Results of a criss-cross serial-dilution analysis (for optimization of secondary and tertiary reactant concentrations) of an antibody-sandwich ELISA to detect antigen. The numbers in columns 1 to 11 and rows B to G represent relative fluorescence units observed for each well on a 96-well microtiter plate.

Plates were coated overnight with the capture antibody at 2 µg/ml. The secondary reactants, 4-fold serial dilutions of the homologous antigen and a non-cross-reactive heterologous antigen, were incubated on the plate 2 hr. The tertiary reactant, 2-fold serial dilutions of specific antibody–alkaline phosphatase conjugates, were incubated on the plate 2 hr. After 1 hr of incubation with the substrate MUP, the fluorescence was read in a microtiter plate spectrofluorometer.

Reagent concentrations depend upon individual assay variables that are set by the investigator. If the time of hydrolysis is set at 1 hr, the relative fluorescence at ~1000 relative fluorescence units, and the sensitivity at 780 pg/ml of homologous antigen, then 500 ng/ml of enzyme-antibody conjugate must be used in the ELISA. If, however, the assay has to detect only 3.12 ng/ml of homologous antigen, then the concentration of conjugate can be reduced to 125 ng/ml. It should be noted by comparing the homologous with the heterologous reactions (wells B5 versus B11 and D4 versus D10) that both the specificity and the signal-to-noise ratio for this assay are excellent.