False Positive Spots and High Background (ELISPOT)

Question

Accepted Answer

Non-specific spots appear in negative control wells or throughout the plate. Background signal is elevated, making spot discrimination difficult. Common causes: 1) Dust or microbial contamination in cell culture 2) Contaminating platelets when using PBMC prepared from blood samples 3) Cell debris or cells remaining on membrane after culture 4) Secondary antibody aggregates due to improper storage 5) Mitogens or other stimulating factors in non-heat-inactivated serum 6) Insufficient washing allowing reagent leakage between membrane and plate bottom Solutions: 1) Maintain sterile technique; filter reagents using 0.2 µm low protein-binding syringe filters 2) Optimize PBMC preparation efficiency; wash plate thoroughly after cell culture 3) Ensure complete cell removal with PBS-Tween 20 before secondary antibody incubation 4) Filter secondary antibody before use; store antibodies properly 5) Heat-inactivate serum to eliminate mitogenic factors 6) Wash both sides of membrane with distilled water before and after color development