The IL-2 ELISA duplicate wells for cell supernatant show large discrepancies—is it an operational issue or a plate issue?

First, perform an exclusion experiment at the operational level: take the same cell supernatant, aliquot it into multiple wells, and process it with different sample addition sequences and plate washing techniques to see whether the CV still exceeds 15%. If the CV decreases after optimizing the operation for the same sample, it is a technique issue; if it is still high at the edges and low in the middle, then suspect the plate. Also calibrate the pipettes, especially the 10/100 µL settings, because sample volume errors will be amplified.

Long sample loading time is a common cause of high replicate well CV. Edge wells are often loaded first or last, resulting in inconsistent reaction times, and manual single-channel pipetting is more likely to cause well-to-well variation. It is recommended to switch to a multichannel pipette and reagent reservoir so that all wells are completed within 5-10 minutes; replicate wells should not be placed adjacent to each other and can be distributed randomly or symmetrically across the plate; during plate washing, the wash solution dwell time should be consistent for each well, and the plate-tapping direction and force should be uniform to avoid inconsistent residual liquid in edge wells.

Switching to imported plates or using reagent reservoirs can help, but it depends on whether the issue is operational or related to the quality of the pre-coated plates. The plates in ready-to-use kits generally cannot be replaced with imported plates alone, unless you purchase antibodies and coat the plates yourself. Using low-adsorption disposable reservoirs can reduce sample loading time. If edge effects and high intra-assay CV persist after ruling out operational and instrument factors, record the lot number and contact the manufacturer's technical support, providing the raw OD values and plate layout. They will generally arrange a retest or replacement.

Elevated edge wells are most commonly caused by uneven temperature during incubation and edge evaporation. In an incubator, if the plate is placed against the wall or near the air outlet, the temperature in the edge wells may be slightly higher, resulting in stronger reactions; if no plate sealing film is applied or the film is not firmly pressed down, evaporation of liquid from the edge wells can also increase the concentration. It is recommended to check that all four sides of the plate sealing film are firmly pressed before incubation, place the plate in the center of the incubator rather than against the wall, and place a preheated metal plate or flat tray underneath to reduce temperature differences.