CN1439650A - Preparation of multi-antigen from waterfowl - Google Patents
Preparation of multi-antigen from waterfowl Download PDFInfo
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- CN1439650A CN1439650A CN 02106193 CN02106193A CN1439650A CN 1439650 A CN1439650 A CN 1439650A CN 02106193 CN02106193 CN 02106193 CN 02106193 A CN02106193 A CN 02106193A CN 1439650 A CN1439650 A CN 1439650A
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- polyclonal antibody
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Abstract
A process for preparing multiple strains of antibody from waterfowls includes such steps as injecting antigen into muscle of laying duck to induce antibody, getting egg yolk, purifying protein, chromatographing with hydrophobic colloid, dialysing, desalting, concentrating, having electrophoresis with SDS-acrylamide, and analysing to obtain high-purity immunoglobulin of egg yolk. Its advantages are high specificity and no pseudo-positive reaction.
Description
The present invention is one and divides an application that the applying date of original application is: on 07 01st, 1999; Application number is: 99109616.9; Invention and created name is: utilize aquatic bird to prepare the method for polyclonal antibody.
Technical field
The present invention relates to a kind of method of utilizing immune response to prepare antibody, particularly relate to the method for utilizing palmipeds to prepare polyclonal antibody for the intermediate host.
Background technology
Polyclonal antibody (polyclonal antibldy) is a most important parts in the immunological test reagent.Be widely used in every biomedical sector now.The preparation of polyclonal antibody in the past all utilizes mammal (as small white mouse, rabbit, ox, sheep etc.) and makes.Yet, the event of biological species poor (Phylogenetic distance), usually need a large amount of antigens can produce ideal antibody, but with the made immunity inspection reagent of the polyclonal antibody of this kind method gained when the clinical application, the puzzlement of generation false positive reaction (false positive) is arranged more.The generation major cause of this reaction is that the antibody (IgG in the animal serum) of gained is all mammal with its source of a corpse or other object for laboratory examination and chemical testing (IgG in the human serum), have due to the biologically so-called homogeneity (homogenecity), especially when human body has autoimmune disease (autoimmune disease), such as the rheumatoid factor (Rheumatoid factor) being arranged when existing, the easier interference that causes this kind false positive reaction.Be immune intermediate host abroad in recent years, in it is laid eggs, make polyclonal antibody with laying hen (flying bird).Discovery is far away because of the biological species difference, can reduce to the interference of false positive reaction minimum in clinical application.And if when being antigen with the human body protein also because of the biological species difference is far away, and immunity is preferably arranged.Because Taiwan is quite flourishing aspect industries such as foster duck and every product processing thereof, it is the intermediate host that the present invention mainly utilizes egg duck (palmipeds) to replace laying hen, produces polyclonal antibody to reach the purpose of widespread use.
Summary of the invention
Purpose of the present invention is to provide a kind of and utilizes aquatic bird to prepare the method for polyclonal antibody for the intermediate host, and this antibody has high specific (specificity) and dilution capacity, more can effectively avoid false positive reaction.This kind method is with the advantage that with the laying hen is intermediate host's comparison: 1, the immune response of laying hen only reaches 1/3 of egg duck; 2, the weight of duck's egg is to two times of egg, and this means can produce one to the duple polyclonal antibody; 3, the mortality ratio of egg duck is low far beyond laying hen, and this is a special character of the present invention.
The present invention relates to a kind of method of utilizing aquatic bird to prepare polyclonal antibody, it comprises:
(a) antigen (immunogen) with purifying is injected to aquatic bird;
(b) strengthen reaction with Freund again;
(c) after 5-15 days, promptly there is polyclonal antibody to produce in the laying eggs of aquatic bird.
Utilize in the method that aquatic bird prepares polyclonal antibody of the present invention, wherein said injection comprises methods such as muscle, subcutaneous, intracutaneous, intravascular injection; Described aquatic bird is selected from duck, female goose; Described antigen comprises the human immunoglobulin;
Utilize in the method that aquatic bird prepares polyclonal antibody of the present invention, wherein said antigen is selected from:
(a) from protein, the hormone of mammal;
(b) various viruses;
(c) bacterium;
(d) recombinant protein for preparing with genetically engineered, synthetic peptide (syntheticpeptide);
The protein conjugate of (e) various medicines for treatment, drug abuse medicine.
