Optimizing ELISA assay components
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.
Ensuring well-to-well consistency
ELISA are typically performed using multiwell plates. If you decide to pipette your ELISA manually make sure that all your pipettors are well calibrated. It is good practise to inspect visually that the liquid level in the pipette tip as well as in the wells is at an identical level while pipetting your assay to avoid inconsistent performance.
If you´re looking for higher-throughput and faster performance plate washers or ELISA assay workstations can be useful additions to your workflow. These instruments will help you to maximise your productivity and optimize well-to-well consistency.