1. Begins with an antibody bound to a polystyrene well.
2. Plus a test sample containing an antigen mixture to which an antigen-enzyme conjugate is added.
3. At this point, competitive inhibition occurs between the antigen-enzyme conjugate and an unlabeled antigen, depending on which antigen type is in excess, two different outcomes can follow when binding to a specific antibody occurs. After the formation of an immune complex from an antigen-antibody binding.
4. The reagents are separated by washing. Next a substrate is added to the immune complex
5. If the antigen-enzyme conjugate is the antigen in excess a color change will occur indicating that the substrate was chemically changed as a result of the enzyme conjugate being bound to the immune complex
6. If it is the unlabeled antigen that is in excess there will be little to no change in color because the test sample contains antibody-type-specific antigen
Principle for Indirect Competitive Antibody Capture ELISAs
1. Immobilization reactionA standard analyte (antigen) is immobilized on a solid phase (microtiter plate well). 2. Competition reactionThe sample is added together with a primary antibody specific for the analyte. The analyte in the sample competes with the standard analyte on the solid phase for binding to the antibody. Unbound components are washed away.
3. Binding of enzyme-labelled antibodyAn enzyme-labelled antibody binding to the primary antibody is added. Unbound antibody is washed away.
4. Chromogenic reactionA non-coloured substrate is added, and the substrate is converted to a coloured product by the enzyme bound to the antigen-antibody complex.
5. Quantitative analysisThe colour intensity is measured with a microplate reader and is inversely proportional to the concentration of analyte in the sample. The relationship between absorbance and analyte concentration is obtained from a standard curve created from a reference material.
