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  • Incorrect dilutions or pipetting errors
  • Improper incubation times
  • Improper mixing of the 2-component TMB substrate. Each component is mixed in equal parts or according to manufacturer¡¯s instructions.
  • Wrong filter on microtiter reader. Wavelength should be 450 nm for TMB.
  • Set materials or reagents are contaminated or expired.
  • Incorrect reagents used.
  • Dilute the HRP Detection Antibody less.

1

  • Cross contamination from other samples or positive control
  • Incorrect dilutions or pipetting errors
  • Improper washing
  • Wrong filter on microtiter reader. Wavelength should be 450 nm for TMB.
  • Contaminated buffers or enzyme substrate
  • Improper incubation times
  • Set materials or reagents are contaminated or expired.
  • Dilute the HRP Detection Antibody more.

2

  • Poor mixing of specimens
  • Incorrect dilutions or pipetting errors
  • Technical error
  • Inconsistency in following ELISA protocol
  • Inefficient washing

3

  • Contaminated buffers or enzyme substrate
  • Incorrect dilutions or pipetting errors
  • Set materials or reagents are contaminated or expired.
  • Inefficient washing

5

  • Procedure not followed correctly
  • Contaminated buffers or enzyme substrate
  • Contaminated Conjugate
  • Set materials or reagents are contaminated or expired.

6

  • When preparing coating buffer from the gel capsule, break the capsule apart and pour ingredients into water. Do not place gel capsule into water. The gelatin from the capsule interferes with the binding of the coating antibody to the plate.
  • Capture antibody diluted with coating buffer should be added to wells immediately.
  • Coated (covered) plates are stable overnight at 4oC.
  • Check all buffers for contamination and expiration. When trouble shooting, it may be helpful to start with all new buffers. Make buffers in new or properly cleaned vessels.
  • Sodium Azide should not be added to any of the buffers.
  • Dilutions should be made shortly before application and immediately applied to appropriate wells. Do not save extra diluted standards or samples for future assays.
  • Wash buffer should be aspirated from wells. Pouring/Dumping wash buffer from wells may lead to cross contamination.
  • Excess antibody/analyte should be wiped from pipettes tips when making dilutions.
  • Incubation time of the TMB Substrate will depend on the intensity of the color change. The high standard should have an O.D. reading of 1.8 ¨C 2.2 and the O.D. reading of the low standard should be above background. Stop solution should be added to the plate in the same order as the TMB Substrate.