Preparation And Analysis Of Anti-AFB1 Anti-idiotypic Polyclonal Antibody

Aflatoxin B1 (AFB1), which has been considered as the most important and toxic mycotoxin of more than ten species of isolated aflatoxin until now, is the toxic secondary metabolites mainly produced by the fungi of Aspergillus flavus and A. parasiticus. AFB1 mainly contaminates rice, corn, peanuts, peanut oil, animal liver, dried salted fish, walnuts, coconut milk and other agricultural products. Almost all the countries and regions in the world have regulated the maximum limit for AFB1 in foods and feedstuffs as a mandatory limit standard for detection and control. The mainly detection methods of AFB1 now contain TLC,HPLC, immunoassay such as enzyme-linked immunosorbent-based immunoassay and the bio-sensor method. Although different detecting AFB1 methods have their advantages and disadvantages, a common disadvantage of these methods is to use AFB1 as the standard substance, and many operators have a strong dread to AFB1 owing to its high carcinogenicity. At the same time, AFB 1 standard substance need to be imported from abroad, high prices lead to high testing costs.But a new type of immune reagent--anti-idiotypic antibody(Ab2), because of its internal image relationship with AFB1 toxic molecular on molecular conformation, may be used to substitute AFB 1 for the immunological detection of AFB 1, which can achieve a drug-free detection of AFB 1 and reduce testing costs.The purpose of this study was to prepare anti-idiotypic antibody to replace toxin standard substance AFB1 to establish drug-free ELISA detection system. Firstly, pepsin was used to digest rabbit polyclonal antibody into F(ab’)2, which was purified by sepharose protein A affinity column and identified by SDS-PAGE and agarose double diffusion test. Secondly,fragments and intact rabbit antibody were injected into BALB/c mice, respectively. At last, the variability of sera from different immunogen and the substitution relationship between anti-idiotypic antibody(Ab2) and AFB1 molecule was studied. The main results were as follows:1 The purifying of rabbit polyclonal antibody and optimization for the conditions of pepsin digestion.Saturated ammonium sulfate was utilized for purifying rabbit polyclonal antibody, which derived from one Japanese White Rabbit immunized by AFB1O-BSA. Then the purified antibody was identified by SDS-PAGE and ELISA. Pepsin was used to digest rabbit polyclonal antibody into F(ab’)2, which was purified by sepharose protein A affinity column and identified by SDS-PAGE and agarose double diffusion test.50%,33% and 33% of saturated ammonium sulfate was used to depurate rabbit polyclonal antibody in order, respectively. The recovery was 25.7%. After purified, the titer of antibody was reduced from 7.88×105 to 2.56×105 without any voluminal variance, only 3 times lower than the raw antibody. And also, the sensibility is about 20 ng/mL as same as the undisposed antibody. SDS-PAGE showed that the bulk of hybridprotein was removed, and most of the remaining part was IgG, which achieved electrophoretic purity on the whole.The optimization conditions for the digestion of IgG by pepsin were 0.4 mol/L NaAc, pH 4.0, l:10(w/w) between pepsin and IgG, reaction in 37℃for 4 h. It was only a little decline in the proportion between titer and quality of IgG before or after digestion.2 Preparation and characterization of anti-idiotypic antibodyThe anti-AFB1 polyclonal antibody(Abl) purified previously and F(ab’)2 fragments were used to immunize BALB/c mice to obtain anti-idiotypic antibodies(Ab2). When Ab1 was used as immunogen, the immune dose and immune period were 50μg per mouse and 14 d for three BALB/c mice (namely No.1, No.2 and No.3), respectively. While F(ab’)2 was used as immunogen, the immune dose and immune period were 100μg per mouse and 14 d for three BALB/c mice (namely No.4, No.5 and No.6), respectively. The analysis on process of antibodies production indicated that good immune responses were produced to all the BALB/c mice and Ab2 against Abl or F(ab’)2 were obtained. The properties of Ab2 were studied and the results were as follows.Coating conditions for Abl at different pH values of phosphate buffered saline (PBS) (pH 6.0, pH 7.4 and pH 8.0, separately) and of carbonate buffer solution (CBS) (pH 9.6) were studied. Results showed that pH 8.0 of PBS was the best pH value for coating Abl on ELISA plate and the best coating concentration of Abl was 0.25μg/mL through analysis by indirect imcompetitive ELISA of Ab2 antiserum titers under this pH. Under the coating condition mentioned above, the titer of Ab2 from each BALB/c mouse was investigated. Results showed that the titers of Ab2 from No.1, No.2 and No.3 BALB/c mice were 1:1.02×106, 1:1.02×106, 1:2.05×106, respectively, while the titers of Ab2 from No.4, No.5 and No.6 mice were 1:3.2×104,1:6.4×104,1:1.6xl04, separately, through analysis by indirect imcompetitive ELISA. The results also indicated that the titers of Ab2 immunized with complete antibodies (Ab1) were better than that immunized with F(ab’)2 fragments, which suggested that Fc fragment of antibody might have an effect of enhancing the immune response.In further studies, AFB1-cOVA was chosen to be coating antigen and Abl was used as antibodies to study the properties of Ab2 through indirect competitive ELISA. Results showed that the sensitivity (IC50 value) was 6.82 ng/mL in competitive inhibitory curve when AFB1 was used as inhibitor, while the sensitivity (IC50 value) was 85.51 ng/mL when Ab2 was used as inhibitor, which was equivalent to 6.82 ng/mL of AFB1. In addition, the detection limit of AFB1 and the linear range were 0.01μg/mL and 0.01～100μg/mL, separately, when Ab2 was used as inhibitor. Furthermore, the study on the internal image relationship between Ab2 and AFB1 showed that the structure similarity of AFB1 and Ab2 from No.4 mouse was better than from No.2 mouse through titers analysis, which indicated that Ab2 from F(ab’)2 fragments immunized was better than Ab2 from Abl immunized, as reported in previous research articles.