CN101293912A - Conjugate for contructing cationised carrier protein and aflatoxin B1 with one-step method - Google Patents

Abstract

The invention utilizes amine methylation reaction principle to synthesize a conjugate of aflatoxin B1 and a cationized carrier protein by one step method via condensation reaction between the alpha-reactive hydrogen of the carbonyl group in the structure of the aflatoxin B1 and the aminoethyl group in a cationized protein by using formaldehyde as a coupling agent. Compared with the two-step method in the prior art, which firstly converts the aflatoxin B1 to a carboxyl active substance and then condensates and couples with a carrier protein, the inventive method has the advantages of less steps, simple operation, and high utilization ratio.

Description

Single stage method makes up cationization carrier proteins and AFB 1Conjugate

Technical field

The present invention relates to proteic preparation of cation carrier and AFB ₁The single stage method of-cationic protein conjugate makes up.The conjugate that makes up as the immunogen immune animal, can be improved the immunne response level of body, prepare high-quality aspergillus flavus resisting toxin B ₁Antibody.

Background technology

(Aflatoxin is the poisonous secondary metabolite that a class is mainly produced by Aspergillus parasiticus (Aspergillus parasiticus) and the malicious flavus (A.flavus) of product AFT), wherein with AFB to aflatoxin ₁(aflatoxin B ₁, AFB ₁) the harm maximum, be one of carcinogens of present known toxicity maximum.International cancer research institution has classified it as I level carcinogens.Because AFB ₁The approach of contaminated food products is a lot, therefore will stop AFB fully ₁Pollution be impossible, improve measures for the supervision and adopt the science detection method can reduce AFB to the full extent ₁Harm to human health.Present AFB ₁Detection method mainly contain based on physics and chemistry and the immunity analytical procedure, the former comprises thin layer chromatography, high performance liquid chromatography, mass spectrum and capillary electrophoresis etc., thin layer chromatography detects AFB at present as China ₁National standard method, though easy and simple to handle, with low cost, owing to be semi-quantitative method, sensitivity is low, has been difficult to satisfy the needs of low detectability; Though and highly sensitive, the favorable reproducibility of other instrument analytical methods exists consuming time, shortcomings such as detecting instrument is expensive, complicated operation simultaneously, can not satisfy the demand of field quick detection; And immunological detection methods such as enzyme linked immunological absorption reaction, immune microtrabeculae and immuno-chip because save time, cost is low, easy and simple to handle, be fit to batch detection and on-the-spot the detection, just progressively be subjected to the attention of each quality inspection relevant departments of China.

AFB ₁As haptens, need with its with carrier protein couplet after, just can possess the immunogenicity that stimulates animal generation antibody.But since complete antigen preparation process complexity, AFB ₁Utilization ratio is low, therefore usually needs to use relatively large AFB ₁Just can prepare corresponding antibody; Yet, after the September 11 attacks, AFB ₁Biological reagent as a kind of severe toxicity is controlled by the strictness of developed countries such as the U.S..Not only price is high but also be difficult to buy for these toxin standard substance on the market, and this gives research AFB ₁Immunological detection method bring very big difficulty, also hindered the application and the popularization of immunological method to a certain extent.

AFB is thought in early stage research ₁Character torpescence own does not possess the active group that is connected with carrier proteins, after it need being derived, again with carrier protein couplet, promptly earlier with AFB ₁Be converted into AFB ₁-activated carboxylic thing (AFB ₁-Oxime, AFB ₁-O), then, carbodiimide (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide, EDC) wait under the catalysis of coupling agent, with carrier proteins generation condensation reaction, form stable AFB ₁-O-carrier protein couplet thing.Wherein, the first step AFB ₁Transformation efficiency be 70%～80%, AFB ₁The purifying yield of-O is 80%～90%, and only there was 30%～40% AFB in second step ₁-O is coupled on the carrier proteins.Therefore, AFB ₁Overall utilization about 20%, utilization ratio is lower.

Comprehensive above-mentioned reason, research improves AFB ₁The conjugate preparation method of utilization ratio is very important.

