Analysis and application of substrate hydrolysis rates in indirect ELISA of a purified plant virus.
The transient colorimetric signal in a microtiter plate is used to quantify a purified plant virus, cowpea mosaic virus (CPMV), over five concentration decades in a single plate. The method involves the coating of the polystyrene microtiter plate wells directly with the CPMV antigen, followed by incubation with a rabbit-derived CPMV-specific antibody, and lastly by incubation with a commercially available antibody against rabbit immunoglobulin which has been pre-labeled with alkaline phosphatase. The rate of p-nitrophenylphosphate hydrolysis, both non-specific and that which was catalyzed by this enzyme, was measured spectrophotometrically at 405 nm. Enzyme-catalyzed hydrolysis rates followed first order kinetics at all antigen coating concentrations, and the 1 degree rate constants, which ranged from 2 X 10(-6) min-1 to 1 X 10(-3) min-1, were found to increase with increasing antigen concentration.
Vilker
V L
VL
Department of Chemical Engineering, University of California, Los Angeles 90024.
Verduin
B J
BJ
D000956:Antigens, Viral / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D006868:Hydrolysis; D007700:Kinetics; D009029:Mosaic Viruses / Q000032:analysis* / Q000276:immunology; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D013053:Spectrophotometry
D000956:Antigens, Viral; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate