ELISA Troubleshooting Guide:
Contents
1. ELISA Troubleshooting Guide Problem Signal is high stan dard curves have saturated O D s Sample readings are out of range High variation in samples and or standards Probable Cause Standard reconstituted with less volume than required Plate incubation was too long Detection antibody incuba tion time is too long Avidin HRP incubation time is too long Substrate solution incuba tion time is too long Samples contain no or below detectable levels of analyte Samples contain analyte concentrations greater than highest standard point Multichannel pipette errors Plate washing was not adequate or uniform Non homogenous samples Samples may have high particulate matter Insufficient plate agitation Cross well contamination Solution Reconstitute lyophilized standard with correct volume of solution recommended in the protocol Decrease incubation time Decrease detection antibody incubation time Decrease Avidin HRP incubation time Decrease substrate solution incubation time If samples are below detectable levels it may be possible to use higher sample volume Check with technical support for appropriate protocol modifications Samples may require dilution and reanalysis Calibrate the pipettes Make sure pipette tips are tightly secured Confirm all reagents are removed completely in all wash steps Thoroughly mix samples before pipetting Remove the particula2. rate solution TMB Substrate Solution should be clear and colorless prior to addition to wells Use a clean container prior to pipetting substrate solution into wells Add appropriate Detection Antibody and continue Add Avidin HRP according to protocol and continue Add substrate solution and continue Avoid sodium azide in the Wash Buffer Reconstitute standard according to protocol Store reconstituted standard in appropriate vials Store reconstituted standard at 70 C Check for pipetting errors and correct reagent volume Assay conditions need to be checked BioLegend The path to legendary discovery
3. te matter by centrifugation The plate should be agitated during all incubation steps using an ELISA plate shaker at a speed where solutions in wells are within constant motion without splashing When reusing plate sealers check that no reagent has touched the sealer Care should be taken when using the same pipette tips used for reagent additions Ensure that pipette tips do not touch the reagents on the plate W BioLegend The path to legendary discovery ELISA Troubleshooting Guide Background is high No signal Low or poor signal for the standard curve Probable Cause Background wells were contaminated Matrix used has en dogenous analyte or interference Insufficient washes TMB Substrate Solution was contaminated Incorrect or no Detection Antibody was added Avidin HRP was not added Substrate solution was not added Wash buffer contains sodium azide Standard was incompletely reconstituted or was inappropriately stored Reagents added to wells with incorrect concentra tions Incubations done at inappropriate temperature timing or agitation Avoid cross well contamination by using the sealer appropriately Use multichannel pipettes without touching the reagents on the plate Check the matrix ingredients for cross reacting components e g interleukin modified tissue culture medium Increase number of washes Increase soaking time between washes prior to addition of subst
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