CN106366191A - Method for producing anti-ApoA1 antiserum through goats - Google Patents
Method for producing anti-ApoA1 antiserum through goats Download PDFInfo
- Publication number
- CN106366191A CN106366191A CN201610854017.2A CN201610854017A CN106366191A CN 106366191 A CN106366191 A CN 106366191A CN 201610854017 A CN201610854017 A CN 201610854017A CN 106366191 A CN106366191 A CN 106366191A
- Authority
- CN
- China
- Prior art keywords
- pin
- injection
- goat
- apoa1
- groin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a method for producing anti-ApoA1 antiserum through goats. The method includes the steps that goats with the higher immune response capacity and the low serum fat content are selected as raw materials, an ApoA1 antigen is subcutaneously injected into the outside of the goat groin to carry out multiple times of booster immunization, seven times of immune injection are carried out, and the quantities of the injected ApoA1 antigen are gradually increased; responses of T cells and B cells can be continuously activated, effective antibodies are better generated, and the valence of the generated efficient goat anti-ApoA1 antibodies can be 1:32 or above. The valence of the antibodies is greatly improved, the stability and the sensitivity are increased, raw materials are provided for biological reagent plants, convenience is brought to medical diagnoses, and goats generate high application value and benefits accordingly; meanwhile, the cost of reagent factories is reduced by 50%, and the economic value and the social value are remarkable.
Description
Technical field
The present invention relates to the preparation method field of antigen, more particularly, to one kind utilize Goat Raising anti-apoa1 polyvalent antibody
Method.
Background technology
Genetyping 1 (apoa1) is a kind of most component of apoa apoa1 race, and apoa1 is Single polypeptide chain, by 243
Individual amino acid residue composition, molecular weight is 28300d, is the major apolipoprotein of high density lipoprotein (hdl).It is biological reagent
Important source material, existing production method adopts traditional immunization wayses, and yield is relatively low with potency, cannot meet growing
Use demand.
Chinese patent cn103467600a (method producing anti-apoa1 polyvalent antibody using sheep) utilizes sheep, obtains
The anti-apoa1 polyvalent antibody up to 1:16 for the antibody titer, but have that antibody titer is not enough, Antibody stability is bad, clever
The not high defect of sensitivity.
Accordingly, it would be desirable to a kind of new technical scheme solves the above problems.
Content of the invention
Goal of the invention: in order to overcome defect present in prior art, the present invention propose a kind of operational approach simple and
The method increasing substantially the utilization Goat Raising anti-apoa1 polyvalent antibody of yield and potency.
Technical scheme: in order to solve above-mentioned technical problem, the technical solution adopted in the present invention is: one kind is using goat life
The method producing anti-apoa1 polyvalent antibody, comprises the following steps:
(1) preparation of immunogen emulsion: by the serum 0.1ml containing apoa1 antigen 1 0mg, aseptic pbs1.9ml, Freund
Adjuvant 2ml mixing emulsifying, obtaining immunogen emulsion is immune first pin amount；Apoa1 amount of antigen is increased as 20mg by the second pin,
3rd pin increases as 30mg, and the 4th pin increases as 40mg, and the 5th pin increases as 50mg, and the 6th pin increases as 60mg, and the 7th pin increases
Add as 70mg, remaining same operation；It is stepped up the responsing reaction that amount of antigen can constantly activate t cell and b cell, more preferably produce
Raw potent antibodies；
(2) goat immune first pin: groin before and after goat, every groin divides 2 regional injection, altogether 12 notes
Exit point, subcutaneous injection, cervical region both sides 2 injection point mean injection；
(3) goat immune second pin: be spaced 15 days injection the second pins with the first pin, groin divides 2 regions in front and back
Injection, 12 injection points altogether, 2 points about the lymph node of cervical region both sides；It is spaced 15 days and can preferably activate with the first pin
In animal body, immunocyte produces responsing reaction. do more preferable preparation for immunity next time.
(4) goat immune the 3rd pin: be spaced ten days injection the 3rd pins with the second pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(5) goat immune the 4th pin: be spaced ten days injection the 4th pins with the 3rd pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(6) goat immune the 5th pin: be spaced ten days injection the 5th pins with the 4th pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(7) goat immune the 6th pin: be spaced ten days injection the 6th pins with the 5th pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(8) goat immune the 7th pin: be spaced ten days injection the 7th pins with the 6th pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(9) with the 7th pin injection interval ten days to immune goat arterial blood drawing, blood is put in water tank, in 40 DEG C of temperature
Lower preservation 1h, treats that serum fully separates out, isolates antiserum through centrifugation, puts refrigerator into and is preserved, and storage temperature is 4-8
℃；
(10) titration is carried out to step (9) gained antiserum.