The present invention has utilized the cation carrier proteinogenic amines principle that methylates to make up AFB ₁The proteic conjugate of-cation carrier.At AFB ₁Structure in because the α-reactive hydrogen of its carbonyl under mild acid conditions, exists with enol form, can be by the coupled action of formaldehyde, amine-methylated coupling takes place with the amine ethyl of carrier proteins.Because the proteic carboxyl of cation carrier is almost all replaced by the amine ethyl, thereby whole protein band positive electricity, during as the complete antigen immune animal, than common proteic conjugate, cation carrier albumen is easier to be combined with the surface of cell membrane of the weak negative electricity of band in the body, and then discerned and the intravital immunne response of stimulation machine by antigen presenting cell, simultaneously, also improved body to covalent coupling at cation carrier albumen haptens (AFB ₁) identification and reply level, can the stimulator antigen presenting cells to the propagation of offering, promote T lymphocyte and bone-marrow-derived lymphocyte of haptenic group.

AFB among the present invention ₁Overall utilization about 50%.Experiment showed, that the conjugate by this method preparation has better immunogenicity, can stimulate body to produce high-quality AFB ₁Antibody.Therefore, the present invention is with respect to prior synthesizing method, has that operation steps is few, a toxin utilization ratio height, antibody titer advantages of higher.

This patent inventor has invented with one step of cation carrier albumen structure AFB at the problems that two-step approach exists ₁The method of-carrier protein couplet thing.Correlative study does not appear in the newspapers both at home and abroad as yet.

The main reference document

[1]Apple，R.J.，Domen，P.L.，Muckerheide，A.and?Michael，J.G.(1988)Cationization?of?protein?antigens.IV.Increased?antigen?uptake?by?antigen-presenting?cells.J?Immunol?140，3290-5.

[2]Barbiroli，A.，Bonomi，F.，Benedetti，S.，Mannino，S.，Monti，L.，Cattaneo，T.and?Iametti，S.(2007)Bindingof?aflatoxin?M1?to?different?protein?fractions?in?ovine?and?caprine?milk.J?Dairy?Sci?90，532-40.

[3]Chu，F.S.，Lau，H.P.，Fan，T.S.and?Zhang，G.S.(1982)Ethylenediamine?modified?bovine?serum?albumin?asprotein?carrier?in?the?production?of?antibody?against?mycotoxins.J?Immunol?Methods?55，73-8.

[4]Domen，P.L.，Muckerheide，A.and?Michael，J.G.(1987)Cationization?of?protein?antigens.III.Abrogation?oforal?tolerance.J?Immunol?139，3195-8.

[5]Fusheng，C.，Xinchang?Luo，Qi?Zhou.(1999)Immunological?detection?of?afltatoxin?B1?I：preparation?ofaflatoxin?B1?antigen.Mycosystema?18，316～320.(in?Chinese)

[6]Gathumbi，J.K.，Usleber，E.，Ngatia，T.A.，Kangethe，E.K.and?Martlbauer，E.(2003)Application?ofimmunoaffinity?chromatography?and?enzyme?immunoassay?in?rapid?detection?of?aflatoxin?B1?in?chickenliver?tissues.Poult?Sci?82，585-90.

[7]Jaimez，J.，Fente，C.A.，Vazquez，B.I.，Franco，C.M.，Cepeda，A.，Mahuzier，G.and?Prognon，P.(2000)Application?of?the?assay?of?aflatoxins?by?liquid?chromatography?with?fluorescence?detection?in?food?analysis.Journal?of?Chromatography?A?882，1-10.

[8]Muckerheide，A.，Apple，R.J.，Pesce，A.J.and?Michael，J.G.(1987a)Cationization?of?protein?antigens.I.Alteration?of?immunogenic?properties.J?Immunol?138，833-7.

[9]Muckerheide，A.，Domen，P.L.and?Michael，J.G.(1987b)Cationization?of?protein?antigens.II.Alteration?ofregulatory?properties.J?Immunol?138，2800-4.

