| Aflatoxin B1 (AFB1), a secondary metabolite of Aspergillus parasiticus and A. flavus, is highly carcinogenic, mutagenic, and teratogenic to human beings and animals. AFBi is considered as a unavoidable, naturally occurring contaminant of agricultural products such as corn, beans, peanuts and dried fruits, so the maximum levels of AFB1 in foodstuffs have been legislated in almost all the countries in the world to reduce AFB1 exposure. Many methods for AFB1 detection have been developed, among which the immununoassay has gained wide application due to its sensitivity, specificity and rapidity in the past 20 years. Standard analytes of AFB1 are needed in almost all assays, which leads an exposure to AFB1. Moreover, AFB1 is expensive and hard to obtain, therefore, it is of great significance to find a substitute for AFB1.With reference to Jerne’s immune network theory, anti-idiotypic antibody (Ab2), classified into four types (Ab2α, Ab2β,Ab2Υ, Ab2δ), is a kind of antibody produced against idiotype of antibody (Abl). Ab2p, which is capable of inhibiting the binding of antigen to Abl, bears an idiotype that mimics the structure of antigen epitope and resembles an internal image of antigen. Consequently, Ab2p can be employed for substituting AFB1 as the standard substance to develop innocuous immunoassay without using AFB1. Several relevant researches have been reported, however, they were mainly focused on anti-idiotypic polyclone antibody (PcAb2).In the present study, PcAb2 was generated by immunizing mice with Fab fragment, which was prepared by papain digestion of rabbit antibody against AFB1. Afterward, anti-idiotypic monoclonal antibody (McAb2) was prepared and its application for AFB1 substitution was subjected to analysis. The results were as follows:1. Preparation of Fab fragmentsIn this research, ammonium sulfate precipitation with saturation at 50% and 33% was applied to purify the anti-AFB1 IgG from rabbit. According to the SDS-PAGE, the IgG obtained was electrophoretically pure, and a slight decrease of the sensitivity from 2.41 ng/mL to 6.55 ng/mL after purification was observed in indirect competitive ELISA.Afterward, papain was employed to digest the purified anti-AFB1 IgG and parameters like quantity of papain, cysteine addition, pH and hydrolysis time were analyzed to optimize the digestion. The results indicated that the optimum digestion condition was as follows:enzyme/antibody (w/w) at 1/50,18 mmol/L cysteine, pH 6.5,37℃ water bath for 30 minutes.Papain digestion was performed following the conditions mentioned above, and then protein A affinity column was used to purify the digestion products. The final yield of Fab is 0.35-0.45 g/g IgG. SDS-PAGE confirmed that the Fab fragment obtained was electrophoretically pure. What’s more, the indirect competitive ELISA results implied that the sensitivity of the Fab fragment was 0.77 ng/mL, which increased by 7.5 times against the purified anti-AFB1 IgG (6.55 ng/mL).2. Preparation and characterization of PcAb2PcAb2 was produced by immunization of mice with either the whole anti-AFB1 IgG (Ab1) (mouse No.1-3) or the Fab fragment (mouse No.4-6). The indirect non-competitive ELISA was performed to monitor the antibody production and results showed that the titer of antiserum in mouse No.2 was the highest (4.7×104) after the fourth immunization with IgG, while the titer of antiserum in mouse No.6 was the highest (2.5×105) after the fourth immunization with Fab.Indirect competitive ELISA was performed to investigate the internal image relationship between PcAb2 and AFB1, and the results implied that both PcAb2 and coating antigen AFB1-O-OVA could bind with the determinant of Abl, which verified their internal image relationship.3. Preparation and characterization of McAb2Fab fragments of Ab1 were employed to immunize mice, then their spleen cells after the fourth immunization and myeloma cells (SP2/0) were fused. The cell fusion rate was 63% and indirect non-competitive ELISA was performed to isolate the positive clone cells capable of secreting specific antibodies, the positive rate was approximately 2.5%. One positive clone cell line was obtained by the limiting dilution method, which was labeled as 2F5.McAb2 was produced by monolayer cell culture method and animal inducing method, the titer of culture supernatant was 2800 with affinity constant of 4.88×109 mol/L and the titer of ascites supernatant was 24500. Indirect competitive ELISA was performed to investigate the internal image relationship between McAb2 from ascites and AFB1 and the results revealed that both the McAb2 and coating antigen AFB1-O-OVA could competitively bind with the determinant of Ab1. By comparing the concentration of AFB1 and McAb2 with the same inhibition rate in competitive ELISA, the linear regression equation between the alignment of AFB1 and McAb2 was:y= 6.10 ×10-5x-1.56 (R2= 0.981) (x:the concentration of McAb2, ng/mL; y:the concentration of AFB1, ng/mL).With the analysis of AFB1 spiked recovery experiment by employing AFB1 and McAb2 as competitive antigen, respectively, the results showed that there was no significant statistics difference when using AFB1 or McAb2 as competitive antigen. Therefore, AFB1 can be substituted by McAb2 as a standard in ELISA of AFB1. |
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