Selection of single chain fragment variables with direct coating of aflatoxin B1 to enzyme-linked immunosorbent assay plates.
Most selections of antihapten recombinant antibodies from antibody libraries were against hapten-carrier conjugates, which are different from free haptens in antigen specificity and often mislead the screening due to immunodominant epitopes in carriers. In the present study, aflatoxin B(1) (AFB(1)) was directly coated to enzyme-linked immunosorbent assay (ELISA) plates and anti-AFB(1) single chain fragment variables (scFvs) were selected with the AFB(1)-coated plates. Compared to the selection against AFB(1)-bovine serum albumin conjugate (31 positives out of 46 random clones), the isolated scFvs against AFB(1) (44 positives out of 46 random clones) showed higher specificities for AFB(1). The clone H4 with K(D) of 9.8 x 10(-11) M to AFB(1)-BSA and K(D) of 1.2 x 10(-12) to AFB1 was sequenced. In addition, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was applied to describe the biopanning for the first time. Our research presents a quick and robust selection technique for antihapten recombinant antibody from large naive libraries.
Yang
Lian
L
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, PR China.
Ding
Husheng
H
Gu
Zhennan
Z
Zhao
Jianxin
J
Chen
Haiqin
H
Tian
Fengwei
F
Chen
Yong Q
YQ
Zhang
Hao
H
Chen
Wei
W
D016604:Aflatoxin B1 / Q000032:analysis* / Q000276:immunology*; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D012710:Serum Albumin, Bovine; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D020349:Surface Plasmon Resonance
D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D012710:Serum Albumin, Bovine; D016604:Aflatoxin B1