CN1042940A - Sick horse of equine infectious anemia and the serum antibody differential diagnosis reagent of inoculating the attenuated vaccine horse - Google Patents
Sick horse of equine infectious anemia and the serum antibody differential diagnosis reagent of inoculating the attenuated vaccine horse Download PDFInfo
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- CN1042940A CN1042940A CN 88108085 CN88108085A CN1042940A CN 1042940 A CN1042940 A CN 1042940A CN 88108085 CN88108085 CN 88108085 CN 88108085 A CN88108085 A CN 88108085A CN 1042940 A CN1042940 A CN 1042940A
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- equine infectious
- infectious anemia
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Abstract
The present invention is sick horse of a kind of equine infectious anemia and the serum antibody appreciation diagnostic reagent of inoculating the attenuated vaccine horse, this reagent is to utilize the hybridoma technology, obtained to secrete anti-equine infectious anemia virus donkey-leukocyte low virulent strain and be the hybridoma cell strain of special monoclonal antibody (McAb), again monoclonal antibody (McAb) has been prepared and purifying.Adopt the sodium periodate oxidation style, prepared enzyme receipts or other documents in duplicate clonal antibody (McAb) reagent, come sick horse of differential diagnosis equine infectious anemia and inoculation attenuated vaccine horse with immunodotting (DB) test method(s).
Description
The present invention is a kind of medicinal preparation that contains not clear structural antigens or antibody, is used for the differential diagnosis reagent of the serum antibody of sick horse of equine infectious anemia and inoculation attenuated vaccine horse.
Equine infectious anemia (abbreviation equine infectious anemia) is one of formidable enemy of various countries' horse keeping industry, is the transmissible disease of the extensive horse that exists in the world.In order to prevent this disease, China's initiative equine infectious anemia virus donkey-leukocyte attenuated vaccine is used for the horse immune prevention.But suffer from these sick horses and all serological reaction can occur with the horses that inoculated attenuated vaccine.As checking that according to the existing immunodiffusion method in various countries, neutralization test method, complement combined techniques then both can't distinguish, and diagnostic work can't be carried out.
The objective of the invention is to adopt the hybridoma technology in the Modern High-Tech, develop is special monoclonal antibody (McAb) to the equine infectious anemia virus donkey-leukocyte low virulent strain only, and make enzyme connection McAb reagent, with sick horse of immunodotting (DB) test method(s) differential diagnosis equine infectious anemia and inoculation attenuated vaccine horses.
Describe the manufacture method of the serum antibody differential diagnosis reagent of sick horse of equine infectious anemia and inoculation attenuated vaccine horse below in detail.
One, the foundation of hybridoma cell strain
With malicious immune BALB/C mouse a little less than the equine infectious anemia virus donkey-leukocyte,, adopt the hybridoma fusion method to carry out cytogamy with the splenocyte and the SP2/0 myeloma cell of immunized mice.To hybridoma growth hole supernatant liquor, the detection of secreting McAb with ELISA and IF method, cell to the positive hole of culture supernatant antibody test, make three time cloning cultivation and propagation with 96 porocyte culture plates with limiting dilution assay again, change the cultivation of going down to posterity in the Tissue Culture Flask then over to, and 60 generations of cultured continuously, obtained to secrete anti-equine infectious anemia virus donkey-leukocyte low virulent strain the hybridoma cell strain that is special McAb with this.
Two, the preparation of McAb and purifying
1, the preparation of McAb
Get hybridoma (1 * 10
7/ ml) 0.5ml is inoculated into the intraperitoneal of the BALB/C mouse of abdominal injection 0.5ml depreciation alkane in advance, through 10-14 days, after the belly of BALB/C mouse expanded, aseptic extraction ascites inoculated the abdominal cavity of s-generation BALB/C mouse, so after BALB/C mouse abdominal cavity passes the three generations, large quantities of inoculation the 4th generation mouse, aseptic collection ascites, centrifugal through 200g centrifugal force, after 10 minutes, it is standby to collect supernatant liquor.
2, the purifying of McAb
(1) ammonium sulfate precipitation method: with acquisition ascites with 50% saturated ammonium sulphate repeated precipitation 3 times, with the PBS dialysis of PH7.2,0.02M, till not having an ammonium ion.
(2) column chromatography: with Sephadex G200 gel-filtration, collect first peak, concentrate, deposit usefulness under the freeze-drying postposition-20 ℃ condition with sucrose.
Three, the preparation of enzyme connection McAb reagent: adopt the periodate oxidation method
1, take by weighing horseradish peroxidase 5mg and be dissolved in 1ml, the 0.3M sodium hydrogen carbonate solution, add 0.1ml, 1% dinitrofluorobenzene ethanol solution under the induction stirring, the room temperature lucifuge stirred 1 hour.
2, add 1ml, 0.06M sodium periodate solution, room temperature lucifuge induction stirring 30 minutes.
3, add 1ml, 0.16M ethylene glycol solution, room temperature lucifuge induction stirring 1 hour.
4, to 1000ml, the dialysis of 0.01M PH9.5 carbonate buffer solution, 4 ℃ are spent the night, and liquid is changed three times in the centre.
