Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 48, Issue 6, Pages 2614-2624Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jf991208a
Keywords
recombinant Fab antibody; PCB; polychlorinated biphenyl; conger-specific; ELISA
Funding
- NIEHS NIH HHS [ES01896] Funding Source: Medline
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Recombinant Fab antibodies (rFabs) specific for coplanar polychlorinated biphenyls (PCBs) were derived from a hybridoma cell line (Chiu et al. Anal. Chem. 1995, 67, 3829-3839). Immunoglobulin V-H-C-H1 and V-L-C-L sequences from S2B1 messenger RNA were amplified by PCR and cloned into the M13 phagemid vector pComb3H. Phage displaying rFab were enriched by panning on a PCB hapten conjugate and expressed as soluble rFabs in Escherichia coli XL-1 Blue. Two rFab clones competitively bound PCBs 77 and 126 with half-maximal inhibition (I-50) of 10-13 ppb in indirect and direct enzyme immunoassays (EIAs), with selectivity nearly identical to that of whole S2B1 IgG and its Fab fragments prepared by papa-in digestion. These results, and comparison of N-terminal amino acid sequences of MAb S2B1 and the rFab, indicated that rFab S2B1 is a functional copy of the MAb. The rFab S2B1 sequences have 75-89% sequence identity with antibodies that bind nitrophenyl haptens and are being used to construct a three-dimensional computational model of the PCB binding site.
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