Epitope Mapping by Competition Assay
Ed Harlow and David Lane
ABSTRACT
The simplest way to determine whether two monoclonal antibodies bind to distinct sites on a protein antigen is to carry out a competition assay. The assay can be used with antibodies that bind both conformational and linear epitopes, and it is most useful in the analysis of monoclonal antibody specificity because polyclonal sera typically recognize multiple different epitopes. This simple, ELISA-based protocol examines the ability of two hypothetical, newly derived monoclonal antibodies, XH1 and XH2, to compete with the well-characterized ZO-1 anti-YFP1 antibody for binding to the target antigen YFP1; hypothetical antibody PC10 is used as a control. First, 96-well plates are coated with the YFP1 antigen and direct binding assays are run to determine that all of the antibody preparations are active and able to react with the immobilized antigen. Second, the competition analysis itself is carried out. Note that the names of antibodies and antigens should be considered as generic.
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