Radioimmunoassay RIA and chemiluminescence - .pptx

this section talk about immunoassay methods

1. TYPE OF LABELS MEASURED CHARACTERISTIC RADIONUCLIDES ENZYMES FLUORESCENT MOLECULES Light generating MOLEC. RADIOACTIVITY ENZYME ACTIVITY FLUORESCENCE Light

2. Radioimmunoassay is more sensitive than ELISA but it is not safe There are two types of immunoassay according to the labeled reagent: 1- RADIOIMMUNOASSAY (RIA) COMPETITION LABELED ANTIGEN 2. IMUNORADIOMETRIC ASSAY (IRMA) LABELED ANTIBODY

3. ANTIBODY LABELED ANTIGEN UN- LABELED ANTIGEN +

4. Y- cpm X- CONCETRATION In RIA there is an inverse relationship between radioactivity and the hormone (antigen) concentration

5. CHEMILUMINESCENCE Measurement of light produced by chemiluminescence during certain chemical reactions provides a convenient and highly sensitive alternative to absorbance measurements in ELISA assays. In this versions of the ELISA using chemiluminescence, a luxogenic (light- generating) substrate takes the place of the chromogenic substrate in conventional ELISA reactions.

6. For example, oxidation of the compound luminol by H ₂ O ₂ and the enzyme horseradish peroxidase (HRP) produces light: Ab- HRP Ag → Ab- HRP- Ag → light The advantage of chemiluminescence assays over chromogenic ones is enhanced sensitivity. CHEMILUMINESCENCE is more sensitive than ELISA In general, the detection limit can be increased at least ten-fold by switching from a chromogenic to a luxogenic substrate