A solid-phase immunosorbent assay for the detection of protein kinase-specific monoclonal antibodies.
We describe two solid-phase immunosorbent assays that detect monoclonal antibodies against a cyclic AMP-dependent protein kinase from Paramecium tetraurelia without the need for pure kinase preparations. A radiometric immunosorbent assay (RISA) previously described by E.A. Pierce, M. C. Dame, and H. F. De Luca (1986, Anal. Biochem. 153, 67-74) was adapted to detect monoclonal antibodies against cyclic AMP-binding proteins. The RISA identified antibodies against the regulatory subunit of the enzyme, but failed to detect antibodies against the catalytic subunit. We therefore developed a solid-phase assay for immunoadsorbed protein kinase activity (IPKA) in 96-well plates. Antibodies were adsorbed from hybridoma supernatants to wells coated with anti-immunoglobulin antibodies. The wells were then incubated with protein kinase, and bound protein kinase activity was assayed with histone as a substrate. Monoclonal antibody concentrations of 1 micrograms/ml were reliably detected in hybridoma supernatants. As little as 10 histone-phosphorylating units (picomoles of phosphate incorporated per minute) were required per assay. The IPKA detected not only catalytic subunit-specific antibodies, but also antibodies directed against the regulatory subunit of the cyclic AMP-dependent protein kinase. Since crude preparations of protein kinase can be used in the IPKA, monoclonal antibodies raised against impure protein kinase can be identified.
Hochstrasser
M
M
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Nelson
D L
DL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism; D006825:Hybridomas / Q000276:immunology; D007163:Immunosorbent Techniques; D010247:Paramecium / Q000201:enzymology; D011487:Protein Conformation; D011494:Protein Kinases / Q000276:immunology* / Q000378:metabolism; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000242:Cyclic AMP; D011494:Protein Kinases