CN104987417A - Method for producing anti-human C-reaction protein antiserum for sheep - Google Patents
Method for producing anti-human C-reaction protein antiserum for sheep Download PDFInfo
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Abstract
The invention provides a method for producing anti-human C-reaction protein antiserum for sheep. The method comprises the following steps of: (1) preparing an antigen; (2) performing injection for the first time: using the antigen emulsion to inject the sheep, wherein injection parts are soles of feet, inguinal regions, armpits, necks, backs and subcutaneous multipoint injection; (3) performing injection for the second time; (4) performing injection for the third time; (5) performing injection for the fourth time; (6) performing injection for the fifth time; and (7) detecting serum titer: drawing blood from the vein of the sheep after 8-10 days from the injection each time, separating the serum and detecting dual-extending titer. Compared with the prior art, the method for producing the anti-human C-reaction protein antiserum for the sheep, disclosed by the invention, is low in cost, and simple in process; the method not only can stably produce the antibodies, but also is high in antibody producing speed; the antibody level is high, and the dosage of the antigen is small.
Description
Technical field
The present invention relates to a kind of sero-fast method of production goat-anti hCRP, belong to antiserum prepare technical field.
Background technology
C reactive protein (C-reactive protein; CRP) refer to body be infected or tissue injury time blood plasma in some protein sharply risen; CRP can activating complement and strengthen cytophagous phagocytic function; thus remove the histocyte of the invasion pathogenic micro-organism of body and damage, necrosis, apoptosis, in the natural immunity process of body, play important provide protection.Its molecular weight is 105KD.
CRP is first and is considered to Acute reaction protein, content denier under normal circumstances, and at acute injury with when infecting, it sharply raises at Plasma, and CRP is acute-phase response index the most frequently used clinically.In blood plasma, CRP concentration increases rapidly significantly when acute myocardial infarction, wound, infection, inflammation, surgical operation, cancer cell infiltration, can reach 2000 times of normal level.Monitoring plasma C RP concentration, and in conjunction with clinical medical history, contribute to the course of disease and follow up a case by regular visits to, especially in inflammatory process, follow up a case by regular visits to rheumatosis, systemic lupus erythematous, leukemia etc.
After Human C-reactiveprotein antigen and adjuvant emulsion, by certain program immunity animal, anti-human CRP antibody can be produced in animal serum.Although current production method can produce a certain amount of antibody, it is slow that antibody produces speed, and level is lower, and antigen consumption is high.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, the invention provides a kind of sero-fast method of production goat-anti hCRP.
Technical scheme: for achieving the above object, the invention provides a kind of sero-fast method of production goat-anti hCRP, comprises the following steps:
(1) antigen prepares: select hCRP antigen, carries out emulsification obtain antigen emulsion before injection with freund's adjuvant;
(2) first time injection: use above-mentioned antigen emulsion to inject sheep, injection site is vola, inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 10-16 point;
(3) second time injection: first time injects and carries out second time injection for latter 20 days, injection site is that lymphoglandula is interior, inguinal region, armpit, neck, dorsal sc multi-point injection, and each position 1-2 point, amounts to 10-16 point;
(4) third time injection: carry out third time injection after second time injection for 9-11 days, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 8-16 point;
(5) the 4th injections: carry out the 4th injection after third time injection for 9-11 days, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 8-16 point;
(6) the 5th injections: carry out the 5th injection after the 4th injection for 9-11 days, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 8-16 point;
(7) serum titer detects: blood sampling in 8-10 days after per injection, separation of serum, detects its pair of expansion and tire.
As preferably, before described first time injection, the freund's adjuvant of emulsification is complete Freund's adjuvant, and for the second time, the freund's adjuvant of emulsification is incomplete Freund's adjuvant for the third time, before the 4th time and the 5th injection.
Preferred as another kind, the dosage of described first time injection is that every sheep amounts to 0.5mg antigen, each injection amount evenly distribute.
Preferred as another kind, the dosage of described second time injection is that every sheep amounts to 1.0mg antigen, each injection amount evenly distribute.
