SAN HQ ELISA FAQ

  1. Can you expect to get it right at first trial?
  2. What is important for a good result?
  3. What mistakes should I be aware of?
  4. Why is the absorbance in some wells too high?
  5. Why is the overall signal recovery quite low?
  6. Why do I get insufficient signal in some wells, but not all?

1. Can you expect to get it right at first trial?

  • Yes. If the instructions and notes are carefully read and followed.
  • When working with immunoassays, inexperienced operators could have problems performing some assay steps leading to inaccurate results.

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2. What is important for a good result?

  • Good laboratory practice
  • Shake and incubate as described in the manual
  • The described pipetting volumes (100 µL for the samples, 150 µL for SAN HQ standards), improves the robustness of the assay.

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3. What mistakes should I be aware of?

  • The blank value is for control only (background signal) and must not be divided by the measured values. “Blanked” absorbance data for creating the calibration curve may cause error in the generated standard curve. Never subtract the BLANK E450 value from the other measurement values.
  • Pipetting errors during preparation of the SAN HQ standards (liquid transfer, mixing, residual liquid in the pipette tips)
  • Improper plate washing steps, including
    • residual liquid in the wells after washing
    • plate wells run dry after removal of liquid due to time delays during assay development
    • air or particles in the liquid-channels when using an automated plate washer
    • damage of the wells surface by pipette tips or needles from automated washers during washing
  • Well-to-well cross-contaminations (liquid drops on the plate cover foil)
  • 8-well strips not properly fixed during plate washing and/or reading of the colored dye (might cause imprecise results due to insufficient washing and light scattering effects)
  • Preparation of dilutions (calibrators/ samples) within the antibody-precoated assay plate (might cause imprecise and inaccurate results)
  • Inaccurate and uneven well filling volumes during incubation steps (common source of errors when using multi-channel pipettes by inexperienced operators)

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4. Why is the absorbance in some wells too high?

  • This is an indicator of improper assay performance which could be caused by
    • Well-to-well cross-contamination
    • Insufficient washing after incubation steps
    • Dirt on the bottom of the plate.
    • Air bubbles within the wells during measuring the absorbance.

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5. Why is the overall signal recovery quite low?

  • To isolate the cause, it is required to evaluate the measurement results from the controls. It could result from
    • Error within the initial dilution of SAN HQ standard and control.
    • Issues during setting up the assay: insufficient shaking speed, wrong dilution of detection antibody concentrate and/or enzyme conjugate, buffer residues within wells after washing (especially before adding the TMB substrate).

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6. Why do I get insufficient signal in some wells, but not all?

  • This could result from dilution and/or sample transfer errors.
  • Inaccurate preparation of the 2-fold serial dilution.

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