Troubleshooting ELISA

Troubleshooting ELISA

 

The OD of my sample falls outside the standard curve range. What should I do?
The variability between my standard or sample replicates is too high. What could have happened?
I am getting very low or no color development with the standards. What went wrong?
I am getting high background values (OD blank > 0.3). What could have happened?
The linearity and/or dynamic range of the standard curve are poor. What went wrong?

My question is not mentioned here, how can I contact U-CyTech?

 

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The OD of my sample falls outside the standard curve range. What should I do?
Samples showing an OD below the lowest concentration of the standard curve should be re-analyzed at a lower dilution. Please keep in mind that the sample can also contain an analyte concentration below the detection limit of the assay. Samples showing an OD that exceeds the highest concentration of the standard curve should be re-analyzed at a higher sample dilution.

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The variability between my standard or sample replicates is too high. What could have happened?
There could be several reasons for poor consistency of replicates in ELISA:

  1. Inaccurate pipetting: ensure accurate pipetting of volume (check pipettes) and avoid air bubbles.
  2. Inadequate mixing of reagents: mix reagents adequately.
  3. Inadequate washing: increase the stringency of wash steps (particularly after the 'detection antibody' incubation step). Check the operation of the automated plate washer.
  4. Evaporation of solutions: ensure precise sealing of the ELISA plate during each incubation step.
  5. Non-homogenous samples or samples with high particulate matter: mix samples thoroughly and remove particulates by centrifugation.
  6. Temperature differences between wells: do not stack the ELISA plates during reagent incubation.

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I am getting very low or no color development with the standards. What went wrong?
There could be several reasons for no or low OD values for the standards:

  1. Improper storage of reconstituted SPP conjugate: avoid storage at room temperature and prolonged exposure to light and heat.
  2. Incorrect incubation times and/or temperatures: ensure correct incubation times and allow reagent solutions to reach room temperature before use. Check function of the 37 °C incubator.
  3. Improper quality of distilled water used for preparing PBS and reconstitution of the lyophilized contents of the vails: use distilled water (no tap water) and check the quality of distilled water. Ensure there are no residues of any cleaning products in the water system.
  4. Improper antibody, conjugate or standard dilution: ensure correct dilution of antibodies, conjugate and standard.
  5. Degradation of antibodies or conjugate: follow recommended storage conditions. Keep SPP conjugate protected from light and avoid prolonged storage at ≥4 °C. 
  6. Overly high washing/aspiration pressure from automated plate washer: check function of washing system or utilize manual washing.
  7. Working solutions contained sodium azide: avoid adding sodium azide in solutions, as this is a 'peroxidase activity' inhibitor.

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I am getting high background values (OD blank > 0.3). What could have happened?
There could be several reasons for high background values:

  1. Working solutions were contaminated: solutions should be clear and colorless. Solutions should not be used when they have changed color, become turbid or if there is an indication of bacterial of fungal growth. Use a clean petri dishes, tubes and pipet tips for working solutions.
  2. Detection antibody or conjugate dilution was too concentrated or has stayed too long on the plate: ensure proper dilution of detection antibody or conjugate and incubation time.
  3. Overdeveloped plate: shorten the incubation time of TMB substrate.
  4. Incubation temperature of TMB substrate is too high: ensure the incubation temperature is between 20 °C and 26 °C.
  5. TMB substrate has been exposed to light: ensure the TMB solution is not exposed to light and protect the plate from light during TMB substrate incubation.

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The linearity and/or dynamic range of the standard curve are poor. What went wrong?
There could be two mean reasons for poor linearity and/or dynamic range of the standard curve:

  1. Improper standard dilutions: ensure proper dilution of standards (two-fold dilutions for the standard curve is recommended).
  2. Inaccurate pipetting: ensure accurate pipetting of volume (check pipettes) and avoid air bubbles.

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My question is not mentioned here, how can I contact U-CyTech?
The researchers of U-CyTech build on >20 years of experience on the development and performance of the ELISAs for human, monkey, mouse and rat species. They are more than happy to provide you with additional advice and to share their experiences.

Please contract our customer service:
E-mail: cs @ ucytech.com

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