CN101165069A - Preparation for self-protein used as carrier protein for hapten-antibody - Google Patents
Preparation for self-protein used as carrier protein for hapten-antibody Download PDFInfo
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- CN101165069A CN101165069A CNA2007100466425A CN200710046642A CN101165069A CN 101165069 A CN101165069 A CN 101165069A CN A2007100466425 A CNA2007100466425 A CN A2007100466425A CN 200710046642 A CN200710046642 A CN 200710046642A CN 101165069 A CN101165069 A CN 101165069A
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Abstract
The present invention relates to hapten antibody preparing process, and is especially the process of first preparing complete antigen through coupling animal's autologous protein to hapten and subsequent preparing antibody through immunizing animal with the complete antigen. The antibody preparing process has no introduced foreign protein, body produced antibody targeting to the small molecular weight compound and small peptide hapten, and no antibody to autologous protein. Therefore, the prepared hapten antibody prepared with autologous protein as carrier protein has high valence and high specificity.
Description
Technical field
The present invention relates to a kind of preparation method of hapten antibody, particularly the animal autologous protein is used for the preparation method of hapten antibody as carrier proteins.
Background technology
Haptens is to have with antibody binding capacity still to stimulate body to produce the molecule of antibody.Antigen is to have macromolecular three bioelements of heterology, structural complexity and certain molecular weight to form, though and haptens has heterology and structural complexity, but because molecular weight is big inadequately, so being not enough to separately in vivo be discerned by immunocyte and offering can not activated lymphocyte, produce antibody, but the antibody that can have been generated is discerned, and promptly has immunoreactivity but does not have immunogenicity.Want to prepare the small molecules hapten antibody, the first step will allow low-molecular-weight small molecules haptens become macromole exactly, prepares antibody according to the method for complete antigen more afterwards.Small molecules becomes macromole generally two kinds of methods, and the one, small molecules self is cross-linked into the congeneric elements aggressiveness mutually, the 2nd, with small molecules and macromole coupling.First method is not introduced new compound, can not bring new antigenic structure into, but be not easy chemically crosslinked sometimes, and the complete antigen lowest molecular weight requires also to want thousands of to 10,000, small molecules will just can reach requirement through aggressivenessization repeatedly, also might make the epi-position group be changed and shield in the polymerization repeatedly.Second method uses large molecular weight protein as carrier, with the small molecules hapten conjugation to protein molecular surface composition complete antigen.This cross-linking method is fairly simple, and weak point is to introduce new protein structure, unwanted epitope occurs, produces a large amount of unwanted antibody.
Usually carrier proteins uses bovine serum albumin (bovie serum albumiu BSA), ovalbumin (ovalbumin OV), key hole chirp hemocyanin albumen (Hemocyanin KLH) and ox, horse, sheep gamma Globulin etc.These proteic characteristics are that molecular weight ratio is bigger, and the commercialization sample obtains easily, so be widely used.But simultaneously because above-mentioned albumen is not oneself protein usually for immune host, bigger in addition molecular weight, itself be exactly that a complete antigen causes that antibody produces, and the epitope on the carrier proteins is considerably beyond haptenic epitope number, and the antibody that immunized animal produces carrier proteins is than being primarily aimed at the also many of haptens itself.Like this to specific needs antibody tire and specificity cause very adverse influence.
