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HORMONES ASSAYS

HORMONES ASSAYS. NABIL BASHIR 2009. R adio I mmuno A ssay ( RIA ) E nzyme- L inked I mmunosorbent A ssay ( ELISA ) F luorescence P olarization I mmuno A ssay ( FPIA ) C hemi l uminescence enzyme i mmuno a ssay ( CLIA ). Radioimmunoassay. Hormone* + Antibody + Cold hormone.

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HORMONES ASSAYS

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  1. HORMONES ASSAYS NABIL BASHIR 2009

  2. RadioImmunoAssay (RIA) • Enzyme-Linked Immunosorbent Assay (ELISA) • Fluorescence Polarization Immuno Assay (FPIA) • Chemiluminescenceenzyme immunoassay (CLIA)

  3. Radioimmunoassay Hormone* +Antibody + Cold hormone .

  4. The unlabelled hormonecompetes with Hormone* for binding to the antibody and displaces a proportional amount of it. • The unbound hormone is separated away (by centrifugation, for example) and the amount of radioactivity remaining is measured (with a Geiger counter). • standard binding curve is constructed.

  5. Enzyme-Linked Immunosorbent Assay (ELISA) immobilized antibody molecules + Hormone + The antibody-enzyme conjugate WASHING + SUBSTRATE STOP colored product formed Spectrophotometer The intensity of color is proportional to the concentration of bound hormone

  6. Fluorescence Polarization Immuno Assay • fluorescently labeled hormone • antibody. • unlabeled patient hormone

  7. Small fluorescent molecules have low degree of fluorescence polarization (FP).(well #1).

  8. FA bound to its antibody represents a large fluorescent molecule, which exhibits a high degree of fluorescence polarization (FP).

  9. polarization is quantified as milli-polarization units, or mP. • A fluorescence polarization reader is required to make this measurement.

  10. fluorescent hormone+ hormone+antibody

  11. FPIAs are based on the competition of FA (flourescent hormone) with free (i.e. unlabeled) hormone in the samples or standards for the high affinity binding site an antibody. • FA:antibody complex will give a high FP reading. • Addition of a increasing amounts of unlabeled hormone will result in a competition between the unlabeled FA for the antibody.

  12. As the competition happens, some of the FA will be released from the antibody.This will cause a detectable loss of FP in the well (well #4).

  13. The addition of large amount of hormone will result in a much larger reduction in the mP of the well (well #5). • Plotting mP versus hormone concentration allows the construction of a standard curve.

  14. CHEMILUMINESCENCEENZYME IMMUNOASSAY (CLIA)

  15. The intensity of the emitting light is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled hormone in the sample. • By reference to a series of hormonestandards assayed in the same way, the concentration of hormone in the unknown sample is quantified.

  16. Example of Standard Curve

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