Preferred as another kind, the dosage of described third time, the 4th time and the 5th time injection is every sheep and amounts to 2.0mg antigen, each injection amount evenly distribute.
Preferred as another kind, the method that described serum titer detects is two expansion immunizations.
Preferred as another kind, described sheep is goat or sheep.
In the present invention, complete Freund's adjuvant is containing the cell wall constituent of mycobacterium tuberculosis, can stimulate division and the they produce antibodies of primed lymphocyte, cause the enlargement of lymphoglandula, strengthen the antibody response to antigen.
In addition, the present invention, by specific antigen dosage, immunization time, injection site and frequency injection, carries out immunity to sheep, finally obtains higher antibody horizontal.Emphasis of the present invention is that the antigen dose of every sheep injection in beginning three immunity is multiplied, with enhancement antigen gradually to the stimulation of body.
Beneficial effect: relative to prior art, the present invention produces the sero-fast method of goat-anti hCRP, and cost is low, and process is simple, can not only stably manufactured antibody, and it is fast to produce antibody speed, and antibody horizontal is high, and antigen consumption is little.
Embodiment
Below in conjunction with embodiment, the present invention is further described.
Embodiment 1
Select 5 adult healthy goats, carry out the following step:
(1) antigen prepares: select hCRP antigen, carries out emulsification obtain antigen emulsion before injection with freund's adjuvant;
(2) first time injection: get 2.5mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml complete Freund's adjuvant again and make antigen emulsion, this antigen emulsion is used to inject sheep, injection site is vola, inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 10-16 point, every sheep injected dose is that 2ml contains 0.5mg antigen, each injection amount evenly distribute;
(3) second time injection: first time injects and carries out second time injection for latter 20 days, get 5.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is that lymphoglandula is interior, inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 10-16 point, every sheep injected dose is that 2ml contains 1.0mg antigen, each injection amount evenly distribute;
(4) third time injection: second time is injected and carried out third time injection for latter 9 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(5) the 4th injections: third time injects and carries out the 4th injection for latter 9 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(6) the 5th injections: the 5th injection is carried out in the 4th injection for latter 9 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(7) serum titer detects: blood sampling in 8 days after per injection, separation of serum, detects its pair of expansion and tire.
Embodiment 2
Select 5 adult healthy sheep, carry out the following step:
(1) antigen prepares: select hCRP antigen, carries out emulsification obtain antigen emulsion before injection with freund's adjuvant;
(2) first time injection: get 2.5mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml complete Freund's adjuvant again and make antigen emulsion, this antigen emulsion is used to inject sheep, injection site is vola, inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 10-16 point, every sheep injected dose is that 2ml contains 0.5mg antigen, each injection amount evenly distribute;
(3) second time injection: first time injects and carries out second time injection for latter 20 days, get 5.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is that lymphoglandula is interior, inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 10-16 point, every sheep injected dose is that 2ml contains 1.0mg antigen, each injection amount evenly distribute;
(4) third time injection: second time is injected and carried out third time injection for latter 11 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(5) the 4th injections: third time injects and carries out the 4th injection for latter 11 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(6) the 5th injections: the 5th injection is carried out in the 4th injection for latter 11 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(7) serum titer detects: blood sampling in 9 days after per injection, separation of serum, detects its pair of expansion and tire.
Embodiment 3
Select 5 adult healthy goats, carry out the following step:
(1) antigen prepares: select hCRP antigen, carries out emulsification obtain antigen emulsion before injection with freund's adjuvant;
(2) first time injection: get 2.5mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml complete Freund's adjuvant again and make antigen emulsion, this antigen emulsion is used to inject sheep, injection site is vola, inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 10-16 point, every sheep injected dose is that 2ml contains 0.5mg antigen, each injection amount evenly distribute;
(3) second time injection: first time injects and carries out second time injection for latter 20 days, get 5.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is that lymphoglandula is interior, inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 10-16 point, every sheep injected dose is that 2ml contains 1.0mg antigen, each injection amount evenly distribute;
(4) third time injection: second time is injected and carried out third time injection for latter 10 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(5) the 4th injections: third time injects and carries out the 4th injection for latter 10 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(6) the 5th injections: the 5th injection is carried out in the 4th injection for latter 10 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(7) serum titer detects: blood sampling in 10 days after per injection, separation of serum, detects its pair of expansion and tire.
