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Diagnostic tests for antibody or antigen Diagnostic tests for antibody or antigen

Diagnostic tests for antibody or antigen - PowerPoint Presentation

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Uploaded On 2022-06-14

Diagnostic tests for antibody or antigen - PPT Presentation

ELISA Immunofluorescence Flow cytometry Western blotting ELISA We use an antibody thats bound covalently to an enzyme to bind antigen direct ELISA antibody that is bound to antigen indirect ELISA ID: 918130

antigen antibody elisa enzyme antibody antigen enzyme elisa cells bound fluorescent sds indirect compound detection light populations separated blot

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Presentation Transcript

Slide1

Diagnostic tests for antibody or antigen

ELISA

Immunofluorescence

Flow cytometry

Western blotting

Slide2

ELISA

We use an antibody that’s bound covalently to an enzyme to bind:

-antigen (direct ELISA)

-antibody that is bound to antigen (indirect ELISA)

After washing, we add a colorless substrate for the enzyme in a reaction that produces color (qualitative detection)

…quantitative measurement can also be done by certain techniques

The enzyme is usually ALK (alkaline phosphatase) or HRP (horseradish peroxidase)

Example: Detection of antibodies to HIV

Slide3

Immunofluorescence

We use antibody that is bound to a fluorochrome (fluorescent compound)…usually fluorescein isothiocyanate (FITC)

Fluorescent compound emits a greenish light when exposed to UV light…we use fluorescence microscope which provides UV light

Usually done on tissues and cells to detect antigens (direct) or antibodies (indirect)

Treponema pallidum

spirochetes

 

Fluorescent

treponemal antibody absorption

 (

FTA-ABS

test

Slide4

Flow cytometry

To enumerate live cells that express certain antigen

Fluorochromes of different colors are used

Counting cell populations with certain fluorochrome label

Separating populations of cells according to physiochemical and fluorescence characteristics

Slide5

Western blotting

Protein antigens (e.g., of known HIV) are separated by

SDS-PAGE (SDS-polyacrylamide gel electrophoresis)

according to molecular weight and charge

The separated proteins are transferred to a nitrocellulose membrane (blot)

The blot is incubated with the antiserum, washed and an enzyme-coupled anti-Ig is then added

The antigen bands that reacted with the antibody are colored