Competitive ELISA

Overview

• Used to determine the concentration of either antigen or antibody in a sample.
• Focus: Determining concentration of antibodies.

Determining Antibody Concentration

1. Example: Determining concentration of anti-A antibodies in a serum sample.
2. Required Components:
   • Specific antigen (e.g., A antigen for anti-A antibodies).
   • Enzyme-linked anti-antibody that binds to the antigen similar to sample antibodies.
     • Enzymes used could be horseradish peroxidase, beta-galactosidase, or alkaline phosphatase.
     • Chromogenic substrates (e.g., TMB for horseradish peroxidase) are used, resulting in a color change measurable by spectrophotometry.

Procedure

1. Microtiter Well Coating:
   • Coat with specific antigen using a coating buffer.
2. Adding Antibodies:
   • Add sample containing anti-antibodies and a fixed amount of enzyme-linked antibodies.
   • These antibodies compete to bind with the antigen in the well.
3. Competition Outcome:
   • Antibody with higher concentration binds more antigens.
   • Two scenarios:
     1. Higher Sample Antibody Concentration:
        • Sample antibodies occupy more antigens.
        • Fewer enzyme-linked antibodies bind, leading to less color product.
     2. Higher Enzyme-linked Antibody Concentration:
        • More enzyme-linked antibodies bind.
        • More color product formed.
4. Washing:
   • Remove unbound antibodies.
5. Color Development:
   • Add chromogenic substrate and incubate.
   • Stop reaction with strong acids.
   • Enzyme converts substrate to color product.
6. Absorbance Measurement:
   • Less absorbance indicates more antibodies in sample.
   • More absorbance indicates fewer antibodies in sample.

Analysis

• Determine antibody concentration by extrapolating absorbance value on a standard graph.

Reverse Procedure

• Used to determine antigen concentration.
  • Similar but opposite approach to antibody determination.