Table I Possible technical factors influencing multiplexed bead-based immunoassay data
Procedure stepLevels at which the technical factors act
Between day or batchBetween plateBetween well or sample
Seed cellsCell counting accuracyCell seeding accuracy (pipetting)
Apply experimental treatmentsConcentrations and specific activities of reagentsTiming of treatments and plate processingMedia volumes per well (pipetting)
Order bias
Process cells (wash, freeze, and lyse)Lysis buffer reagent concentrationsaNumber of adhered, healthy cells present at experiment's end
Lysis buffer volume (pipetting)
Measure total protein concentrations, dilute samples to a common concentrationAccuracy of protein assay standardsAccuracy of protein concentration measurements
Accuracy of assay buffer dilution volume
Perform the assayBead concentrationsaFactors affecting the number of beads and antibody amounts
Antibody concentrationsaEffects introduced by multiple wash & rinse steps
Instrument calibration and performanceaResuspension volume (pipetting)
Liquid evaporation
Spillage, leakage, and/or clogging of the filter plates
Bead carryover between wells (5)
  • a These between-day effects would pertain to cases in which the cell processing and assay themselves were performed on separate days. In our experiment, only the treatments were performed on separate days.