[10]Ramos?Catharino，R.，de?Azevedo?Marques，L.，Silva?Santos，L.，Baptista，A.S.，Gloria，E.M.，Calori-Domingues，M.A.，Facco，E.M.and?Eberlin，M.N.(2005)Aflatoxin?screening?by?MALDI-TOF?massspectrometry.Anal?Chem?77，8155-7.

[11]Saha，D.，Acharya，D.，Roy，D.，Shrestha，D.and?Dhar，T.K.(2007)Simultaneous?enzyme?immunoassay?forthe?screening?of?aflatoxin?B1?and?ochratoxin?A?in?chili?samples.Anal?Chim?Acta?584，343-9.

[12]Sapsford，K.E.，Taitt，C.R.，Fertig，S.，Moore，M.H.，Lassman，M.E.，Maragos，C.M.and?Shriver-Lake，L.C.(2006)Indirect?competitive?immunoassay?for?detection?of?aflatoxin?B1?in?corn?and?nut?products?using?thearray?biosensor.Biosensors?and?Bioelectronics?21，2298-2305.

Summary of the invention

(1) technical problem that will solve

The purpose of this invention is to provide an a kind of step structure AFB ₁The method of-carrier protein couplet thing.

(2) technical scheme

For achieving the above object, the technical solution used in the present invention is:

Enol or contain the carbonyl compound of α-reactive hydrogen under the effect of coupling agent formaldehyde, can carry out coupling with primary amine or secondary amine.

According to this principle, AFB ₁α-the reactive hydrogen of last carbonyl just can react with the cationic protein that contains the amine ethyl under the coupled action of formaldehyde, obtains AFB ₁The conjugate of-cationic protein.Cationization albumen is for simple albumen, has more amine ethyl, thereby whole protein band positive electricity, during as the complete antigen immune animal, than common proteic conjugate, cation carrier albumen is easier to be combined with the surface of cell membrane of the weak negative electricity of band in the body, and then is discerned and the intravital immunne response of stimulation machine by antigen presenting cell.

Concrete steps:

1. the preparation of cationic protein

Carrier proteins comprises: bovine serum albumin (Bovine serum albumin, BSA), oralbumin (Ovalbumin, OVA), horseradish peroxidase (Horseradish peroxidase, HRP), blood hole spoon albumen (Keyhole limpethemocyanin, KLH) and poly-lysine (Ploy-l-lysine, PLL).

Preparation process:

(1) with quadrol (ethylenediamine, (2-(N-morpholino) ethane sulfonic acid MES) in the damping fluid, and regulates pH to slightly acidic with HCl EDA) to join 2 ethane sulfonic aicd base in the ice bath;

(2) carrier proteins is dissolved in MES after, with above-mentioned EDA solution mixing;

(3) drip coupling agent EDC in (2) described solution, behind the room temperature reaction, use the acetic acid termination reaction;

(4) above-mentioned reactant is fully dialysed with deionized water;

(5) with after its freeze-drying, freeze-drying is standby.

Used EDA volume between 20 μ L～100 μ L, used carrier proteins quality between 5mg～100mg, the volume ratio of EDA and MES between 1: 25～1: 5, the pH regulator to 4 of MES damping fluid～6.

Prepare positively charged ion bovine serum albumin (Cationized bovine serum albumin, cBSA), positively charged ion oralbumin (Cationized ovalbumin, cOVA), positively charged ion horseradish peroxidase (Cationized horseradishperoxidase, cHRP), positively charged ion blood hole spoon albumen (Cationized keyhole limpet hemocyanin, cKLH) or the positively charged ion poly-lysine (Cationized ploy-l-lysine, cPLL).

2.AFB ₁The preparation of-cation carrier protein conjugate

AFB ₁-cation carrier protein conjugate comprises AFB ₁-cBSA, AFB ₁-cOVA, AFB ₁-cHRP, AFB ₁-cKLH and AFB ₁-cPLL.

