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Enzyme-Linked Immunosorbent Assay

Enzyme-Linked Immunosorbent Assay. Enzyme-linked immunosorbent assay Antibody molecules used to detect an antigen Enzymes linked to the antibodies to produce colored reactions Used to detect the amount of antigen or antibody in a sample Performed as a solid phase assay

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Enzyme-Linked Immunosorbent Assay

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  1. Enzyme-Linked Immunosorbent Assay

  2. Enzyme-linked immunosorbent assay Antibody molecules used to detect an antigen Enzymes linked to the antibodies to produce colored reactions Used to detect the amount of antigen or antibody in a sample Performed as a solid phase assay Where the antigen/antibody being detected is bound to a solid surface Assay usually done in a microtiter plate or ELISA plate ELISA

  3. Add labeled antibody Wash away unbound antibody Add enzyme substrate and measure absorbance

  4. b-gal b-gal b-gal Types of ELISA • Direct • Detects amount of antigen • One antigen, one antibody • Indirect • Detects amount of antibody • One antigen, two antibodies • Sandwich • Detects amount of antigen • One antigen, two antibodies • Used if the antigen does not bind well to the ELISA plate

  5. Only detects antibody/antigen, does not tell you if an infection is ongoing People may be poor producers of antibody or recently exposed to an antigen = false negative Unrelated antibodies or antigens may react in an unspecific way = false positive Limitations of ELISA

  6. Diagnostic Diagnosing diseases: Herpes, Syphilis Vaccination titers: Chicken pox, Hepatitis The  the amount of antibody  the chance of exposure and/or immunity Research Specificity of an antibody for an antigen Determining antigenic determinants Vaccine development Applications of ELISA

  7. First and most basic test used to determine if a person is positive for HIV Detects for the presence of anti-HIV antibodies Indirect ELISA b-gal Anti-HIV antibodies in a patient’s serum Anti-human IgG labeled with an enzyme Inactivated HIV antigen HIV ELISA (Indirect ELISA)

  8. ELISA-based test Checks a woman’s urine for human chorionic gonadotropin (hCG), Sandwich ELISA Mouse a-hCG Enzyme conjugate Urine sample (hCG) Goat a-mouse IgG Substrate conjugate Mouse a-hCG Substrate conjugate a Wick Location 1 2 3 Handle Pregnancy Test (Sandwich ELISA)

  9. Direct ELISA • Most Common • Solid Phase Assay • Detects the amount of antibody-antigen reaction by the use of a substrate • Take reading in a spectrophotometer, the higher the reading, the more reaction

  10. Many different enzymes can be used in ELISA, which can produce different colored reactions Todays Enzyme Is HRP enzyme substrate Color detection by an ELISA reader, which acts similarly to a spectrophotometer or by eye Substrate

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