Enzyme immunoassays using bispecific diabodies

Abstract

Background: Bispecific antibodies with a first binding specificity to a target antigen and a second to an enzyme have great potential in enzyme immunoassays. As bispecific antibodies are difficult to make, the use of recombinant bispecific antibody fragments may provide a breakthrough. Objectives: To make bispecific antibody fragments directed against an enzyme and to demonstrate their application in enzyme immunoassays. Study design: Bispecific antibody fragments were assembled as diabodies (Holliger P., Prospero T., Winter G. Proc. Natl. Acad. Sci. USA 90, 1993, 6444–6448) directed to an enzyme, E. coliβ-galactosidase, and to each of three target antigens, hen-egg lysozyme (HEL), carcinoembryonic antigen (CEA), and HIV gp120 (HIV). The diabodies were then evaluated in immunoassays. Results: The HEL diabody was shown to recruit β-galactosidase in a microtiter plate immunoassay in which diabody and enzyme were co-incubated with antigen, washed and enzyme substrate added. The CEA diabody was shown to detect CEA by immunocytochemical staining of transfected, CEA-expressing HeLa cells and of adenocarcinoma colon tissue sections, and the HIV diabody to detect gp120 in immunoblots of total cell extracts. Conclusion: The results illustrate the diagnostic potential of diabodies in enzyme immunoassays.