Preparation process:

(1) with cation carrier albumen and AFB ₁Respectively with after deionized water and the dimethyl formamide dissolving, with AFB ₁Solution dropwise joins mixing in the carrier proteins liquid;

(2) in above-mentioned reaction solution, add coupling agent formaldehyde, promptly obtain AFB after the reaction ₁-cation carrier protein conjugate;

(3) fully dialyse with deionized water after, freeze-drying is standby.

Wherein, cation carrier albumen and AFB ₁Mass ratio between 1: 1～5: 1, the volume ratio of dimethyl formamide and formaldehyde is between 1: 1～5: 1.

(3) beneficial effect

The present invention is with AFB ₁Be haptens, utilize the cationic protein single stage method to synthesize AFB ₁-carrier protein couplet thing, this method have simple, the AFB of step ₁The utilization ratio advantages of higher has been avoided shortcomings such as present used two-step approach step is many, utilization ratio is low.

Description of drawings

Below in conjunction with drawings and Examples patent of the present invention is further specified.

Figure 1A FB ₁Structure

The cationization of Fig. 2 carrier proteins

Fig. 3 AFB ₁With the proteic coupling mechanism of cation carrier

1. AFB ₁Enolization in weak acidic buffer;

2. the imines ionic forms in the cation carrier albumen;

3. the imines ion in the cation carrier albumen is under the formaldehyde effect, with enolization AFB ₁Coupling

Embodiment

Embodiment 1AFB ₁The preparation of-cBSA conjugate

1.1cBSA preparation:

50～100 μ LEDA are joined in the 500 μ L ice bath MES damping fluids, and with HCl with pH regulator to 5.5 about, again 5～100mg BSA is dissolved in the MES damping fluid of 50 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.

1.2AFB ₁The preparation of-cBSA conjugate:

CBSA and 2mg AFB with 5～100mg ₁Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB ₁Solution dropwise joins mixing in the above-mentioned protein soln, adds 50～1000 μ L formaldehyde then rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.

Embodiment 2AFB ₁-cOVA conjugate preparation

2.1cOVA preparation:

50～100 μ L EDA are joined in the 500 μ L ice bath MES damping fluids, and with HCl with pH regulator to 5.5 about, again 5～100mgOVA is dissolved in the above-mentioned damping fluid of 100 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.

2.2AFB ₁The preparation of-cOVA conjugate:

COVA and 1～50mg AFB with 5～100mg ₁Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB ₁Solution dropwise joins mixing in the above-mentioned protein soln, adds 50～1000 μ L formaldehyde then rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.

Embodiment 3AFB ₁The preparation of-cHRP conjugate

3.1cHRP preparation:

50～100 μ L EDA are joined in the 500 μ L MES damping fluids of ice bath, and with HCl with pH regulator to 5.5 about, again 5～100mgHRP is dissolved in the above-mentioned MES damping fluid of 500 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.

3.2AFB ₁The preparation of-cHRP conjugate:

CHRP and 1～50mg AFB with 5～100mg ₁Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB ₁Solution dropwise joins in the above-mentioned protein soln behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.

Embodiment 4AFB ₁The preparation of-cKLH conjugate

4.1cKLH preparation:

50～100 μ L EDA are joined in the 500 μ L MES damping fluids of ice bath, and with HCl with pH regulator to 5.5 about, again 5～100mg KLH is dissolved in the above-mentioned MES damping fluid of 800 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.

4.2AFB ₁The preparation of-cKLH conjugate:

CKLH and 1～50mg AFB with 5～100mg ₁Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB ₁Solution dropwise joins mixing in the above-mentioned protein soln, adds 50～1000 μ L formaldehyde then rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.

Embodiment 5AFB ₁The preparation of-cPLL conjugate

5.1cPLL preparation:

50～100 μ L EDA are joined in the 500 μ L MES damping fluids of ice bath, and with HCl with pH regulator to 5.5 about, again 5～100mg PLL is dissolved in the above-mentioned MES damping fluid of 1000 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.

5.2AFB ₁The preparation of-cPLL conjugate:

CPLL and 1～50mg AFB with 5～100mg ₁Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB ₁Solution dropwise joins mixing in the above-mentioned protein soln, adds 50～1000 μ L formaldehyde then rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.