The present invention relates to a kind of polyclonal antibody, it is characterized in that, it is to utilize to comprise that the method for above-mentioned steps (a)-(c) is prepared.
The invention still further relates to the purposes of described polyclonal antibody, it is applied to various clinical immunologic function test reagents and research is used in the preparation and food and feed of immunoreagent.
The invention further relates to a kind of in aquatic bird is laid eggs the method for purifying polyclonal antibody, it comprises:
(1) with water is solvent, roughing out IgY in the yolk that this is laid eggs, can use tap water, purifying distilled water, deionized water, or for little acid of PH 〉=5.1 or contain nonionic detergent (non-ionic detergent) aqueous solution or 0.15-1.0M common salt aqueous solution below 0.05%, phosphate buffered saline buffer or tris buffer (Tris-buffer) etc.;
(2) add ammonium sulfate, make the saturation ratio that reaches 30-50%, or add sodium sulfate, make the concentration that reaches 14-20% (W/V), under its concentration, repeat to saltout;
(3) use and to contain hydrophobic group colloid (hydrophobic ligand coupled to matrices) and carry out chromatography IgY:
(a) described hydrophobic group colloid is selected from:
Butyl-agarose (Butyl sepharose), octyl sepharose (Octyl sepharose), phenyl sepharose (Phenyl sepharose), alkyl agarose (Alkyl sepharose), methyl methacrylate (Methyl methacrylate) and methacrylic acid butyl ester (T-Butylmethacrylate);
(b) operational condition of chromatography tubing string:
(i) the tubing string level pad is at PH7.0-8.0;
(ii) level pad is ammonium sulfate or the sodium sulfate of 1.0M, and its conductivity is 110-200ms/ml;
(iii) type of elution can be with gradient elution or single liquid wash-out: gradient elution: A liquid be aforementioned b (ii),
B liquid is for the damping fluid of salt,
Single liquid wash-out: in advance A liquid and B liquid being mixed and adjusts its conductivity is 45-80ms/ml.
In aquatic bird is laid eggs in the method for purifying polyclonal antibody, wherein said laying eggs laid eggs or through the cold storage egg of 4 ℃ of preservations for fresh of the present invention.
Description of drawings
Brief description of drawings:, can have more fully the present invention and understand via following description and diagram.Wherein:
Fig. 1: utilizing 10%SDS-acryloyl electrophoresis (SDS-PAGE) to cooperate Kumasi orchid (commassie blue, trade(brand)name) is dyestuff, decision antibody molecule amount size and purity assay.Molecular weight unit kda is 1000 dalton (dalton) among the figure.
1.-9. meaning is as follows in Fig. 1:
1. salt precipitation, 100 times of dilutions, not reduced states.
2. salt precipitation, 10 times of dilutions, reduced state.
3. salt precipitation, 10 times of dilutions, not reduced states.
4. colloid chromatography, secondary peak top gleanings, reduced state.
5. colloid chromatography, secondary peak top gleanings, not reduced state.
6. concentrate finished product, reduced state.
7. concentrate finished product, not reduced state.
8. concentrate finished product, reduced state.
9. standard molecular weight reagent (Molecular Marker), 29～200kda
Fig. 2: enzyme immunoassay is measured the antibody dilution ability.
In Fig. 2 the meaning of ordinate zou OD450 for the optical density(OD) that under the 450nm wavelength, records (optical density, OD).
Fig. 3: agar immunoelectrophoresis figure.This measures the specificity and the purity of antibody.
The meaning of each mark is as follows in Fig. 3:
Ab
1The IgY antibody of → duck antagonism human IgG (full molecule)
Ab
2The IgY antibody of → duck antagonism human IgG (full molecule)
Ab
3The antibody of → rabbit antagonism duck IgG (W)
Ag
1→ human IgG (w)
Ag
2→ human albumin
Ag
3→ human whole serum
Ag
4→ rabbit whole blood is clear
Ag
5→ it is Ab
1
Ag
6→ human IgG (w)
Embodiment
Following embodiment is used to further specify the present invention, but these embodiment only are not in order to restriction the present invention as preferred example.
Embodiment 1: the preparation of polyclonal antibody
The egg duck is a palmipeds, and the difference of highly significant is arranged with mammals on the biological species difference, so when being antigen with the protein in mammals source, easily obtaining good immune response (Immuneresponse) and obtain better antibody.Getting the human immunoglobulin (human IgG) that healthy egg duck (as dish duck, Beijing duck, kind duck and change duck etc.) finishes with purifying in advance is antigen with Freund's complete adjuvant (Complete Freund ' s adjuvant) with 1: 1 mixed, with the muscle injection mode injection, generate with induce antibody.Afterwards, strengthen reaction (booster dose) with Freund (IncompleteFreund ' s adjuvant) with 1: 1 ratio again.About ten day after tomorrow, promptly there is antibody to generate in the duck's egg.
Embodiment 2: the antibody list is from reaching purifying
Yolk accounts for duck 1/3 weight, and its main component is a lipoprotein, accounts for 85%.Contained Yolk immune globulin (Yolk Immunologlobulin in the yolk; IgY) genus is water-soluble, therefore can utilize this characteristic, is solvent with water, and IgY is single from coming out in yolk.Get one piece in duck's egg, this duck's egg is fresh lay eggs or through the cold storage egg of 4 ℃ of preservations, break to take out the water that yolk adds the about 6-20 of its volume times, utilizes general agitator or magnetic stirrer (magnetic stirrer) that it is mixed.Then with high-speed refrigerated centrifuge under the state of 4 ℃ and 5000rpm, separated, and collected the supernatant liquor that is rich in IgY.
Embodiment 3: saltout
Add ammonium sulfate (Ammonium sulfate (NH
4)
2SO
4) or sodium sulfate (sodiumsulfate Na
2SO
4) in the supernatant liquor that is rich in IgY, make to reach 30-50% ammonium sulfate saturation ratio, or the concentration of 14-20% (w/v) sodium sulfate, to saltout.After full and uniform stirring, utilize high-speed refrigerated centrifuge equally under the state of 4 ℃ and 5000rpm, separated, and collected its precipitation.Generally speaking, repeat to saltout and centrifugal two steps, can effectively improve product purity.The protein of different molecular weight has different binding abilities with the colloid of band hydrophobic group (hydrophobicligand) under the environment of different concns.The hydrophobic group colloid is selected from following material: butyl-agarose, octyl sepharose, phenyl sepharose, alkyl agarose, methyl methacrylate and methacrylic acid butyl ester.
By fill the tubing string (column) form with the hydrophobic group colloid, then with band salt (as ammonium sulfate or sodium sulfate etc.) and be not with two kinds of damping fluids of salt with the alternately washing of gradient mode, type of elution can be with gradient elution or single liquid wash-out with the salt precipitation thing.Promptly with ammonium sulfate or the sodium sulfate of 1.0M to 1.5M, its conductivity is 110-200ms/ml to gradient elution A liquid, and B liquid is the damping fluid of not being with salt.And single liquid wash-out promptly in advance A liquid and B liquid to be mixed and adjusts its conductivity be 45-80ms/ml.Collect the secondary peak of washings, with phosphate buffered saline buffer (Phosphate buffer saline) dialysis desalting.After treating that desalination is finished, dialyzate is condensed into includes the 1.0mg/ml protein soln to concentrate film (ultrafiltration membrane), this is the antibody behind the purifying.
Embodiment 4: purity testing and antibody ability are measured
The molecular weight of IgY is about 170,000 (170kDa), and removing its molecular weight of Fc end back is 110,000 (110kDa).Electrophoretic technique is at present in order to the method for the most normal use of decision molecular weight size and purity assay aspect.Therefore the present invention utilizes SDS-acrylamide electrophoresis (SDS-PAGE) to cooperate the Kumasi orchid to be dyestuff, with various antibody samples colloid dyeing sentence read result such as Fig. 1 after electrophoretic analysis.The sample of the 9th row is a standard molecular weight 29-200kDa reagent among Fig. 1.
Embodiment 5: the mensuration of antibody dilution ability and antibodies specific
Utilize enzyme immunoassay (enzyme immuno assay; EIA) dilution capacity of analysis antibody, experimental result such as Fig. 2.Symbolic representation among Fig. 2 is at the different resulting antibody of time the method according to this invention.
