Principles of ELISA

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Introduction:

  • ELISA¬†(enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones
  • Most common substrate used: TMB (Tetramethylbenzidine)
  • Most common enzyme used: HRP (Horseradish peroxidase).

Types & Principles:

  1. Direct ELISA:
    • Detect Ag using specific Ab
    • Specificity is high.
  2. Indirect ELISA:
    • Detect Ab using conjugates
    • Conjugate = Anti-human immunoglobulin labelled with enzyme
    • Sensitivity is high.
  3. Competitive ELISA:
    • Detects Ab using specific Ab labelled with enzyme
    • There is a competiton between patient’s Ab &¬†specific Ab with enzyme, hence the name ‘Competitive ELISA’.
  4. Capture ELISA:
    • Detect specific isotype of Ab in patient’s serum (IgM/ IgG) using specific monoclonal Ab (also known as ‘capture Ab‘)
    • Dye used: FITC (Fluorescein Isothiocyanate).

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