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.
Harmer
I J
IJ
Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, London, U.K.
Samuel
D
D
D000469:Alkaline Phosphatase / Q000032:analysis; D000915:Antibody Affinity; D001426:Bacterial Proteins; D001710:Biotin; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019809:Streptavidin; D013861:Thiocyanates
D001426:Bacterial Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D001710:Biotin; D019809:Streptavidin; D000469:Alkaline Phosphatase; D016650:Fluorescein-5-isothiocyanate