Embodiment 4 (control group)
Select 5 adult healthy goats, carry out the following step:
(1) antigen prepares: select hCRP antigen, carries out emulsification obtain antigen emulsion before injection with freund's adjuvant;
(2) first time injection: get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml complete Freund's adjuvant again and make antigen emulsion, this antigen emulsion is used to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(3) second time injection: first time injects and carries out second time injection for latter 20 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(4) third time injection: second time is injected and carried out third time injection for latter 10 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(5) the 4th injections: third time injects and carries out the 4th injection for latter 10 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute;
(6) the 5th injections: the 5th injection is carried out in the 4th injection for latter 10 days, get 10.0mg hCRP antigen, with PBS or normal saline dilution to 5ml, add 5ml incomplete Freund's adjuvant again and make antigen emulsion, use this antigen emulsion to inject sheep, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amount to 8-16 point, every sheep injected dose is that 2ml contains 2.0mg antigen, each injection amount evenly distribute; (7) serum titer detects: blood sampling in 9 days after per injection, separation of serum, detects its pair of expansion and tire.
The two immunization that expands of experimental example detects antiserum titre
Vein adopts sheep blood 1ml, and separation of serum is about 0.5ml; By sheep blood serum PBS or physiological saline doubling dilution, be not respectively 1:2,1:4 to 1:2048 not etc.; Beat plum blossom hole on a lbmc agar plate, aperture 2mm, pitch-row 3mm; Be sequentially added into 1:2,1:4 to 1:2048 sheep blood serum 20 μ l not etc. in the hole outer Saturday often organizing plum blossom hole respectively, add at medium pore the hCRP antigen 20 μ l that concentration is 1.0mg/ml; The agar plate having added serum and antigen being put into 37 DEG C and hatches 12-24 hour, there is the final position of precipitation line in observation antigen and serum, and two expansion theing be serum is tired.
Control group carries out immunity according to embodiment 4 method, and the antigen dose of its each immunity is every sheep 2.0mg, and per injection position is inguinal region, armpit, neck, dorsal sc multi-point injection, does not carry out injection in vola and lymphoglandula;
Embodiment 1 group, 2 groups and 3 groups carry out immunization according to the embodiment of the present invention 1,2 and 3 prescription method respectively.
Detect each group of sero-fast two expansion of final gained by above method to tire, the results are shown in Table 1.
Table 1 respectively sero-fast two expansion of group gained is tired
Can be obtained by upper table 1 result, the antiserum(antisera) of control group gained, only have one to reach 1:512 only, other be 1:256; And in the present invention's three embodiment groups, 1:512 and 1:1204 can be reached, gained antibody horizontal improves further greatly on the basis of control group, and antigen amount minimizing 2.5mg (25%) that every sheep uses.
Claims (7)
1. produce the sero-fast method of goat-anti hCRP, it is characterized in that, comprise the following steps:
(1) antigen prepares: select hCRP antigen, carries out emulsification obtain antigen emulsion before injection with freund's adjuvant;
(2) first time injection: use above-mentioned antigen emulsion to inject sheep, injection site is vola, inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 10-16 point;
(3) second time injection: first time injects and carries out second time injection for latter 20 days, injection site is that lymphoglandula is interior, inguinal region, armpit, neck, dorsal sc multi-point injection, and each position 1-2 point, amounts to 10-16 point;
(4) third time injection: carry out third time injection after second time injection for 9-11 days, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 8-16 point;
(5) the 4th injections: carry out the 4th injection after third time injection for 9-11 days, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 8-16 point;
(6) the 5th injections: carry out the 5th injection after the 4th injection for 9-11 days, injection site is inguinal region, armpit, neck, dorsal sc multi-point injection, each position 1-2 point, amounts to 8-16 point;
(7) serum titer detects: blood sampling in 8-10 days after per injection, separation of serum, detects its pair of expansion and tire.