In some embodiments, the Freund adjuvant that the first pin described in step (1) uses is Freund's complete adjuvant.
In some embodiments, the Freund adjuvant that second pin the-the seven pin described in step (1) uses is that Freund is incomplete
Adjuvant.
In some embodiments, the method for the titration described in step (10) is fine jade expanding method.
Beneficial effect: a kind of method of the anti-apoa1 polyvalent antibody of utilization Goat Raising that the present invention provides, anti-from immunity
Should be able to power higher, the low goat of serum fat content is raw material, and apoa1 antigen is outer and carry out through being subcutaneously injected into goat groin
Repeatedly booster immunization, carries out seven inoculations, per injection is relatively gone up a pin apoa1 amount of antigen and is gradually increased, can be continuous altogether
Activation t cell and the responsing reaction of b cell, more preferably produce potent antibodies so as to produce efficient goat-anti apoa1 antibody titer up to
More than 1:32, is greatly improved antibody titer, increases stability and sensitivity, provides raw material for biological chemical reagent work, for curing
Treat diagnosis provides convenient, make goat create high using value and benefit, reduce the cost of reagent producer half, warp simultaneously
Ji is worth and social value is notable.
Specific embodiment
With reference to embodiment, the present invention is described in further detail:
Embodiment:
Experimental subject:
From goat ram 50, select the Healthy Sheep of 35 kilograms about of body weight, all stamp ear tag before immunity, flock of sheep are adopted
With stable breeding, in conjunction with putting in a suitable place to breed, keep normal body condition, it is to avoid sheep overfertilization, enhance immunity, automatic drinking bowl, daily herbage supply,
Appropriate interpolation concentrate, vinegar, wheat bran and vitamin etc..
Experimental technique:
A kind of method of the anti-apoa1 polyvalent antibody of utilization Goat Raising, comprises the following steps:
(1) preparation of immunogen emulsion: by the serum 0.1ml containing apoa1 antigen 1 0mg, aseptic pbs1.9ml, Freund
Adjuvant 2ml mixing emulsifying, obtaining immunogen emulsion is immune first pin amount；Apoa1 amount of antigen is increased as 20mg by the second pin,
3rd pin increases as 30mg, and the 4th pin increases as 40mg, and the 5th pin increases as 50mg, and the 6th pin increases as 60mg, and the 7th pin increases
Add as 70mg, remaining same operation；
Apoa1 inoculation 0.1ml (closing antigen 1 0mg) and 1.9ml aseptic pbs Freund adjuvant that wherein apoa1 antigen is behaved
It is standby that 2ml makes immunogen emulsion；The Freund adjuvant adopting during the immunogen emulsion emulsifying that the wherein first pin inoculation uses
It is Freund's complete adjuvant, the Freund adjuvant adopting during the immunogen emulsion emulsifying that in remaining immunity, i.e. the second to seven pin is injected is not
Family name's Freund's incomplete adjuvant；
(2) goat immune first pin: groin before and after goat, every groin divides 2 regional injection, altogether 12 notes
Exit point, subcutaneous injection, cervical region both sides 2 injection point mean injection；
(3) goat immune second pin: be spaced 15 days injection the second pins with the first pin, groin divides 2 regions in front and back
Injection, 12 injection points altogether, 2 points about the lymph node of cervical region both sides；
(4) goat immune the 3rd pin: be spaced ten days injection the 3rd pins with the second pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(5) goat immune the 4th pin: be spaced ten days injection the 4th pins with the 3rd pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(6) goat immune the 5th pin: be spaced ten days injection the 5th pins with the 4th pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(7) goat immune the 6th pin: be spaced ten days injection the 6th pins with the 5th pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(8) goat immune the 7th pin: be spaced ten days injection the 7th pins with the 6th pin, groin divides 2 region notes in front and back
Penetrate, altogether 12 injection points；
(9) with the 7th pin injection interval ten days to immune goat arterial blood drawing, blood is put in water tank, in 40 DEG C of temperature
Lower preservation 1h, treats that serum fully separates out, isolates antiserum through centrifugation, puts refrigerator into and is preserved, and storage temperature is 4-8
℃.
Titration:
Using the method that fine jade expands titration carried out to step (9) gained antiserum:
1. prepare agar: weigh 1.2g agar powder, add in 100ml normal saline, after heating in water bath dissolving, with 115 DEG C
Sterilizing 10 minutes；
After sterilizing terminates, take out agar solution, be cooled to 70 DEG C about, draw agar solution with suction pipe, add clean putting down
In ware, every piece of plate 16ml, packs up plate after cooling, is put in 2～8 DEG C of refrigerators and preserves, can preserve 2 weeks；
Flos Mume card punch is used to punch on plate with front, interstitial hole adds antigen, and outer perimeter holes add the serum to be checked after dilution.
