Frequently Asked Questions

Useful Hints

Carbohydrate Detection Experts

Troubleshooting ELISA Assays

Browse through the categories below.  If you don’t see your specific issue listed, please contact our technical service at info@lifespantech.com  for further assistance.

• Contamination of reagents. Use fresh reagents and pipette carefully.
• Splashing between wells. Use a plate sealer during incubations.
• Insufficient washing of plate. Use the proper 1X wash solution. Check that the plate washer is functioning properly and that the correct number of washes are being done.

  • Conjugate left on too long. Follow instructions for conjugate incubation.
  • If using lyophilized conjugate, was it reconstituted correctly? Reconstituted conjugate should be added immediately after reconstitution.  Check reconstitution instructions.
  • TMB Substrate is too old.
  • Substrate reaction allowed to go too long before adding stop reagent. Reaction should be stopped as soon as a medium blue color has developed.
  • Substrate incubation not done in the dark.
  • Incubation temperature too high. Try to avoid temperatures above 28 degrees C.
  • Stop solution is contaminated. It should be a clear, colorless liquid.
  • Plate not read soon after stop reagent is added. (Color may continue to develop at a slow rate even if stop reagent has been added.)
  • Re-using plate sealers, reservoirs, or pipette tips causing cross-well contamination.
  • Insufficient washing of plate.  Check that plate washer is functioning correctly and that the correct numbers of washes are being done.
  • Sample values too high. In competitive ELISAs, a low signal corresponds with a high concentration of target. You may need to dilute samples, or change to a wider sensitivty range assay. Consult with technical service.
  • Incubation times too long. Check that you are using the recommended length of time.
  • Incubation temperature too low or high. Make sure to bring all reagents to room temperature (20-25 degrees C) prior to use.
  • Stop solution not added.
  • Plate washed after substrate incubation. After substrate incubation, do not wash. Add stop solution and directly read the plate at 450 nm. 
  • Incorrect wavelength set on plate reader.
  • Concentration of samples is too high and out of range for sensitivity of assay. Re-assess the assay you are using (consult with technical service) or dilute your samples. Remember to take into account the dilution factor when calculating final results.
  • Pipetting inconsistent. Ensure pipettes are working correctly and are calibrated. Check that tips are the correct size and are properly seated to create a good seal. Ensure you are not using a volume outside the range of the pipette. Watch to make sure tips are aspirating and delivering the correct amount of fluid.
  • Standards and Reagents are not well mixed prior to adding to plate. Mixing ensures a consistent concentration across the plate.
  • Wells are allowed to dry out. Ensure that new plate sealers are used during all incubations and that they are adhering to all sides of the plate.
  • Inadequate washing. Ensure correct amount of wash is being dispensed into all wells.
  • Bottom of the plate is dirty on the outside. Clean the bottom of the plate carefully before reading plate.  Gloves should be worn whenever handling the plate to prevent smudges on the bottom.
  • Check reagents for any deterioration. Substrate should be clear and colorless.
  • Check that storage conditions for all reagents are correct.
  • Ensure that wash solution is diluted with deionized or more pure water.
  • Check that the correct wavelength was used to read plate.
  • Inadequate washing.
  • Conjugate or substrate may be bad.

• Ensure that standards were made correctly. Double-check this.

  • Possible causes are evaporation in wells or inconsistent incubation temperature across the plate. Use a plate sealer that is adhered to all sides of the plate. Avoid incubating plate in an area with temperature fluctuations.
  • Insufficient washing. Check that the appropriate numbers of washes are being done. Ensure that the correct amount of wash solution is being dispensed into all wells. Check that none of the dispense outlets are plugged.
  • Variation in assay protocol. Ensure the same protocol is being followed each time.
  • Standard degradation. Check standards and make fresh ones if necessary.
  • Plate sealers reused. Use a fresh plate sealer for each step.
  • Improper calculation of standard curve. Check math and recalculate curve. Refer to kit technical data sheet for help with assay analysis and curve equation.
Carbohydrate Detection Experts

Useful Hints

The protocols provided on Lifespan’s technical data sheets are straight forward and easy to follow.  If you are new to ELISA assays or having problems, the following topics will provide further hints to ensure the best results.

