Immunological Pregnancy Testing

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Presentation transcript:

Immunological Pregnancy Testing ELISA What does it stand for?

Human Chorionic Gonadotropin (hCG) = glycoprotein hormone Trophoblast (= embryo) cells produce hCG, structurally similar to LH. Purpose? Detectable in urine 6-8 days after conception Old ways of detection?

Early Pregnancy Tests Time consuming. Patient’s urine injected into female laboratory rabbit. High concentrations of hCG  ovulation and development of the rabbit's uterus. ”The rabbit died” meant women is pregnant.  misnomer!

ELISA – the New Way of Pregnancy Testing ELISA and the use of antibodies How do you make antibodies? What are conjugated antibodies? Indirect Sandwich Assay

Indirect Sandwich ELISA uses two antibodies ( more sensitive) Inactivated substrate Secondary antibody conjugated to enzyme (e.g.: HRP) Activated substrate changes color Sandwich assay: measures amount of antigen between two layers of antibodies (most common case of indirect ELISA) Target protein (hCG) Primary antibody Need picture of this type of assay Surface molecules of ELISA plate plastic wells

Production of 1 and 2 Antibodies Inject rabbit (mouse, goat etc.) with a human protein (specific ab / human hormone etc.) What does rabbit do? Polyclonal antibodies: mixed population of antibodies (produced by many clones) that recognize numerous epitopes Monoclonal antibodies: bind to only 1 epitope

Monoclonal Antibody Production

96-well microtiter plate hCG ELISA Review only 1st part