2. Sample serum dilution: take out 96 porocyte culture plates or blood-coagulation-board, add in every hole 25 μ l normal saline or
pbs；
Every row the 1st row hole in add 25 μ l Sample serum, from the 1st hole backward doubling dilution to the 6th hole.
3. sample-adding detection: take out the agar plate accomplishing fluently plum blossom hole, be sequentially added into the sample of doubling dilution in outer perimeter holes
Product serum, every Kong Yuejia 20 μ l, is filled it up with hole and is advisable；
The above-mentioned antiserum isolated is added in the medium pore of plum blossom hole；
Excellent agar plate will be added put in wet box, put 37 DEG C overnight, and observe outer perimeter holes and occur with medium pore precipitation line
Position, calculates the potency of Sample serum.
4. serum titer evaluation: recording the 4th hole has precipitation line (agglutination) to illustrate to measure the sero-fast potency of this batch
For 1:32.
Can be seen that the present invention produces efficient goat-anti apoa1 antibody titer up to more than 1:32 from above-mentioned titration result,
Antibody titer is greatly improved, increases stability and sensitivity, provide for biological chemical reagent work offer raw material, for medical diagnosiss
Convenient, make goat create high using value and benefit, reduce the cost of reagent producer half, economic worth and society simultaneously
Can be worth notable.
It should be pointed out that above specific embodiment is only illustrative of the invention and is not intended to limit the scope of the invention,
After having read the present invention, the modification of the various equivalent form of values to the present invention for the those skilled in the art all falls within power appended by the application
Profit requires limited range.
Claims (4)
1. a kind of method using Goat Raising anti-apoa1 polyvalent antibody is it is characterised in that comprise the following steps:
(1) preparation of immunogen emulsion: by the serum 0.1ml containing apoa1 antigen 1 0mg, aseptic pbs1.9ml, Freund adjuvant
2ml mixing emulsifying, obtaining immunogen emulsion is immune first pin amount；Apoa1 amount of antigen is increased as 20mg by the second pin, and the 3rd
Pin increases as 30mg, and the 4th pin increases as 40mg, and the 5th pin increases as 50mg, and the 6th pin increases as 60mg, and the 7th pin increases and is
70mg, remaining same operation；
(2) goat immune first pin: groin before and after goat, every groin divides 2 regional injection, altogether 12 injections
Point, subcutaneous injection, cervical region both sides 2 injection point mean injection；
(3) goat immune second pin: it is spaced 15 days injection the second pins with the first pin, groin divides 2 regional injection in front and back,
12 injection points altogether, 2 points about the lymph node of cervical region both sides；
(4) goat immune the 3rd pin: be spaced ten days injection the 3rd pins with the second pin, groin divides 2 regional injection in front and back, always
Totally 12 injection points；
(5) goat immune the 4th pin: be spaced ten days injection the 4th pins with the 3rd pin, groin divides 2 regional injection in front and back, always
Totally 12 injection points；
(6) goat immune the 5th pin: be spaced ten days injection the 5th pins with the 4th pin, groin divides 2 regional injection in front and back, always
Totally 12 injection points；
(7) goat immune the 6th pin: be spaced ten days injection the 6th pins with the 5th pin, groin divides 2 regional injection in front and back, always
Totally 12 injection points；
(8) goat immune the 7th pin: be spaced ten days injection the 7th pins with the 6th pin, groin divides 2 regional injection in front and back, always
Totally 12 injection points；
(9) with the 7th pin injection interval ten days to immune goat arterial blood drawing, blood is put in water tank, protects at 40 DEG C of temperature
Deposit 1h, treat that serum fully separates out, isolate antiserum through centrifugation, put refrigerator into and preserved, storage temperature is 4-8 DEG C；
(10) titration is carried out to step (9) gained antiserum.
2. the anti-apoa1 polyvalent antibody of utilization Goat Raising according to claim 1 method it is characterised in that: step (1)
The Freund adjuvant that the first described pin uses is Freund's complete adjuvant.
3. the anti-apoa1 polyvalent antibody of utilization Goat Raising according to claim 2 method it is characterised in that: step (1)
The Freund adjuvant that second described pin the-the seven pin uses is incomplete Freund's adjuvant.
