Comparative sensitivity of immunoassays for haptens using monomeric and dimeric antibody fragments.
A single-chain anti-atrazine antibody fragment, scAb (single-chain Fv with a CK domain), was expressed in Escherichia coli, and monomeric and dimeric species were preferentially purified from periplasmic extracts by chromatography upon nickel chelate immunosorbent columns or by immunoaffinity purification using a constant domain (CK) tag. Recombinant monomeric and dimeric antibody fragments, Fab, and intact monoclonal antibodies were compared in assays by competition between free atrazine in solution and (a) immobilized atrazine-bovine serum albumin conjugate (indirect assay) or (b) atrazine-alkaline phosphatase (direct assay). Recombinant antibody fragments provided a lower detection limit than either Fab or intact monoclonal antibody in both assay formats. Monomeric fragments displayed a sensitivity of detection down to 0.1 ppb, compared to 1.0 ppb for dimeric fragments and the parental monoclonal.
Grant
S D
SD
Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, U.K.
Porter
A J
AJ
Harris
W J
WJ
D001280:Atrazine / Q000276:immunology; D019281:Dimerization; D006241:Haptens / Q000032:analysis*; D007118:Immunoassay / Q000592:standards*; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D012680:Sensitivity and Specificity
D006241:Haptens; D007128:Immunoglobulin Fragments; D001280:Atrazine