Labeled immunoassays
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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. Labeled Immunoassays.

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Labeled Immunoassays

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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


Labeled Immunoassays

  • Is there separation of Ag-Ab complexes from free Ag & free Ab?

    • Homogeneous assay: no

    • Heterogeneous assay: yes


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


Labeled Immunoassays

Homogeneous Competitive EIA

Enzyme is inactivated if bound to an antibody

Patient Ag

Color rxn

Due to free enzyme


Labeled Immunoassays

Sandwich EIA

A wash is performed to remove unattached test reactants after each step

Patient Ag

Color rxn


Labeled Immunoassays

Indirect EIA

A wash is performed to remove unattached test reactants after each step

Patient Ab

Color rxn


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


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


Labeled Immunoassays

Immunofluorescent Microscopy Techniques


Labeled Immunoassays

Immunofluorescent Microscopy Techniques

A “wash” is done to remove unattached antibody between each step


Summary of Methods

  • Directly visualize

    • Precipitation / Western blot analysis

    • Flocculation

    • Agglutination

  • Indirectly visualize

    • Neutralization

    • Complement fixation

    • Labeling techniques


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