4. the anti-apoa1 polyvalent antibody of utilization Goat Raising according to claim 1 method it is characterised in that: step
(10) method of the titration described in is fine jade expanding method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610854017.2A CN106366191A (en) | 2016-09-27 | 2016-09-27 | Method for producing anti-ApoA1 antiserum through goats |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610854017.2A CN106366191A (en) | 2016-09-27 | 2016-09-27 | Method for producing anti-ApoA1 antiserum through goats |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106366191A true CN106366191A (en) | 2017-02-01 |
Family
ID=57897027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610854017.2A Pending CN106366191A (en) | 2016-09-27 | 2016-09-27 | Method for producing anti-ApoA1 antiserum through goats |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106366191A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107216386A (en) * | 2017-05-27 | 2017-09-29 | 海奥斯生物科技镇江有限公司 | A kind of goat-anti human serum prepares the accurate determination method of antigen immune dosage |
| 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 |
| CN112375144A (en) * | 2020-11-18 | 2021-02-19 | 上海钹乐诗生物技术有限公司 | Preparation method of rabbit anti-human ApoA1 polyclonal antiserum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103509109A (en) * | 2013-09-26 | 2014-01-15 | 镇江万山红遍农业园 | Method for improving quality of blood serum |
| CN104211806A (en) * | 2014-08-20 | 2014-12-17 | 镇江万山红遍农业园 | Method for gradient production of goat anti-Cys-C multiple resistant serum by means of amount of antigens |
-
2016
- 2016-09-27 CN CN201610854017.2A patent/CN106366191A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103509109A (en) * | 2013-09-26 | 2014-01-15 | 镇江万山红遍农业园 | Method for improving quality of blood serum |
| CN104211806A (en) * | 2014-08-20 | 2014-12-17 | 镇江万山红遍农业园 | Method for gradient production of goat anti-Cys-C multiple resistant serum by means of amount of antigens |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107216386A (en) * | 2017-05-27 | 2017-09-29 | 海奥斯生物科技镇江有限公司 | A kind of goat-anti human serum prepares the accurate determination method of antigen immune dosage |
| 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 |
| CN107216386B (en) * | 2017-05-27 | 2021-02-12 | 海奥斯生物科技镇江有限公司 | Method for accurately determining antigen immunization dose prepared from sheep anti-human serum |
| CN112375144A (en) * | 2020-11-18 | 2021-02-19 | 上海钹乐诗生物技术有限公司 | Preparation method of rabbit anti-human ApoA1 polyclonal antiserum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103467600A (en) | Method for producing anti-apolipoprotein A1 (ApoA1) multi-antibody serum by using Zhejiang sheep | |
| Do Carmo et al. | Food poisoning due to enterotoxigenic strains of Staphylococcus present in Minas cheese and raw milk in Brazil | |
| Brüssow | Nutrition, population growth and disease: a short history of lactose | |
| CN106366191A (en) | Method for producing anti-ApoA1 antiserum through goats | |
| CN103467601A (en) | Method for producing anti-apolipoprotein B (ApoB) multi-antibody serum by using Zhejiang sheep | |
| CN101147506A (en) | Tibetan mushroom yoghourt and its producing technique method, by-product in production course | |
| CN102007968A (en) | Lactobacillus casei-containing active lactobacillus beverage and preparation method thereof | |
| Sahlu et al. | ASAS Centennial Paper: Impact of animal science research on United States goat production and predictions for the future | |
| CN110218250A (en) | A kind of preparation method of grass carp component 5 polyclonal antibody | |
| CN101538545B (en) | Lactobacillus salivarius and food composition thereof | |
| CN102093954B (en) | Cell culture device and cell culture method | |
| CN103027123A (en) | Frozen sour soybean milk processing process | |
| CN105198994A (en) | Preparation method for beta-casein monoclonal antibody resistant to milk and free of cross reaction with buffalo milk protein | |
| CN102590522B (en) | Plasmodium antibody colloidal gold detection kit and preparation method thereof | |
| Eteng et al. | Storage beyond three hours at ambient temperature alters the biochemical and nutritional qualities of breast milk | |
| CN109507421B (en) | Anti-buffalo IgG monoclonal antibody cell strain and preparation method and application thereof | |
| Marks | The standardization of staphylococcal α-antitoxin, with special reference to anomalous haemolysins including δ-lysin | |
| CN109430380A (en) | A kind of source of people mixing lactic acid bacteria acidified milk and preparation method thereof reducing cholesterol effect with auxiliary | |
| CN104177498A (en) | Method for producing anti-goat IgA multi-resistant serum by using multi-immune pathway | |
| CN102206612A (en) | Hybridoma cell and preparation method and application thereof | |
| CN107056941A (en) | A kind of specific recognition goat IFN γ method for preparing monoclonal antibody | |
| CN114409776A (en) | Preparation method of mycoplasma gallisepticum monoclonal antibody | |
| CN104211806A (en) | Method for gradient production of goat anti-Cys-C multiple resistant serum by means of amount of antigens | |
| CN104211805A (en) | Method for producing multi-antibody serum for resisting complement C3 by utilizing sheep raised in Zhejiang province | |
| CN106543285B (en) | Anti- Ttyh1 monoclonal antibodies and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170201 |
|
| RJ01 | Rejection of invention patent application after publication |