Non-specific Secondary Antibody Binding Causing High Background (ELISA (High Background))

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Accepted Answer

Elevated background signal across wells, including negative controls. Signal appears uniformly high rather than specific to target-containing wells. Common causes: 1) Secondary antibody binds non-specifically to plate surface or sample components 2) Secondary antibody species matches sample species causing cross-reactivity 3) Secondary antibody not pre-adsorbed against sample species immunoglobulins 4) Insufficient or inappropriate blocking buffer composition Solutions: 1) Run no-primary-antibody control to confirm secondary antibody non-specific binding 2) Use secondary antibody raised in different species than sample origin 3) Select pre-adsorbed secondary antibody against sample species Ig 4) Change blocking buffer to 5-10% normal serum from detection antibody species 5) Extend blocking incubation period