High Variability Between Experimental Runs (ELISA (R&D Guide))

Question

Accepted Answer

Standard curves or sample values differ significantly between experiments run on different days, despite using the same reagents and protocol, showing poor reproducibility. Common causes: 1) Solutions degraded or contaminated (precipitates in buffer, loss of buffering capacity) 2) Biological samples prepared differently (varying freeze-thaw cycles, different dilutions, treatment conditions) 3) Variations in incubation temperature or time between runs; plates in areas with varying environmental conditions 4) Plate sealer reused causing HRP contamination turning TMB blue 5) Improper calculation of standard curve dilutions; lack of internal controls Solutions: 1) Prepare fresh solutions for each experiment; check for precipitates before use 2) Standardize sample preparation: use same treatment, buffers, dilutions; limit freeze-thaw cycles to maximum 3 3) Strictly adhere to protocol incubation times and temperatures; avoid variable-environment areas 4) Use fresh plate sealer for each step of protocol 5) Verify standard curve dilution calculations; include internal controls in every run