Above-mentioned used MES buffer concentration is 0.1mol/L, and HCl is 1mol/L, and acetic acid is 4mol/L.

AFB ₁The evaluation of-cationic protein conjugate:

Adopt ultraviolet spectrophotometer to cation carrier albumen, AFB ₁Mark product and conjugate scan, and determine according to its charateristic avsorption band whether coupling is successful.

The result shows, AFB ₁-cation carrier protein conjugate has two main absorption peaks at 270nm and 366nm, and previous absorption peak is carrier proteins (280nm) and AFB ₁Stack peak (266nm), and the latter is AFB ₁Another feature absorption peak (363nm).According to the coupling ratio formula,, can calculate AFB in conjunction with absorption peak wavelength and absorbancy on the uv-spectrogram ₁Coupling ratio with cationic protein.

Formula:

Wherein: AFB in the conjugate ₁Molar mass;

M _{Carrier proteins}: the molar mass of carrier proteins in the conjugate;

A _{Conjugate 278nm}: conjugate is in the absorbancy at 278nm place;

A _{Conjugate 366nm}: conjugate is in the absorbancy at 366nm place;

AFB ₁Molar absorptivity at the 278nm place;

AFB ₁Molar absorptivity at the 366nm place;

ε _{Carrier proteins 278nm}: carrier proteins is at the molar absorptivity at 278nm place

Through calculating AFB ₁With the coupling ratio of cationic protein about 6.1, proved successfully to prepare AFB ₁-cationic protein conjugate.

Claims (6)

1. cationic protein is characterized in that structural formula:

Wherein, the carboxyl of carrier proteins is almost all replaced by the amine ethyl, whole protein band positive electricity, thereby during as the complete antigen immune animal, cation carrier albumen is easier to be combined with the surface of cell membrane of the weak negative electricity of band in the body, and then is discerned and the intravital immunne response of stimulation machine by antigen presenting cell.

2.AFB ₁-cationic protein conjugate is characterized in that its molecular structural formula is:

Wherein, carrier proteins can be positively charged ion bovine serum albumin (Cationized bovine serum albumin, cBSA), positively charged ion oralbumin (Cationized ovalbumin, cOVA), positively charged ion horseradish peroxidase (Cationizedhorseradish peroxidase, cHRP), positively charged ion blood hole spoon albumen (Cationized keyhole limpet hemocyanin, cKLH) or positively charged ion poly-lysine (Cationized ploy-L-lysine, cPLL), respectively with AFB ₁In conjunction with obtaining AFB ₁-cBSA, AFB ₁-cOVA, AFB ₁-cHRP, AFB ₁-cKLH and AFB ₁-cPLL.

3. prepare the proteic method of the described a kind of cation carrier of claim 1, it is characterized in that comprising step:

(1) preparation cBSA, step is as follows:

With 20～100 μ L quadrol (ethylenediamine, EDA) join 500 μ L 0.1mol/L 2 ethane sulfonic aicds (2-(N-morpholino) the ethane sulfonic acid of ice bath, MES) in the damping fluid, and with 1mol/L HCl with pH regulator to 4～6, then, after 5～100mg BSA being dissolved in the above-mentioned MES damping fluid of 50 μ L, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add again 2～50mg the inferior diamines of carbonization (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide, EDC), stirring at room 1～12h is with the acetic acid termination reaction of 4mol/L, fully dialyse behind 48～72h with deionized water, its cold storage is standby.

(2) preparation cOVA, step is as follows:

20～100 μ L EDA are joined in the 500 μ L 0.1mol/L MES damping fluids of ice bath, and with 1mol/L HCl with pH regulator to 4～6, then, after 5～100mg OVA being dissolved in the above-mentioned MES damping fluid of 100 μ L,, in above-mentioned mixed solution, add the EDC of 2～50mg again with above-mentioned EDA solution mixing, stirring at room 1～12h, with the acetic acid termination reaction of 4mol/L, fully dialyse behind 48～72h with deionized water, its cold storage is standby.