Summary of the invention
In order to address the above problem, we propose to adopt the animal autologous protein to overcome this drawback that above universal method produces as the technology of immune carrier protein.Because same individual animal proteinum injects in health, there is not immunogenicity, body only produces immune response to the haptens on the carrier proteins, thereby produces anti-specific haptenic antibody.The animal that is used for the specific antisera preparation, generally select the bigger animal of the scale of construction for use, so that once immunity obtains the antiserum(antisera) of capacity, as rat, rabbit, goat, sheep etc., the weight of animals of these kinds generally restrains 30 kilograms from 300 and does not wait, although body weight differs greatly, all possesses the condition that 2-20 milliliter blood can once be extracted and do not influence animal health.Utilize the 2-20 milliliter blood that extracts, purifying obtains 10-100 milligram gamma Globulin or 10-100 milligram serum albumin respectively, and the albumen of quantity enough as the proteic required dosage of hapten-carrier, is used for the immunity of same individual animals subsequently like this.Main summary of the invention is as follows:
A kind of preparation method of hapten antibody, it is characterized in that, may further comprise the steps: at first, adopt the animal autologous protein to get the coupling complete antigen, make hapten antibody with coupling complete antigen immune animal then as carrier proteins and haptens generation linked reaction.
Described carrier proteins is to take from the autologous protein that is used to immune animal.
Described autologous protein is the high-abundance proteins in the serum, and concrete can be albumin or gamma Globulin.
Described preparation method is a Polyclonal Antibody Preparation, also can be used for MONOCLONAL ANTIBODIES SPECIFIC FOR, and immune animal comprises mouse, rat and rabbit.
Preparation polyclonal antibody (antiserum(antisera)) overwhelming majority selects immunizing rabbit at present, prepare serum albumin from rabbit anteserum, the rivanol complex extractions method that method can adopt equally, but also can obtain gamma Globulin (40% saturated ammonium sulphate adds the albumin A affinity chromatography again) and serum albumin (40% saturated ammonium sulphate) step by step with ammonium sulfate precipitation method.
Description of drawings
Fig. 1 is the BSA electrophorogram of embodiment 1,
Wherein 1: self-control albumin rabbit serum 2: commercialization albumin rabbit serum (sigma purity＞99%);
Fig. 2 is the structural formula of FK506;
Fig. 3 is the measurement result of antiserum titre behind FK506 and the different carriers albumen coupling;
Fig. 4 is the measurement result of antiserum titre behind BNP and the different carriers albumen coupling.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.
Adopt the rivanol method purifying to obtain albumin rabbit serum in the present embodiment.Concrete steps are as follows:
1) the rabbit auricular vein is got blood 10ml, the placement of spending the night under 4 ℃;
2) 4000rpm/ minute, 4 ℃ were descended centrifugal 15 minutes, and getting supernatant is rabbit anteserum, and volume is 4.5 milliliters;
3) rabbit anteserum adds with 50% (NH
4)
2SO
4The precipitation, will precipitate the removal after, be 4.3-4.4 with 1mol/L salt acid for adjusting pH value, place after one hour, use the canvas natural filtration, scrape off throw out;
4) throw out is wrapped in the glassine paper dialysis band, leaves and takes suitable volume and drive away air, tying is dialysed to flowing water, when ammonium sulfate content is lower than 2g/L till, dialyzate;
5) merge dialyzate, measure volume, add the sodium chloride stirring and dissolving, mass concentration reaches 8.5g/l, uses the canvas natural filtration, gets filtrate and measures protein content, and the mass concentration that adds apirogen water diluted protein matter again is to 12-20g/l.Add the solid Sodium octoate and make proteinic mass concentration reach 2g/l, to 5-5.2, after the stirring and dissolving, place down for 0-5 ℃ and spend the night with 1mol/l salt acid for adjusting pH value.Weigh adjust pH next day to 5-5.2, put when being heated to 67 ℃ in the water-bath, add 2g/l (0.2%) activated carbon, stir 15min and be warmed up to 69 ℃, insulation 1h is cooled to below 45 ℃ then, uses the canvas natural filtration, gets filtrate;
6) adjust proteinic mass concentration to 100mg/ml, filtration sterilization, gel electrophoresis is identified its purity＞95%, sees shown in Figure 1;
7) ℃ fluid preservation-20.
FK506 belongs to macrolide antibiotics, and by one and half ketone groups, α, β diketone base and 23 rings are formed, and its molecular formula is C
44H
69NO
12H
2O, molecular weight are 822.Its structural formula such as Fig. 2.It can not obtain specific antibody as antigen-immunized animal separately, need obtain immunogenicity with a carrier protein couplet.We adopt the acid anhydrides method to obtain the derivative of FK506, use EBC method and the coupling of autoserum albumin then.Concrete steps are as follows:
1) FK506 of 20mg adds the 50mg Pyroglutaric acid with the dissolving of 5ml pyridine, 80 ℃ of back flow reaction 4 hours, and carry out degree with the silicon thin-layer chromatography detection reaction, stop back flow reaction during extremely near complete reaction;
2) underpressure distillation removes the pyridine that desolvates, and obtains the FK506 glutaric acid monoester;
3) 10mg FK506 glutaric acid monoester is dissolved with 0.5ml DMF (dimethyl formamide); 20mg self-control albumin rabbit serum dissolves with the PBS (pH7.2) of 2ml 0.05M.
4) 2ml contains and adds fully mixing reaction 10 minutes of 50mg EDC (carbodiimide) in the solution of 20mg self-control albumin rabbit serum, slowly be added dropwise to 0.5ml FK506 glutaric acid monoester solution in the albumin solution, reacted 30 minutes, and added 4 ℃ of reactions of 50mg EDC again and spend the night.
5) above-mentioned reaction solution was fully dialysed 24 hours with the PBS (pH 7.2) of 0.05M.Electrophoretic method certification mark effect.
Adopt the same bovine serum albumin of coupling FK506 that uses the same method, use as immunity.
Adopt the same KLH of coupling FK506 that uses the same method, use as detecting.
3 immunity back antibody titerss are measured
Adopt the ordinary method immune animal, immunity divides two groups, three rabbits of every group of immunity.
Albumin rabbit serum group: adopt and go immune animal (being respectively RSA-1#, RSA-2# and RSA-3#) from body albumin and FK506 link coupled antigen.
Bovine serum albumin group: adopt bovine serum albumin and FK506 link coupled antigen to go immune animal (being respectively BSA-1#, BSA-2# and BSA-3#).
After three immunity, every rabbit auricular vein is got blood 5ml, and directly the ELISA method is surveyed antiserum titre, and method is as follows:
1) bag quilt: dilute FK506-KLH to 2 μ g/ml with pH 9.6,0.05M CBS (carbonate buffer solution), every hole 100 μ l, 4 ℃ of bag quilts that spend the night;
2) use the 5%PBST skim-milk, 37 ℃ were sealed one hour down;
3) antiserum(antisera) dilution, two groups of serum carry out the twice gradient dilution all since 1: 1000 with the 5%PBST skim-milk, have 12 extent of dilution, and each extent of dilution is set up two multiple holes.As negative control, do same gradient dilution with the serum before the immunity;
4) behind the application of sample, 37 ℃ were reacted one hour down;
5) two anti-do dilution in 1: 10000, reacted one hour for 37 ℃ times behind the application of sample with the 5%PBST skim-milk;
6) add TMB colour developing 10 minutes, 2M sulfuric acid stops microplate reader 450nm place colorimetric.
The results are shown in Figure 3, table 1, as can be seen from the chart with the autologous protein coupling after immune animal, the anti-FK506 of acquisition is sero-fast to tire and is higher than the antiserum(antisera) that adopts bovine serum albumin coupling FK506 immune animal to obtain.
Table 1
| OD 415 | ||||||
| The serum thinning ratio | RSA-1# | RSA-2# | RSA-3# | BSA-1# | BSA-2# | BSA-3# |
| 1∶1000 | 2.131 | 2.172 | 2.208 | 1.73 | 1.655 | 1.86 |
| 1∶2000 | 1.938 | 1.966 | 1.995 | 1.4 | 1.344 | 1.42 |
| 1∶4000 | 1.692 | 1.638 | 1.713 | 0.98 | 1.021 | 1 |
| 1∶8000 | 1.222 | 1.098 | 1.318 | 0.7 | 0.666 | 0.764 |
| 1∶16000 | 0.748 | 0.687 | 0.837 | 0.62 | 0.6 | 0.512 |
| 1∶32000 | 0.501 | 0.402 | 0.52 | 0.36 | 0.352 | 0.356 |
| 1∶64000 | 0.368 | 0.38 | 0.388 | 0.23 | 0.224 | 0.27 |
| 1∶128000 | 0.292 | 0.294 | 0.255 | 0.12 | 0.118 | 0.14 |
| 1∶256000 | 0.22 | 0.202 | 0.224 | 0.13 | 0.13 | 0.13 |
| 1∶512000 | 0.122 | 0.112 | 0.135 | 0.11 | 0.077 | 0.122 |
| 1∶1024000 | 0.065 | 0.068 | 0.061 | 0.098 | 0.073 | 0.116 |
| (-) | 0.068 | 0.062 | 0.063 | 0.1 | 0.065 | 0.106 |
The aminoacid sequence of BNP (B-type natriuretic peptide, brain natriuretic peptide) is followed successively by SSKMYQGSGCPGFKMDRLSSSSGLGCKVLRRR from the aminoterminal to the carboxyl, be the peptide that contains 32 amino-acid residues, and the BNP relative molecular mass is 3.46 * 10
3, it also is not enough to go immune animal as a complete antigen, need with carrier protein couplet to obtain immunogenicity.Adopt bi-functional cross-linking agent method and serum albumin coupling.Concrete steps are as follows:
1) gets brain and receive element (BNP) 1mg, be dissolved in the 1ml bi-distilled water; The homemade albumin rabbit serum of 10mg is dissolved in the PBS that 1ml concentration is 1mol/L (pH 7.5);
2) haptens solution and albumin rabbit serum solution vibration mixing;
3) be added dropwise to 0.25% (volume ratio) glutaraldehyde 1ml while stirring.Finish back restir 5～10min, after at room temperature continuing to react 2～3h, identify coupling efficiency, promptly can be used for immune animal and use with electrophoretic method.
Adopt the coupling bovine serum albumin that uses the same method and receive element, use as animal immune with the reorganization brain.
Adopt the ordinary method immune animal, immunity divides two groups, three rabbits of every group of immunity.
Albumin rabbit serum group: adopt and go immune animal from body albumin and BNP link coupled antigen;
Bovine serum albumin group: adopt bovine serum albumin and BNP link coupled antigen to go immune animal;
Two groups packet numbering is the same.
After three immunity, every rabbit auricular vein is got blood 5ml, and directly the ELISA method is surveyed antiserum titre, and method is as follows:
1) bag quilt: with pH 9.6, concentration is that the CBS of 0.05M dilutes BNP to 2ug/ml, every hole 100 μ l, 4 ℃ of bag quilts that spend the night;
2) 5%PBST skim-milk, 37 ℃ were sealed one hour down;
3) antiserum(antisera) dilution, two groups of serum carry out the twice gradient dilution all since 1: 1000 with the 5%PBST skim-milk, have 12 extent of dilution, and each extent of dilution is set up two multiple holes.Negative serum is done same dilution as negative control;
4) behind the application of sample, 37 ℃ were reacted one hour down;
5) two is anti-with the dilution in 1: 10000 of 5%PBST milk powder, and 37 ℃ were reacted one hour down behind the application of sample;
6) add TMB colour developing 10 minutes, 2M sulfuric acid stops microplate reader 450nm place colorimetric.
The results are shown in Figure 4, table 2, as can be seen from the chart with the autologous protein coupling after immune animal, the anti-BNP of acquisition is sero-fast to tire and is higher than the antiserum(antisera) that adopts bovine serum albumin coupling BNP immune animal to obtain.
