Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

Abstract

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml (R2 > 0.99) and from 1 to 100 ng/ml (R2 > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

Design of Hapten Synthesis and Antibody Characterization of G-group Aflatoxins

“…With the development of new disciplines such as molecular immunology, quantum chemistry, and molecular mechanics, as well as the popularization and application of molecular information technology, molecular simulation technology, and computer-aided technology, these provide theories and methods for reference to improve the rationality, timeliness and predictability of hapten molecular design and antigen synthesis (Xu et al, 2009;Kortkhonjia et al, 2013;Sevy & Meiler, 2014). Kim et al (2011) used computer-aided technology to prepare TAFs mAbs with strong specificity and broad recognition spectrum. The IC50 for AFB1, AFB2, AFG1, and AFG2 were 4.36, 7.22, 6.61, 29.41 μg/kg -1 , respectively, ELISA detection technique to achieve TAFs mode.…”

Design of antigen synthesis and identification of its artificial antigen for zearalenone

“…2.6 Preparation of ZEN pAb. Referring to the method of Kim S. H. et al (Kim et al, 2011) and improve it. Five female BALB/c mice aged 6-8 weeks were immunized with ZEN-BSA and injected subcutaneously with 50 μg/mouse.…”

Advances in Antibody Preparation Techniques for Immunoassays of Total Aflatoxin in Food

“…The CR values of 1C11 mAb for AFB1, AFB2, AFG1, and AFG2 were 100%, 92.3%, 54.5%, and 6.7%, respectively. Kim et al [ 37 ] used this method to screen the 8H10 hybridoma line with IC50 value for AFB1, AFB2, AFG1, and AFG2 of 4.36, 7.22, 6.61, and 29.41 μg/kg. The CR values of the 8H10 mAb for AFB1, AFB2, AFG1, and AFG2 were 100%, 60.47%, 65.97%, and 14.83%, respectively.…”

“…These continue to be improved with molecular immunology, computational science, quantum chemistry, molecular dynamics, and molecular simulations. Kim et al prepared TAF mAb with strong specificity and a broad recognition spectrum through computer-aided technology and realized TAF immunoassay via ELISA [ 37 ].…”