Assessment of the Effect of Increased Fluorophore Labelling on the Binding Ability of an Antibody

Abstract

Abstract The effect of the fluorophore-to-protein labelling ratio on the binding ability or 'affinity' of an antibody preparation, using two immunosensors, the BIAcoreTM (Pharmacia Biosensor AB) and a fibre optical fluoroimmunosensor based on the evanescent wave technique (EWIS), constructed in our laboratory, was assessed. The BIAcoreTM was used to determine the effect of increased labelling on the 'affinityy' of the FITC (fluorescein isothiocyanate)-labelled anti-LDH antibodies, while the EWIS was used to assess how the fluorescence intensity of the conjugates may compensate for loss of binding ability in, for example, fluoroimmunoassay applications. The optimal labelling ratio was determined to be 2-5 FITC molecules per antibody protein molecule. Key Words: FluorophoreAntibodyLabellingAffinityBIAcoreTM Fibre Optical Evanescent Wave Fluoroimmunosensor