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.
Erikaku
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T
Yokohama Research Center, Chisso Corporation, Japan.
Zenno
S
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Inouye
S
S
D000331:Aequorin; D000911:Antibodies, Monoclonal; D002850:Chromatography, Gel; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008301:Maleimides; D015394:Molecular Structure; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D008301:Maleimides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D000331:Aequorin; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester