Poor Assay-to-Assay Reproducibility (ELISA Development)

Question

Accepted Answer

Inconsistent results between different assay runs with same samples Common causes: 1) Insufficient washing 2) Automatic plate washer ports clogged 3) Variations in incubation temperature 4) Variations in protocol between runs 5) Reused plate sealers with residual HRP 6) Improper calculation of standard curve dilutions 7) Contaminated buffers Solutions: 1) Follow washing procedure from ELISA Development Guide page 4 2) If using automatic plate washer, check all ports are clean and free of obstructions 3) Adhere to recommended incubation temperature 4) Avoid incubating plates in areas where environmental conditions vary 5) Adhere to same protocol from run to run 6) Use fresh plate sealer for each step 7) Check calculations and make new standard curve 8) Use internal controls 9) Make fresh buffers