Labeled Immunoassays

1. Labeled Immunoassays • Indicator • Known antibody (rarely the antigen) is labeled for detection / visualization of Ag-Ab reaction • Labeling techniques • Radioactive (not used much in clinical lab) • Fluorescence • Enzyme

2. Labeled Immunoassays • Is there separation of Ag-Ab complexes from free Ag & free Ab? • Homogeneous assay: no • Heterogeneous assay: yes

3. Labeled Immunoassays Heterogeneous Competitive EIA Ab may be attached to a solid phase to allow for separation Patient Ag Color rxn Enzyme with substrate develops color reaction

4. Labeled Immunoassays Homogeneous Competitive EIA Enzyme is inactivated if bound to an antibody Patient Ag Color rxn Due to free enzyme

5. Labeled Immunoassays Sandwich EIA A wash is performed to remove unattached test reactants after each step Patient Ag Color rxn

6. Labeled Immunoassays Indirect EIA A wash is performed to remove unattached test reactants after each step Patient Ab Color rxn

7. Labeled Immunoassays HIV Indirect EIA Controls conc. IU/ml 3.0 2.5 1.0 0.0 Patient Serum (3 different patients) #1 #2 #3 What is being tested for? Antibody to HIV Explain the test results. Patient 1 & 3 are positive w/ #3 having a higher concentration of antibody to HIV than #1

8. Labeled Immunoassays Immunofluorescent Microscopy Techniques • Fluorophores or fluorochromes absorb light of a shorter wavelength and emit light at a longer wavelength • These compounds are attached to a known antibody • Observe test reaction with a fluorescent microscope

9. Labeled Immunoassays Immunofluorescent Microscopy Techniques

10. Labeled Immunoassays Immunofluorescent Microscopy Techniques A “wash” is done to remove unattached antibody between each step

11. Summary of Methods • Directly visualize • Precipitation / Western blot analysis • Flocculation • Agglutination • Indirectly visualize • Neutralization • Complement fixation • Labeling techniques