earticle

영어

Aflatoxin B1 (AFB1) is the most toxic component one of mycotoxins present in food and feed. Immunological detection of low molecular weight toxins, such as AFB1 using a single-chain variable fragment (scFv), is a potentially novel and safe method of diagnosing fungal infection and food contamination. Surface plasmon resonance (SPR) studies showed that its cognate scFv (82.7 X 10-7 M) has about 118-fold lower KD than the parental mAb 2c12 (0.7 X 10-8 M). To develop an AFB1 detection protein chip using scFv, a random mutagenic scFv library displayed on the surface of yeast was constructed. Five mutated scFv clones with high binding affinity to AFB1 were sorted from this library by a fluorescence activated cell sorter (FACS). The most sensitive scFv (37M-scFv) contained 6 substitutions in amino acid, compared with the wild-scFv (WT-scFv) and was characterized to retain almost the same specificities to other mycotoxins as its parental antibody 2c12 and have KD (9.2 X 10-8 M) 9-fold lower than WT-scFv. The binding affinity of 37MscFv was more improved by replacing Ala at the position 108 in the heavy chain
with Thr (VH:A108T), which is known to be a framework region-4 (FR-4) of murine antibody. Finally, the BM3-scFv, reverse-mutated to Thr at the position VH:108 has the highest binding affinity (2.7 X 10-8 M) which is approximately 31- fold lower than that of WT-scFv. Even though Thr at the position VH:108, located in FR-4 of the heavy chain, connecting the variable region with the constant region also played an important role for sensitivity of anti-AFB1 scFv in this reverse
mutation study and should be conserved for binding with AFB1.