A Homogeneous Noncompetitive Immunoassay for the Detection of Small Haptens

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Abstract
We describe a noncompetitive homogeneous immunoassay for small haptens based on the antigen-dependent reassociation of antibody variable domains and β-galactosidase (β-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 β-gal (VHΔα) and the light-chain variable-region fragment fused to a C-terminal deletion mutant of β-gal (VLΔω), 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, ∼1000-fold improvement in the sensitivity was attained, probably due to lower background VH−VL association. In addition, the assay required less time, handling, sample volume, and assay reagents.