chemiluminescence immonoassay

Chemiluminescence immunoassay (CLIA)

Chemiluminescence immunoassays are a variation of the standard ELISA detection method. They use chemiluminescent labels which produce light when excited by chemical energy and are a highly sensitive way of quantifying proteins and have extremely low backgrounds. The advantage of this method is that there isn’t a long incubation time required and is more economical compared to ELISA.

Reagents

  • Capture Antibody
  • Detection Antibody-HRP (Enzyme Conjugate Reagent)
  • Hydrogen Peroxide / Luminol (Chemiluminescent Substrate)
  • Blocking Buffer
  • PBS Wash Buffer

Note: The Capture and Detection Antibodies used must be against the target protein of interest present in the sample.

Method

1) Ensure wells are coated with desired Capture Antibody and blocked with an appropriate Blocking Buffer.
2) Wash coated plates 3 times with PBS Wash Buffer.
3) Incubate wells with blocking buffer, then wash plates 3 times with PBS Wash Buffer.
4) Dispense 50μl of the sample into the appropriate wells, then wash plate 3 times in PBS Wash Buffer.
5) Dispense 100μl of Detection Antibody-HRP (enzyme conjugate reagent) into each well.
6) Make sure it is thoroughly mixed by mixing for 30 seconds.
7) Incubate for 45 mins at 37oC.
8) Remove the mixture and wash wells 5 times with PBS.
9) Remove excess water by tapping the plate.
10) Add Hydrogen Peroxide / Luminol (Chemiluminescent Substrate) into each well and mix gently for 5 seconds.
11) Read the wells with a chemiluminescence microplate reader between 5 and 20 minutes after dispensing the substrate.