Factors to consider when developing your own ELISA: Antibody Selection (part 1)

ELISAs have become a popular method used to rapidly screen and quantitate a large number of samples for the presence of analyte or the antibody recognizing it. ELISAs are typically easy to use, can be automated and are relatively inexpensive. The antibody selection is of critical importance when developing an ELISA as they provide the basis for its specificity, accuracy and sensitivity. Optimization criteria include the affinity, specificity and the antibody titer.

Affinity: Antibodies recognize epitopes on an antigen with varying affinities from low to high. The antibodies chosen for an ELISA should bind to the antigen with a high-affinity so that non-specific substances do not interfere by binding to the antibody with a higher affinity than the antigen.

Specificity: Binding differences may occur between recombinant and natural samples due to conformational changes of the antigen after it binds to the antibody, which may affect the binding with the detection antibody. Careful selection and optimization will ensure that the antibodies are recognizing the recombinant and natural antigen in parallel.

Titer: Antibodies must be carefully titered on the plate so that the concentrations chosen provide the most binding and the best precision. Secondary/detection antibodies are titered against the capture antibody on the plate to determine the concentration that gives the best signal-to-noise ratio.