Enzyme immunoassays using bispecific diabodies.
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.


OBJECTIVE
To make bispecific antibody fragments directed against an enzyme and to demonstrate their application in enzyme immunoassays.


METHODS
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. coli beta-galactosidase, and to each of three target antigens, hen-egg lysozyme (HEL), carcinoembryonic antigen (CEA), and HIV gpl20 (HIV). The diabodies were then evaluated in immunoassays.


RESULTS
The HEL diabody was shown to recruit beta-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 gpl20 in immunoblots of total cell extracts.


CONCLUSIONS
The results illustrate the diagnostic potential of diabodies in enzyme immunoassays.
Kontermann
R E
RE
MRC Centre for Protein Engineering, Cambridge, UK.
Martineau
P
P
Cummings
C E
CE
Karpas
A
A
Allen
D
D
Derbyshire
E
E
Winter
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002272:Carcinoembryonic Antigen / Q000235:genetics / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques / Q000295:instrumentation; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000235:genetics / Q000276:immunology
D002272:Carcinoembryonic Antigen; D015699:HIV Envelope Protein gp120; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D001616:beta-Galactosidase