Embodiment 6: the agar bidirectional diffusion is analyzed (gel double diffusion)
(3a) antibody dilution ability: the antibody dilution ability that records during for antigen with the human IgG is more than or equal to 32 times.
(3b) antibodies specific: with the agar immunoelectrophoresis analysis, experimental result such as Fig. 3.With rabbit igg, when goat IgG and mouse IgG are antigen, all do not have precipitation line and occur, this shows that this kind polyclonal antibody is splendid to the specificity of IgG.
Embodiment 7: the purposes of polyclonal antibody
The method according to this invention (embodiment 1), utilize the antigen of different structure, can prepare and have corresponding specific polyclonal antibody, these antibody just can be used for clinical immunologic function test reagent, research immunoreagent, or add in food, the feed and make the eater have immunizing power.
Claims (2)
1, a kind of in aquatic bird is laid eggs the method for purifying polyclonal antibody, it comprises:
(1) with water is solvent, roughing out IgY in the yolk that this is laid eggs: can use tap water, pure water distilled water, deionized water, or for little acid of pH 〉=5.1 or contain the nonionic detergent aqueous solution or 0.15-1.0M common salt aqueous solution below 0.05%, phosphate buffered saline buffer or tris buffer;
(2) add ammonium sulfate, make the saturation ratio that reaches 30-50%, or add sodium sulfate, make the concentration that reaches 14-20% (w/v), under its concentration, repeat to saltout;
(3) use and to contain the hydrophobic group colloid and carry out chromatography IgY:
(a) described hydrophobic group colloid is selected from following material:
Butyl-agarose, octyl sepharose, phenyl sepharose, alkyl agarose, methyl methacrylate and methacrylic acid butyl ester;
(b) operational condition of chromatography tubing string:
(i) the tubing string level pad is at pH7.0-8.0;
(ii) level pad is ammonium sulfate or the sodium sulfate of 1.0M, and its conductivity is 110-200ms/ml;
(iii) type of elution can be with gradient elution or single liquid wash-out:
Gradient elution: A liquid be aforementioned b (ii),
B liquid is for the damping fluid of salt,
Single liquid wash-out: in advance A liquid and B liquid being mixed and adjusts its conductivity is 45-80ms/ml.
2, as claimed in claim 1 in aquatic bird is laid eggs the method for purifying polyclonal antibody, wherein said laying eggs laid eggs or through the cold storage egg of 4 ℃ of preservations for fresh.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02106193 CN1439650A (en) | 2002-04-08 | 2002-04-08 | Preparation of multi-antigen from waterfowl |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02106193 CN1439650A (en) | 2002-04-08 | 2002-04-08 | Preparation of multi-antigen from waterfowl |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99109616A Division CN1092979C (en) | 1999-07-01 | 1999-07-01 | Process for preparing multi-strain antibody from waterfowls |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1439650A true CN1439650A (en) | 2003-09-03 |
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ID=27793175
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02106193 Pending CN1439650A (en) | 2002-04-08 | 2002-04-08 | Preparation of multi-antigen from waterfowl |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100434119C (en) * | 2006-09-04 | 2008-11-19 | 杨建彬 | Method for producing high immunity yolk antibody liquid |
| CN103570829A (en) * | 2012-08-01 | 2014-02-12 | 王玉麒 | Method for extracting gamma-livetin (IgY) from yolk |
| US9605052B2 (en) | 2012-08-01 | 2017-03-28 | National Taiwan Normal University | Method for extracting IgY (γ-livetin) from egg yolk |
-
2002
- 2002-04-08 CN CN 02106193 patent/CN1439650A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100434119C (en) * | 2006-09-04 | 2008-11-19 | 杨建彬 | Method for producing high immunity yolk antibody liquid |
| CN103570829A (en) * | 2012-08-01 | 2014-02-12 | 王玉麒 | Method for extracting gamma-livetin (IgY) from yolk |
| US9605052B2 (en) | 2012-08-01 | 2017-03-28 | National Taiwan Normal University | Method for extracting IgY (γ-livetin) from egg yolk |
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