RRC ID 29141
Author Yokozeki T, Ueda H, Arai R, Mahoney W, Nagamune T.
Title A homogeneous noncompetitive immunoassay for the detection of small haptens.
Journal Anal Chem
Abstract We describe a noncompetitive homogeneous immunoassay for small haptens based on the antigen-dependent reassociation of antibody variable domains and beta-galactosidase (beta-gal) complementation (open sandwich enzymatic complementation immunoassay). As a model system, the reassociation of two fusion proteins, an anti 4-hydroxy-3-nitrophenylacetyl (NP) antibody heavy-chain variable-region fragment fused to an N-terminal deletion mutant of beta-gal (V(H)delta alpha) and the light-chain variable-region fragment fused to a C-terminal deletion mutant of beta-gal (V(L)delta omega), was monitored by the enzymatic complementation between the two. Upon simple mixing of the reagents with the sample, an antigen (NP)-dependent increase in enzymatic activity was observed. When 5-iodo-NP was measured, a 10 times higher sensitivity was observed, probably due to its higher affinity. Compared with our corresponding heterogeneous open sandwich enzyme-linked immunosorbent assay, approximately 1000-fold improvement in the sensitivity was attained, probably due to lower background V(H)-V(L) association. In addition, the assay required less time, handling, sample volume, and assay reagents.
Volume 74(11)
Pages 2500-4
Published 2002-6-1
DOI 10.1021/ac015743x
PMID 12069229
MeSH Electrophoresis, Polyacrylamide Gel Escherichia coli / chemistry Escherichia coli / genetics Haptens / analysis* Immunoenzyme Techniques / methods* Plasmids / chemistry Recombinant Fusion Proteins / analysis beta-Galactosidase / chemistry
IF 6.785
Times Cited 29
DNA material pPCR/delta alpha (RDB02987) pPCR/delta omega (RDB02988)