2. the sero-fast method of production goat-anti hCRP according to claim 1, it is characterized in that, before the injection of described first time, the freund's adjuvant of emulsification is complete Freund's adjuvant, and for the second time, the freund's adjuvant of emulsification is incomplete Freund's adjuvant for the third time, before the 4th time and the 5th injection.
3. the sero-fast method of production goat-anti hCRP according to claim 1, is characterized in that, the dosage of described first time injection is that every sheep amounts to 0.5mg antigen, each injection amount evenly distribute.
4. the sero-fast method of production goat-anti hCRP according to claim 1, is characterized in that, the dosage of described second time injection is that every sheep amounts to 1.0mg antigen, each injection amount evenly distribute.
5. the sero-fast method of production goat-anti hCRP according to claim 1, is characterized in that, the dosage of described third time, the 4th time and the 5th time injection is every sheep and amounts to 2.0mg antigen, each injection amount evenly distribute.
6. the sero-fast method of production goat-anti hCRP according to claim 1, is characterized in that, the method that described serum titer detects is two expansion immunizations.
7. the sero-fast method of production goat-anti hCRP according to claim 1, is characterized in that, described sheep is goat or sheep.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107163145A (en) * | 2017-05-27 | 2017-09-15 | 海奥斯生物科技镇江有限公司 | One kind production sero-fast method of goat-anti human serum amyloid A |
| CN107163141A (en) * | 2017-05-27 | 2017-09-15 | 海奥斯生物科技镇江有限公司 | A kind of purification process of sheep polyvalent antibody specific antibody |
| CN107312086A (en) * | 2017-05-27 | 2017-11-03 | 海奥斯生物科技镇江有限公司 | The preparation method for the animal anti-human serum being precisely controlled based on immune position point |
| CN107383191A (en) * | 2017-05-27 | 2017-11-24 | 海奥斯生物科技镇江有限公司 | Gamut sheep or the preparation method of rabbit-anti Human C-reactiveprotein antibody |
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| CN103014047A (en) * | 2011-09-27 | 2013-04-03 | 深圳市菲鹏生物股份有限公司 | Recombined human cystatin-C protein with natural activity and preparation method thereof |
| CN103467600A (en) * | 2013-09-09 | 2013-12-25 | 镇江万山红遍农业园 | Method for producing anti-apolipoprotein A1 (ApoA1) multi-antibody serum by using Zhejiang sheep |
| CN103941017A (en) * | 2014-03-18 | 2014-07-23 | 北京普恩光德生物科技开发有限公司 | C reactive protein detection kit |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103014047A (en) * | 2011-09-27 | 2013-04-03 | 深圳市菲鹏生物股份有限公司 | Recombined human cystatin-C protein with natural activity and preparation method thereof |
| CN103467600A (en) * | 2013-09-09 | 2013-12-25 | 镇江万山红遍农业园 | Method for producing anti-apolipoprotein A1 (ApoA1) multi-antibody serum by using Zhejiang sheep |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107163145A (en) * | 2017-05-27 | 2017-09-15 | 海奥斯生物科技镇江有限公司 | One kind production sero-fast method of goat-anti human serum amyloid A |
| CN107163141A (en) * | 2017-05-27 | 2017-09-15 | 海奥斯生物科技镇江有限公司 | A kind of purification process of sheep polyvalent antibody specific antibody |
| CN107312086A (en) * | 2017-05-27 | 2017-11-03 | 海奥斯生物科技镇江有限公司 | The preparation method for the animal anti-human serum being precisely controlled based on immune position point |
| CN107383191A (en) * | 2017-05-27 | 2017-11-24 | 海奥斯生物科技镇江有限公司 | Gamut sheep or the preparation method of rabbit-anti Human C-reactiveprotein antibody |
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