(3) preparation cHRP, step is as follows:

20～100 μ L EDA are joined in the 500 μ L 0.1mol/L MES damping fluids of ice bath, and with 1mol/L HCl with pH regulator to 4～6, then, after 5～100mg HRP being dissolved in the above-mentioned MES damping fluid of 500 μ L,, in above-mentioned mixed solution, add the EDC of 2～50mg again with above-mentioned EDA solution mixing, stirring at room 1～12h, with the acetic acid termination reaction of 4mol/L, fully to dialyse behind 48～72h with deionized water, cold storage is standby.

(4) preparation cKLH, step is as follows:

20～100 μ L EDA are joined in the 500 μ L 0.1mol/L MES damping fluids of ice bath, and with 1mol/L HCl with pH regulator to 4～6, after 5～100mg KLH being dissolved in the above-mentioned MES damping fluid of 800 μ L again, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, with the acetic acid termination reaction of 4mol/L, fully dialyse behind 48～72h with deionized water, cold storage is standby.

(5) preparation cPLL, step is as follows:

20～100 μ L EDA are joined in the 500 μ L 0.1mol/L MES damping fluids of ice bath, and with 1mol/L HCl with pH regulator to 4～6, again 5～100mg PLL is dissolved in the above-mentioned MES damping fluid of 1000 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, with the acetic acid termination reaction of 4mol/L, with the deionized water 48～72h that fully dialyses, cold storage is standby at last.

Used EDA volume between 20 μ L～100 μ L, used carrier proteins quality between 5mg～100mg, the volume ratio of EDA and MES between 1: 25～1: 5, the pH regulator to 4 of MES damping fluid～6.

4. as a kind of AFB as described in the claim 2 ₁The preparation method of-cation carrier protein conjugate is characterized in that AFB ₁α-the reactive hydrogen of last carbonyl can be under the coupled action of formaldehyde and the amine ethyl reaction of cationic protein, obtains AFB ₁The proteic conjugate of-cation carrier can prepare the AFB that comprises immunizing antigen, envelope antigen and enzyme labelling thing ₁-cationic protein conjugate obtains AFB ₁Complete antigen.

5. as a kind of AFB as described in the claim 2 ₁The preparation method of-cation carrier protein conjugate is characterized in that coupling agent formaldehyde.

6. method according to claim 4 is characterized in that comprising step:

(1) AFB ₁The preparation of-cBSA, step is as follows: with 5～100mg cBSA and 1～50mg AFB ₁Respectively with 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with AFB ₁Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction ₁-cBSA fully dialyses behind the 48h with deionized water, and freeze-drying is standby.

(2) AFB ₁The preparation of-cOVA, step is as follows: with cOVA and the 1～50mg AFB of 5～100mg ₁Respectively with 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with AFB ₁Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction ₁-cOVA then fully dialyses behind the 48h with deionized water, and freeze-drying is standby.

(3) preparation of AFB1-cHRP, step is as follows: with cHRP and the 1～50mg AFB of 5～100mg ₁Respectively with 500～10000 μ L deionized waters and the dissolving of 500 μ L dimethyl formamides, again with AFB ₁Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction ₁-cHRP then fully dialyses behind the 48h with deionized water, and freeze-drying is standby.

(4) AFB ₁The preparation of-cKLH, step is as follows: with cKLH and the 1～50mg AFB of 5～100mg ₁Respectively with 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with AFB ₁Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction ₁-cKLH then fully dialyses behind the 48h with deionized water, and freeze-drying is standby.

(5) AFB ₁The preparation of-cPLL, step is as follows: with cPLL and the 1～50mg AFB of 5～100mg ₁Respectively with 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB ₁Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction ₁-cPLL then fully dialyses behind the 48h with deionized water, and freeze-drying is standby.

Wherein, cation carrier albumen and AFB ₁Mass ratio between 1: 1～5: 1, the volume ratio of dimethyl formamide and formaldehyde is between 1: 1～5: 1.

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