General Protocol for Sandwich-style Enzyme-Linked Immunosorbent Assay (ELISA)

Principle

Analytes in plasma and other fluids can be measured by capturing onto a microtitre plate coated with a capture antibody. After washing the plate to remove unbound proteins the captured analyte is detected by incubating with another antibody containing a reporter molecule, in this case the enzyme horseradish peroxidase. The unbound detecting antibody is washed away and the plate developed with a solution of peroxidase substrate which produces a coloured end product. After a fixed time the reaction is stopped and the adsorbance of each well in the microtitre plate is determined. As the concentrations of capture antibody and detecting antibody are fixed, the colour generated is proportional to the concentration of analyte present in the sample.

 
Materials Required but Not Provided:

 

Procedure:

 

References:

  • Nix,B, Wild D, in Immunoassays, A Practical Approach, editor J.P. Gosling, pp. 239-261, Oxford University Press, 2000.
  • NCCLS. Evaluation of the Linearity of Quantitative Analytical Methods; Proposed Guidline – Second Edition. NCCLS Document EP6-P2 (ISBN 1-56238-446-5, NCCLS, Wayne, Pennsylvania USA, 2001
  • FDA Guidance for Industry. Bioanalytical Method Validation; May 2001, available on the internet: www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf