Time-resolved fluorescence immunoassay: An experiment for instrumental analysis

CHED 410

Nicole L. Ritzert and Donald C. Zapien, donald.zapien@sru.edu. Department of Chemistry, Slippery Rock University, Slippery Rock, PA 16057
Quantitative immunoassay is a method used widely in the biomedical field and can be a valuable topic to present in instrumental analysis. An experiment performed in the laboratory must be straightforward yet exemplify the tremendous sensitivity of the method, and be non-hazardous to undergraduate operators. The experiment illustrates concepts pertaining to fluorescence spectroscopy, background reduction, and immunological analysis. The experiment discussed here, modeled after a study reported in the literature, entails the analysis of bovine insulin in simulated bovine serum. First, a 96-well plate is coated with a layer of anti-mouse serum. Monoclonal anti-insulin antibody and bovine insulin in simulated bovine serum are then added simultaneously to each well. Biotnamidocaproyl-labeled bovine insulin is added to the wells, and following a wash step, europium-labeled streptavidin is added. The europium probe is released into solution and its fluorescence is measured using a fluorometric plate reader with time-resolution capability.