2. Radioimmunoassay (RIA)
• The technique was first developed in 1960 by two endocrinologists,
S.A. Berson and Rosalyn Yalow, to determine levels of insulin & anti-
insulin complexes in diabetics.
• It soon proved its value for measuring hormones, serum proteins,
drugs and vitamins at very low concentrations (0.001µg/mL).
• The principle of RIA involves competitive binding of radiolabeled
antigen and unlabeled antigen to a high-affinity antibody.
• Similar to Archimedes' principle.
3. • To a limited amount of Ab concentration, saturating amount of
labelled Ag are added.
• Unbound Ag are washed off.
• Test sample of unlabelled Ag are added at high concentration, to
compete with labelled Ag.
• The antibody does not distinguish labeled from unlabeled antigen.
• Amount of labelled Ag gradually decreases.
• Radioactivity level decreases and is measured.
• Standard curve is prepared using known concentration of unlabeled
Ag.
5. Enzyme Linked Immunosorbent Assay (ELISA)
• Performed in microtitre plates, (96)
• Can be used in detection of HIV (Viral proteins in well + serum Ab of patient)
6.
7. • higher the concentration of antigen in the original sample, the lower the absorbance.
8. Immunofluorescence
• In 1944, Albert Coons showed that antibodies could be labelled with
molecules that have the property of fluorescence.
• Fluorescent molecules absorb light of one wavelength (excitation) and
emit light of another wavelength (emission).
• Ab’s attached with fluorochrome or fluorescent dye can be detected
after their exposure to the radiation of suitable wavelength.
• The emitted light can be viewed with a fluorescence microscope,
which is equipped with a UV light source.
• Fluorescein and Rhodamine are commonly used dyes.
9. • These molecules can be conjugated to the Fc region of an antibody
molecule without affecting the specificity of the antibody.
• Pigments like Phycoerythrin are extracted from algae, which show
intense color are also used.
• Fluorescein – Blue light (490 nm), Yellow-green fluorescence (517 nm)
• Rhodamine - Yellow-green (515 nm), Deep Red (546 nm)
10.
11. • Applied to identify a number of subpopulations of lymphocytes,
notably the CD4 and CD8 T-cell subpopulations.
• The technique is also suitable for identifying bacterial species (MTB
virulence factors)
• Detecting Ag-Ab complexes in autoimmune disease.
• Detecting components in tissues, and localizing hormones and other
cellular products stained in situ.
• Flow Cytometry – Surface antigen based cell separation
12. Applications
• Ag-Ab reactions are extensively used in detection of diseases.
a) ELISA – HIV
b) Mantoux Test – Tuberculosis
c) Widal Test – Typhoid
• Cell sorting has been carried out based on surface antigens
• ChIP (Chromatin Immunoprecipitation)
• Hybridoma technology
• Severity or stage of disease
• Respond to treatment
• Epidemiology – distribution and effects of the disease.
