, the washing of pH7.4 phosphate buffer with 1% (volume) new-born calve serum are mixed with red cell suspension with this damping fluid again, after the blood clotting detection is qualified, promptly get.
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 forward indirect hemagg lutination diagnostic reagent with purifying goat capripoxvirus antigen sensibilization hydroformylation sheep red blood cell (SRBC); Detection goat capripoxvirus antibody that can be quick, accurate, easy; In 50 minutes, get final product result of determination, and can detect antibody, have specificity, susceptibility and repeatability preferably than low liter; Can be widely used in quick, a large amount of detections of goat capripoxvirus antibody on the veterinary clinic, 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 adds 10 volumes times PBS, and 2000 rev/mins centrifugal 15 minutes; Wash 5 times with PBS, be made into the red cell suspension of 2.5% (volume) with phosphate buffer (0.1M, pH 7.4); Then this red cell suspension and 1% (volume) glutaraldehyde water solution is mixed 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, get hydroformylation deposition red blood cell.
2, sensitization: hydroformylation is precipitated red blood cell is dissolved in the 10 volumes times phosphate buffer (0.1M, pH 7.4), with equal volume amounts purifying goat capripoxvirus mixed antigen, under room temperature, stir 2h again, 2000 rev/mins centrifugal 15 minutes, must precipitate sensitized erythrocyte.
3, get the deposition sensitized erythrocyte with the phosphate buffer (0.1M that contains 0.1% (weight) NaN3,1% (volume) new-born calve serum; PH 7.4) wash 5 times, be made into the red cell suspension of sensitized erythrocyte concentration 1% (volume), viral antigen content 100 μ g/ml then.
4, detect by conventional reverse indirect hemagglutination assay methods analyst and promptly make the reverse indirect hemagglutination diagnostic reagent after qualified.
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 (7)
1. goatpox forward indirect hemagg lutination diagnostic reagent, it is characterized in that: the erythrocytic content of goat capripoxvirus antigen sensibilization is 0.6～1.2% (volume) in the hemagg lutination diagnostic reagent, purifying goat capripoxvirus antigenic content is 20～200 μ g/ml.
2. according to the said goatpox forward of claim 1 indirect hemagg lutination diagnostic reagent, it is characterized in that: the erythrocytic content of goat capripoxvirus antigen sensibilization is 0.8～1% in the hemagg lutination diagnostic reagent, and purifying goat capripoxvirus antigenic content is 100 μ g/ml.
3. according to claim 1 or 2 said goatpox forward indirect hemagg lutination diagnostic reagents, it is characterized in that: said sensitized erythrocyte is the hydroformylation sheep red blood cell (SRBC) with purifying goat capripoxvirus antigen sensibilization.
4. according to the preparation method of each said goatpox forward indirect hemagg lutination diagnostic reagent of claim 1 to 3; It is characterized in that: gather healthy sheep venous blood, be prepared into red cell suspension, carry out hydroformylation with glutaraldehyde; With purifying goat capripoxvirus antigen sensibilization; Centrifuging is got the deposition red blood cell and is mixed with red cell suspension, after the blood clotting detection is qualified, promptly gets.
5. according to the preparation method of the said goatpox forward of claim 4 indirect hemagg lutination 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; Use the glutaraldehyde hydroformylation, centrifuging gets the hydroformylation red blood cell, with purifying goat capripoxvirus antigen sensibilization; Centrifuging is got the deposition red blood cell with containing NaN
3Phosphate buffer washing with new-born calve serum is mixed with red cell suspension with this damping fluid again, after the blood clotting detection is qualified, promptly gets.
6. according to the preparation method of the said goatpox forward of claim 5 indirect hemagg lutination diagnostic reagent, it is characterized in that: gather healthy sheep venous blood, extract red blood cell with the PBS centrifuging; Be prepared into the red cell suspension of 2.5% (volume) again with the phosphate buffer of 0.1M, pH7.4; With centrifuging after the glutaraldehyde hydroformylation, with washing of the phosphate buffer of 0.1M, pH7.4 the hydroformylation red blood cell, the hydroformylation red blood cell is dissolved in the phosphate buffer of 0.1M that 10 volumes doubly measure, pH 7.4; Again with equal volume amounts purifying goat capripoxvirus mixed antigen; Stir 2h sensitization down in room temperature condition, centrifuging is got the deposition red blood cell with containing 0.1% (quality) NaN
30.1M, the washing of pH7.4 phosphate buffer with 1% (volume) new-born calve serum are mixed with red cell suspension with this damping fluid again, after the blood clotting detection is qualified, promptly get.
7. the arbitrary said diagnostic reagent of claim 1 to 6 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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| Application Number | Priority Date | Filing Date | Title |
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| CN201110166920A CN102313803A (en) | 2011-06-21 | 2011-06-21 | Goat pox indirect hemagglutination diagnosis reagent as well as preparation method and detecting method thereof |
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| CN201110166920A CN102313803A (en) | 2011-06-21 | 2011-06-21 | Goat pox indirect hemagglutination diagnosis reagent as well as preparation method and detecting method thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101460627A (en) * | 2006-06-20 | 2009-06-17 | 特朗斯吉有限公司 | Process for producing poxviruses and poxvirus compositions |
| WO2011003194A1 (en) * | 2009-07-10 | 2011-01-13 | The Governors Of The University Of Alberta | Oncolytic viruses and methods for treating neoplastic disorders |
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2011
- 2011-06-21 CN CN201110166920A patent/CN102313803A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101460627A (en) * | 2006-06-20 | 2009-06-17 | 特朗斯吉有限公司 | Process for producing poxviruses and poxvirus compositions |
| WO2011003194A1 (en) * | 2009-07-10 | 2011-01-13 | The Governors Of The University Of Alberta | Oncolytic viruses and methods for treating neoplastic disorders |
Non-Patent Citations (1)
| Title |
|---|
| 刘忠伟: "山羊痘病毒的血清学检测与PCR鉴定", 《中国优秀博硕士学位论文数据库（硕士）农业科技辑》, no. 11, 15 November 2006 (2006-11-15) * |
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Application publication date: 20120111 |