Optimizing ELISA assay components

Optimization of ELISA assay components (antibodies, buffers, samples) is done by testing different solutions to identify the working concentration of 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. The 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 and some contain BSA. The manufacturers of these buffers usually provide extensive optimization protocols. Try different concentrations of BSA If the blocking buffer is not pre-formulated.

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

Ensuring well-to-well consistency

ELISAs are typically performed using multi-well microplates. If you decide to pipette your ELISA manually make sure that all your pipettors are calibrated. It is good practice to visually inspect the liquid level in the pipette tip as well as in the microplate wells. These must be at identical levels while pipetting your assay to avoid inconsistent performance.

A higher-throughput and fast plate washer and/or an ELISA assay workstation can be a useful addition to enhance your workflow. These instruments will help you to maximize your productivity and optimize well-to-well consistency.