Skip to main content

Table 1 A simple set of steps to validate a sandwich ELISA for research use

From: A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients

Potential problem

Validation step

The essential steps

 

False positive measurements

Measure samples in wells coated with antigen specific antibody and in wells coated with an isotype antibody. Use samples before and after preincubation with immunoglobulins or another blocking agent.

False negative measurements

Spike samples with a known concentration of analyte and calculate the recovery. Use spiked samples before and after preincubation with immunoglobulins or another blocking agent.

Other matrix effects

Measure serial dilutions of samples to test for linearity. Use samples before and after preincubation with immunoglobulins or another blocking agent.

The extra steps

 

Low signal-to-noise ratio

Test different solutions for blocking the non-specific binding sites in the polystyrene wells. Titrate the antibodies. Test an amplification step.

High variability or dissimilarities between laboratories

Use a positive control sample in every plate. Test the effect of repetitive freeze/thaw cycles. Test the effect of different sample anticoagulants. Test different buffers for making the standard curve.