A homogeneous noncompetitive immunoassay for the detection of small haptens.
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.
Yokozeki
Tomoichi
T
Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Japan.
Ueda
Hiroshi
H
Arai
Ryoichi
R
Mahoney
Walt
W
Nagamune
Teruyuki
T
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006241:Haptens / Q000032:analysis*; D007124:Immunoenzyme Techniques / Q000379:methods*; D010957:Plasmids / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis; D001616:beta-Galactosidase / Q000737:chemistry
D006241:Haptens; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase