Potential problem | Validation step |
---|---|
The essential steps | Â |
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 | Â |
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. |