The use of acridinium ester-labelled streptavidin in immunoassays

We describe here the preparation of acridinium ester-labelled streptavidin, as a universal reagent for immunoassay detection. Streptavidin was labelled using an acridinium ester substituted with a N-hydroxysuccinimidyl group which at a pH of 7.4 reacts to form covalent bonds with the streptavidin amino groups. 63% of the added ester is covalently bound, and the chemiluminescence of the acridinium salt is unaffected by this labelling procedure. Labelled streptavidin can be detected down to less than 3 X 10(-19) mol in a detection reaction that takes 10 s. The labelled protein was used in a sandwich assay for mouse IgG using commercially available reagents, including biotin-labelled goat antibody to mouse IgG. In this assay the detection limit with 200 amol of the antigen (mouse IgG) using a 3 h coated tube procedure. The stability of labelled protein was examined under different buffer and temperature conditions. The labelled conjugates lost less than 20% of their original chemiluminescence activity when stored at 4 degrees C in the presence of sheep IgG and human serum albumin. In the absence of these proteins the labelled conjugates will adhere to the walls of the storage container and over 80% of the soluble activity is lost in 40 days. At 37 degrees C the chemiluminescence activity is lost rapidly and this loss is independent of buffer composition.