  • Calibrate pipettes regularly according to manufacturer’s instructions.
  • Use pipette within the range suggested by the manufacturer.
  • Pre-rinse the tip with the reagent to be pipetted
  • Maintain consistent speed while aspirating. Avoid sudden motions.
  • Avoid touching bottom of well with tips.
  • Avoid splashing of sample and reagents.
  • Use a new tip for each sample/reagent addition.
  • Use a multichannel pipette whenever pipetting multiple wells.
  • Pipette quickly and efficiently to minimize timing differences between beginning and end of plate.
  • If an air bubble appears while pipetting, return liquid to the reservoir and re-pipette. If an air bubble continues to appear, replace tip.
  • Check pipette tips are long enough to provide air space between top of liquid and pipette barrel.
  • Check pipette barrel for residual fluid or dried material, remove if present.
  • Ensure pipettes tips are the correct size and are fitted tightly.
  • Watch to ensure tips are aspirating and delivering correct amount of liquid.
  • Use separate reservoirs for each reagent.
  • Follow procedure for preparation of wash buffer. Prepare with clean, pure deionized water.
  • Check washer alignment daily as part of routine instrument start-up procedures.
  • Ensure that the plate is level throughout washing.
  • Make certain well is completely filled when washing, to ensure residual conjugate is removed.
  • When washing, do not allow wells to overflow.
  • Check washers before use to determine they are working properly. Perform routine maintenance.
  • Be certain to wash the specified number of times.
  • Examine the wells for complete aspiration of contents.
  • Upon completion of wash cycle, blot to remove residual fluid.
  • Bring plate pouches to room temperature before opening.
  • Seal unused wells with plate sealer. Store at 4 °C in plastic pouch along with the desiccant pack.
  • Date the pouches when first opened if any wells are to be reused.
  • Make sure plates are level during washing, reagent addition and plate reading.
  • Place plates in dark immediately after addition of substrate solution
  • Wipe the bottom of the plate with a lint-free cloth/ towel before reading.
  • Do not allow wells to become dry once the assay has begun.
  • Bring samples and reagents to room temperature (22-28 ºC) approximately 30 minutes prior to use.
  • Maintain proper incubation temperature:

   — Lower temperature can decrease absorbance values.

   — Higher temperatures can increase absorbance values.

   — Evaporation in wells can cause edge effect.

  • The optimal temperature for incubation is 22-28 ºC.
  • Check temperature against calibrated thermometer.
  • Keep the temperature the same each time the assay is performed.
  • Keep incubation times the same each run.
  • Check calibration of timers.
  • Record time of incubation.
  • Read plate within minutes after adding stop solution.

Rotation of plates while incubating reagents

  • Plates should be rotated during incubation for better binding reaction.
  • The effect of rotating plates is to mix the reactants completely during the incubation steps.
  • Rotating plates produces mixing, which ensures that potentially reactive molecules are continuously coming into contact with the solid-phase.
  • During stationary incubation, mixing only takes place because of diffusion of reagents and is thus temperature dependent.
  • To allow maximum reaction from reagents in stationary conditions, greater incubation times may be required, than if they are rotated.
  • Use a new plate sealer for all incubations and ensure that it has a tight seal around all edges.
  1. Substrate
  • Substrate solution is light-sensitive. Incubate in the dark.
  • The temperature of substrate solution is important because of affects the rate of color reaction. Allow to come to room temperature prior to adding to plate.
  • Do not add fresh substrate to reagent bottle containing old substrate.
  • Ensure that substrate is a clear, colorless solution prior to adding to plate.
  • Tap plate gently after addition of substrate to ensure mixing.
  • Do not wash plate after incubation with substrate.
  1. Conjugate Preparation
  • For lyophilized conjugate, follow instructions on the technical data sheet regarding preparation of working solution.
  • Store at recommended temperature.
  • Always make up the working dilution of conjugate immediately before you need it.
  • Mix conjugate solution prior to adding to plate.
  • Never leave conjugate on the bench for excessive time.
  1. Addition of Samples/Reagents
  • Pay attention to the proper order and volume for addition of samples and reagents.
  • Reagents should always be pipetted in the same order on the plate throughout the assay.
  1. Stop Reagent
  • Stop reagents are added to prevent further enzyme reaction in ELISA.
  • Molar concentration of strong acids or strong bases stops enzyme activity by quickly denaturing enzymes. Sodium azide is a potent inhibitor of HRP.
  • Do not delay in reading plate after addition of stop.
  1. Laboratory conditions
  • Wipe bench down prior to starting ELISA assay to avoid any contamination of plate or reagents.
  • The laboratory should be devoid of any acid fumes.
CARBOHYDRATE DETECTION EXPERTS

Plasma Preparation Tips

For optimum results when using Lifespan's plasma specific kits, it is necessary to utilize an effective cell free plasma preparation protocol. Click on the Learn More button for our recommended protocol.
Learn More

GET IN TOUCH

Lifespan welcomes all inquiries about kit selection for your particular experimental conditions, problems running the assays or getting unexpected results.
Contact Us
Shopping Cart
Scroll to Top