ZAGENO

ELISA Troubleshooting

Introduction

ELISA (Enzyme-linked immunosorbent assay) is an invaluable method of identifying and quantifying proteins due to its use of antibodies, which enable high specificity and sensitivity. The enzyme/conjugated antibody acts as the means of detection; the concentration of antibody-bound protein is proportional to the activity of the enzyme when incubated with the substrate.

However, attaining the required signal is not always easy and can require multiple optimizations. It can prove difficult to gain the necessary signal strength, while the washing stages involved can also be problematic, if not done thoroughly, leading to high background and poor reproducibility.

If this is the case for you, have no fear! This ZAGENO troubleshooting guide is here to help.

ELISA signal is high and standard curves have saturated ODs

  • One or more incubation steps are too long: Decrease incubation time

  • Standard reconstituted with less volume than required: Reconstitute lyophilized standard with the correct amount of solution recommended in the protocol

  • Insufficient washing: Invert plate on some absorbent tissue after each washing step, tapping if necessary

  • Incorrect dilutions: Check calculations and pipetting technique

  • Plate sealers not used or reused: Use fresh sealers for assay plates during every incubation as it will prevent contamination

  • Incubation time is too long: Reduce incubation time, check protocol for reference

ELISA sample readings are out of range

  • The sample contains analyte concentrations greater than highest point: Dilute samples and reanalyze

  • The sample contains no or undetectable level of the analyte: Use higher sample volume and alter protocol as necessary

Weak or no ELISA signal

  • Reagents not at room temperature: Allow reagents to sit on the bench for 15/20 minutes

  • Components stored incorrectly: Check storage conditions on the kit label, generally 2 – 8 °C

  • Reagents have expired: Make sure the reagents are still within the expiration dates

  • Reagents added incorrectly: Inspect the protocol for directions in adding and diluting reagents

  • Incorrect dilutions: Check calculations and pipetting technique

  • Plate read at the incorrect wavelength: Use wavelength or filter recommended by protocol and make sure plate reader is set correctly for the type of substrate

  • Too little detector antibody used: Check protocol for dilution required, use a more concentrated solution if needed

  • Scratched wells: Be careful when dispensing and aspirating into and out of wells not to touch the bottom of the wells

  • The capture antibody did not bind to the plate: Make sure to use an ELISA plate, and dilute the antibody in PBS (Phosphate buffered saline). Also, ensure the preparation and incubation are correct for both coating and blocking steps

High Background

  • Insufficient washing: Increase time for soaking by about 30 seconds and invert the plate onto tissue after washing until dry

  • The substrate was exposed to light beforehand: Store in a dark place and limit exposure during the assay

  • Incubation times were too long: Reduce incubation times closer to that suggested in the protocol or until optimal

  • Standard curve dilutions prepared are incorrect: Check calculations and pipetting technique

  • Wells were contaminated: Use fresh plate sealers for assay plates during every incubation as it will prevent contamination. Use a multichannel pipette

Poor Standard Curve

  • Standard curve dilutions prepared are incorrect: Check calculations and pipetting technique

  • The capture antibody did not bind to the plate: Make sure to use an ELISA plate and dilute the antibody in PBS (Phosphate buffered saline). Also, ensure preparation and incubation are correct for both coating and blocking steps

Poor Replicate Data

  • Insufficient washing: Increase time for soaking by about 30 seconds and invert the plate onto tissue after washing until dry

  • The capture antibody did not bind to the plate: Make sure to use an ELISA plate and dilute the antibody in PBS (Phosphate buffered saline). Also, ensure preparation and incubation are correct for both coating and blocking steps

  • Plate sealers not used or reused: Use fresh sealers for assay plates during every incubation as it will prevent contamination

Inconsistent Results

  • Insufficient washing: Invert plate on some absorbent tissue after each washing step, tapping if necessary

  • Incubation temperature is inconsistent: Be aware of fluctuations due to environmental factors and try to follow protocol guidelines

  • Plate sealers not used or reused: Use fresh sealers for assay plates during every incubation as it will prevent contamination

  • Incorrect dilutions: Check calculations and pipetting technique

Edge Effects

  • Plates stacked on top of each other: Do not stack during incubation

  • Evaporation: Seal the plate during incubations

  • Temperature is uneven: Make sure the plate is sealed during incubations and the plate is in the center of the incubator

Optimization Tips

  • Prepare different concentrations of capture antibody/detection antibody or different blocking solutions etc.

  • Use an equal volume of each variation on the plate

  • Compare their ability to produce a strong signal against their capacity to minimize background

Pipetting Tips

  • Use the pipette that is within the range of the required volume

  • Make sure that the tip is fitted firmly on the pipette

  • While pipetting, make sure no air bubbles are present

  • Use a new tip for each standard, sample, or reagent

  • Use different reservoirs for each reagent

  • Pipette samples onto the side of wells so as to avoid splashing

  • Always run replicate samples/standards in parallel

Washing Tips

  • Gently lower an aspiration tip into the bottom of each well and completely remove liquid from all wells, but do not scratch the inside of the well

  • Use at least 400 µL of diluted wash buffer for each well

  • Allow 15 to 30 seconds for soaking

  • Aspirate wash buffer from wells

  • Repeat washing 3 or 4 times

  • Invert plate on absorbent tissue after washing and tap dry if necessary

Problems in the lab? Learn about methods and experiments in our Knowledge section or look up a specific Troubleshooting guide.

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