5, adding 5mg McAb(and be diluted to 1ml with 0.1M PH9.5 carbonate buffer solution) the room temperature lucifuge stirred 3 hours.
6, add 4 ℃ of effects of 5mg sodium borohydride 3 hours.
7, dress dialysis tubing with the PBS dialysis of 0.02M PH7.4, was placed 24 hours down for 4 ℃, and liquid is changed 2-3 time in the centre.
8, dropwise adding equal-volume 100% saturated ammonium sulphate under the induction stirring left standstill 1 hour for 4 ℃, centrifugal 15 minutes of 400G centrifugal force, abandon supernatant liquor, throw out is washed secondary with 50% saturated ammonium sulphate, centrifugal by preceding method, precipitation is dissolved among the 0.02M PH7.4 PBS of a small amount of sterilization, to 0.02M PH7.4 PBS dialysed overnight, changes liquid for several times till do not have an ammonium ion under 4 ℃.Survey OD 403nm and OD 280nm with ultraviolet spectrophotometer.
9, calculate McAb amount, enzyme amount, the mol ratio of enzymic-labelled antibody.
10, enzymic-labelled antibody is diluted to finite concentration (200 μ g/ml) packing peace bottle ,-20 ℃ of cryopreservation with the 0.02M PH7.4 PBS that sterilizes.
Four, using method: adopt the immunodot test method.
Use nitrocellulose filter or cellulose mixture adipose membrane and make solid phase carrier.
Operation steps:
(1) point sample: with the positive, negative control and examined serum and be stained with about 2.5ml with microsyringe or glass stick, point on the specified location of film, drying at room temperature 15 minutes.
(2) with containing the 0.02M PBS(PH7.2 of 0.05% Tween-20) rinsing three times, each 3-5 minute.
(3) film is immersed in antigen-enzyme connection McAb reagent solution following 1 hour of room temperature, antigen-enzyme connection McAb reagent (2) solution dilution.
(4) repeat (2) item
(5) film is immersed substrate solution (the 0.05M Tris-HCl 20ml of PH7.6; DAB 3mg; 30%H
2O
260ml) 30 minutes.
(6) use the distilled water rinsing.Termination reaction, sentence read result, it is positive to show reddish-brown, i.e. equine infectious anemia virus donkey-leukocyte attenuated vaccine immunity horse.So just distinguish the sick horse of equine infectious anemia and inoculated equine infectious anemia virus donkey-leukocyte attenuated vaccine horse.
With sick horse of reagent differential diagnosis equine infectious anemia of the present invention and the horse that inoculated attenuated vaccine, the result is accurate, and method is simple.
Claims (1)
1, sick horse of a kind of equine infectious anemia and the differential diagnosis reagent of inoculating the attenuated vaccine horse, it is characterized in that, adopt the hybridoma technology, that develops is special monoclonal antibody (McAb) to equine infectious anemia donkey horse white corpuscle low virulent strain only and makes enzyme receipts or other documents in duplicate clonal antibody (McAb) reagent, with the sick horse of immunodotting (DB) test method(s) differential diagnosis equine infectious anemia with inoculated donkey leukocyte attenuated vaccine horse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 88108085 CN1042940A (en) | 1988-12-01 | 1988-12-01 | Sick horse of equine infectious anemia and the serum antibody differential diagnosis reagent of inoculating the attenuated vaccine horse |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 88108085 CN1042940A (en) | 1988-12-01 | 1988-12-01 | Sick horse of equine infectious anemia and the serum antibody differential diagnosis reagent of inoculating the attenuated vaccine horse |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1042940A true CN1042940A (en) | 1990-06-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 88108085 Pending CN1042940A (en) | 1988-12-01 | 1988-12-01 | Sick horse of equine infectious anemia and the serum antibody differential diagnosis reagent of inoculating the attenuated vaccine horse |
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| CN (1) | CN1042940A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059701C (en) * | 1996-04-03 | 2000-12-20 | 中国农业科学院哈尔滨兽医研究所 | Donkey leukocyte hypotoxic strain for equine infection anemia and its culture method |
| CN101044242B (en) * | 2004-06-10 | 2013-10-30 | 维文蒂阿生物技术股份有限公司 | Tumor specific antibody |
| CN113325178A (en) * | 2021-05-27 | 2021-08-31 | 江苏省肿瘤医院 | Detection kit for early diagnosis and prognosis evaluation of ovarian cancer |
-
1988
- 1988-12-01 CN CN 88108085 patent/CN1042940A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059701C (en) * | 1996-04-03 | 2000-12-20 | 中国农业科学院哈尔滨兽医研究所 | Donkey leukocyte hypotoxic strain for equine infection anemia and its culture method |
| CN101044242B (en) * | 2004-06-10 | 2013-10-30 | 维文蒂阿生物技术股份有限公司 | Tumor specific antibody |
| CN113325178A (en) * | 2021-05-27 | 2021-08-31 | 江苏省肿瘤医院 | Detection kit for early diagnosis and prognosis evaluation of ovarian cancer |
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