Derivation and properties of recombinant Fab antibodies to coplanar polychlorinated biphenyls.
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 papain 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.
Chiu
Y W
YW
Department of Nutritional Sciences, University of California, Berkeley 94720, USA.
Chen
R
R
Li
Q X
QX
Karu
A E
AE
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D017931:DNA Primers; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011078:Polychlorinated Biphenyls / Q000032:analysis* / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D011078:Polychlorinated Biphenyls