Bioluminescent immunoassay using a monomeric Fab'-photoprotein aequorin conjugate

The photoprotein aequorin emits light by an intermolecular reaction when mixed with Ca2+. To apply a bioluminescent immunoassay based on the light emission property of aequorin, we prepared aequorin-antibody Fab' conjugates by a chemical cross-linking technique. Recombinant apoaequorin was coupled with human tumor necrosis factor-alpha (TNF-alpha) antibodies (polyclonal Fab' or monoclonal Fab' fragments) using N-succinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate. The luminescent activity of the Fab'-aequorin conjugate was about one-tenth that of aequorin. Using a monomeric conjugate, human TNF-alpha can be measured at an attomole level by a sandwich immunoassay technique.