BM Chemiluminescence ELISA Substrate (AP) Protocol

Protocol

ELISA Protocol

Handling instructions

  • The AP assay can be performed in all automated or manual luminometers in tube and microplate format as well as in liquid scintillation counters (LSC).
  • When using the microplate format, black or white microplates must be used.
  • The kit contains all reagents required for ELISA application.
  • For SEAP reporter gene analysis dilution buffer (TBS) and L-homoarginine are additionally required.

Procedure

Important: All reagents should be fully equilibrated to room temperature (+15 to +25 °C) before starting the test. Reagents with different lot numbers must not be used in one test. Changing relative amounts and concentrations may result in reduced sensitivity.

  • Follow your standard ELISA protocol as optimized for colorimetric detection. Apply sample, primary and secondary antibody in a volume of 150 ml each. Otherwise adapt the volume of assay reagent to the altered conditions.
  • After incubation of the AP-labeled antibody discard the solution and wash three times (e.g., with TBS), leaving the buffer in the wells for 0.5 to 5 minutes between each individual wash.
  • Discard the wash solution and tap the MP on a lintfree, dry, absorbant cloth.

Manual application of the assay reagent

  • Add 150 ml substrate reagent per well.
    Note: For best accuracy the addition of the substrate reagent should be timed in the same interval as the luminometer/LSC reads the samples.
  • Incubate for 10 min at room temperature, while gently rocking.
  • Transfer MP to the luminometer. Integrate light production for 1 to 5 seconds.
    Note: The light emission peaks after 10 min and keeps constant for at least 60 min.

Automatic application of the assay reagent

  • Transfer MP to the luminometer.
  • Inject 150 μl substrate reagent automatically in the same interval as the luminometer reads the samples.
  • Incubate for 10 min at room temperature.
  • Start integration of light reaction for 1 to 5 seconds.

SEAP Reporter gene assay

Additional reagents required

Dilution buffer: 25 mM Tris/HCl, 150 mM NaCl, pH 7,4 (TBS)

Procedure

Important: All reagents should be fully equilibrated to room temperature (+15 to +25 °C) before starting the test. Reagents with different lot numbers must not be used in one test. Changing relative amounts and concentrations may result in reduced sensitivity.

Materials

     
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