ELISA – optimisation tips

Typically, optimization of ELISA assay components (antibodies, buffers, samples) is performed by testing different solutions to identify the ideal working concentration for each component. For the capture antibody, dilutions in coating buffer ranging from 5 to 15 µg/ml for unpurified antibodies and 1 to 12 µg/ml for affinity-purified antibodies are prepared. Affinity-purified antibodies are recommended for optimal signal-to-noise ratio. Sample should be added in a high and a low concentration, reflecting the expected working range. For the detection antibody, a similar dilution setup can be performed in standard diluent, ranging from 1 to 10 µg/ml for unpurified antibodies and 0.5 to 5 µg/ml for affinity-purified antibodies.

There is a wide range of commercial blocking buffers to choose from, some of which contain BSA. The manufacturers of these buffers usually provide extensive optimization protocols. If the blocking buffer is not pre-formulated, try different concentrations of the protein, such as BSA.

Some types of samples can cause matrix-effects, for instance serum or plasma. These can be reduced by diluting the sample. Standard diluents should reflect the matrix of the sample as closely as possible and linearity of the dilutions be checked to ensure a good dynamic range.