Table 2
| |
||||||
| The serum thinning ratio | RSA-1# | RSA-2# | RSA-3# | BSA-1# | BSA-2# | BSA-3# |
| 1∶1000 | 2.79 | 2.731 | 2.531 | 2.991 | 2.991 | 2.841 |
| 1∶2000 | 2.759 | 2.75 | 2.55 | 2.147 | 2.613 | 2.463 |
| 1∶4000 | 2.608 | 2.659 | 2.459 | 1.857 | 1.906 | 1.756 |
| 1∶8000 | 2.173 | 2.121 | 1.921 | 1.269 | 1.214 | 1.064 |
| 1∶16000 | 1.586 | 1.736 | 1.536 | 0.581 | 0.991 | 0.841 |
| 1∶32000 | 0.948 | 1.055 | 0.855 | 0.275 | 0.311 | 0.161 |
| 1∶64000 | 0.452 | 0.58 | 0.38 | 0.142 | 0.198 | 0.048 |
| 1∶128000 | 0.305 | 0.278 | 0.078 | 0.082 | 0.112 | 0.142 |
| 1∶256000 | 0.177 | 0.179 | 0.199 | 0.095 | 0.089 | 0.119 |
| 1∶512000 | 0.111 | 0.11 | 0.13 | 0.073 | 0.071 | 0.101 |
| 1∶1024000 | 0.074 | 0.078 | 0.098 | 0.068 | 0.065 | 0.095 |
| (-) | 0.066 | 0.064 | 0.084 | 0.056 | 0.056 | 0.086 |
From the result as can be seen with the autologous protein coupling after immune animal, its sero-fast tiring is higher than the bovine serum albumin control group.This be since autologous protein with hapten conjugation after immune animal, body is can not produce antibody at the albumen of self, the antibody that is produced is all at micromolecular compound and little peptide.Therefore adopt autologous protein as carrier proteins, prepared hapten antibody is tired higher, and specificity is better.
Claims (6)
1. the preparation method of a hapten antibody, it is characterized in that, may further comprise the steps: at first, adopt the animal autologous protein to get the coupling complete antigen, make hapten antibody with coupling complete antigen immune animal then as carrier proteins and haptens generation linked reaction.
2. according to the preparation method of claim 1, it is characterized in that described carrier proteins is to take from the autologous protein that is used to immune animal.
3. according to the preparation method of claim 2, it is characterized in that described autologous protein is the high-abundance proteins in the serum.
4. according to the preparation method of claim 3, it is characterized in that described high-abundance proteins is albumin or gamma Globulin.
5. according to the preparation method of claim 1, it is characterized in that described preparation method is a Polyclonal Antibody Preparation.
6. according to the preparation method of claim 1, it is characterized in that described preparation method is a MONOCLONAL ANTIBODIES SPECIFIC FOR, immune animal comprises mouse, rat and rabbit.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110317255A (en) * | 2019-07-19 | 2019-10-11 | 北京工商大学 | αs1The monoclonal antibody and milk allergen detection method that the epitope of casein is prepared |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317255A (en) * | 2019-07-19 | 2019-10-11 | 北京工商大学 | αs1The monoclonal antibody and milk allergen detection method that the epitope of casein is prepared |
| CN110317255B (en) * | 2019-07-19 | 2020-07-31 | 北京工商大学 | αs1Monoclonal antibody prepared from casein epitope and method for detecting cow milk allergen |
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Effective date of registration: 20090213 Address after: Room 1011, 501 Harley Road, Shanghai, Pudong New Area: 201203 Applicant after: Shanghai Fuchun Kexin biotech Co. Ltd. Address before: Room 1011, 501 Harley Road, Shanghai, Pudong New Area: 200203 Applicant before: Shanghai Fuchun Zhongnan Biotechnology Co., Ltd. |
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Owner name: SHANGHAI FUCHUN KEXIN BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: SHANGHAI FUCHUN ZHONGNAN BIOTECHNOLOGY CO., LTD. Effective date: 20090213 |
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