Poor standard curve

·        Insufficient vortexing

·        OD₄₅₀ too high for the high standard point

·        Air bubbles in wells.

·        Standard defect or not fully recovered

·        Vortex 30 sec for each of standard dilution steps and vortex again (10 sec) before pipetting to the 96-well plate.

·        Reduce substrate incubation time

·        Remove air bubbles in wells by pipette tip.

·        Change a standard vial or spin down the vial before reconstitution

Low signal

·        Too brief incubation times

·        Inadequate reagent volume or improper dilution

·        Standard defect and sample overdiluted

·        Microplate shaker may improve signals.

·        Insufficient HRP Conjugate. Ensure sufficient incubation time and increase sample incubation to 2 h.

·        Change a Standard vial. Undilute sample or less dilution

·        Air bubbles in wells

·        Stop the reaction by adding 50 µl of Stop Solution when it turns to dark blue in the highest concentration of standard wells.

·        Remove air bubbles in wells

Large CV

·        Improper standard/sample dilutions.

·        Air bubbles in wells.

·        Microplate reader out of calibration

·        It did not turn yellow after adding Stop Solution

·        Ensure accurate pipetting and thorough mixing.

·        Use reverse, instead of forward pipetting.

·        Use the correct dilution buffers

·        Remove air bubbles in wells by pipette tip.

·        Calibrate the microplate reader properly

·        If it did not turn yellow after adding Stop Solution, mix with pipette tip till it turns yellow prior to measurement.

High background

·        Plate is insufficiently washed and air bubbles in wells.

·        Contaminated Assay Buffer

·        Pipet tip contaminated

·        Increase wash to 4 times before adding substrate and ensure plate washer functions normally. Remove air bubbles in wells by pipette tip. Use squirt bottle for washing.

·        Make fresh Assay Buffer and wash thoroughly.

·        Use new pipette tips for blank wells.

No signal detected

·        Failures of spin down the contents in Detection Antibody and Standards.

·        Failure of HRP or Substrate Samples overdiluted

·        Ensure HRP volume. Mix 100 µl of Substrate with 0.5 µl HRP and dark blue color should develop in 5 min.

·        Try a new standard vial and use positive control.

·        Try not dilute samples

Low sensitivity

·        Improper storage of the ELISA kit

·        Store detection antibody at -20°C after reconstitution and others at 4°C. Keep substrate solution protected from light.