The FITC-anti-FITC system is a sensitive alternative to biotin-streptavidin in ELISA

In a model ELISA system alkaline phosphatase (AP) absorbed onto microtitre wells was employed as the target antigen. The antigen was then reacted with a monoclonal antibody to AP either unlabelled or labelled with (a) FITC and (b) biotin. The bound anti-AP was then detected with horseradish peroxidase (HRP) conjugates of polyvalent anti-mouse IgG, and the FITC and biotin anti-AP conjugates with HRP conjugates of a monoclonal anti-FITC or streptavidin respectively. The FITC-anti-FITC system proved to be of similar sensitivity to the biotin-streptavidin system detecting 140 amol compared to 350 amol of antigen. Both these methods of antigen detection were superior to the anti-mouse IgG reagent (2100 amol). In contrast to biotinylated antibodies, FITC-labelled antibodies are highly coloured and fluorescent. These features aid the preparation, purification and characterisation of conjugates. In addition, very low non-specific binding is encountered with enzyme conjugates of anti-FITC and this may confer an advantage over enzyme conjugates of avidin/streptavidin reagents.