Clinical Applications of Ab/Ag Interactions

Question 1

Direct ELISA

Answer 1

1. Unlabeled Ag attached to inert substance.
2. Bound Ag detected by an Ab that has been labeled with an enzyme.
3. Substrate added.
4. Colometric or fluorescent change detected.

Question 2

Indirect ELISA

Answer 2

1. Unlabeled Ag bound to plate.
2. Serum added to plate & Ab/Ag interaction allowed to occur.
3. Anti-Ig conjugated to an enzyme added which binds to previous Ab.
4. Excess or unbound enzyme-anti-Ig washed away.
5. Enzyme substrate added.
6. Color change quantitated.

Question 3

Sandwich ELISA

Answer 3

Used for quantitation of antigen.

1. Ab conjugated to plate.
2. Ag added.
3. Enzyme-labeled Ab added.
4. Substrate added.
5. Color change detected.

Question 4

Serological Diagnosis

Answer 4

A definitive serological diagnosis for acute infection shows a four-fold increase in antibody titer between two serum samples drawn at least 10-14 days apart.

Referred to as acute and convalescent serum samples.

Question 5

Immunohistochemistry

Answer 5

Used to detect and localize antigens in tssues and cells.

Direct

Uses a labeled Ab to a specific antigenic component.

1. Sample immobilized on a slide and treated with a tagged primary Ab.
2. Sample washed to remove unbound Ab.
3. Sample viewed by LM or fluorescent microscopy.

Indirect

Secondary antibody has been labeled which allows for detection of lower Ag concentrations.

1. Sample immobilized on a slide and treated with primary Ab or serum.
2. Sample washed to remove primary Ab.
3. Secondary Ab added which reacts with primary Ab (eg anti-Ig Ab)
4. Sample washed to remove unbound secondary Ab.
5. Sample viewed by LM or fluorescent microscopy.

Question 6

Flow cytometry

and

Fluorescence-activated cell sorting (FACS)

Answer 6

Useful to identify status of a single cell in a mixed population.

Utilizes tagged Ab to identify different molecules on the cell surface or expressed in the cytoplasm allowing visually identical cells to be differentiated.

1. Cells stained with fluorescently labeled Ab specific for the cell type analyzed.
2. Suspension forced through nozzle which seperates into droplets each containing a single cell.
3. Droplet passed through a laser beam.
4. As each cell scatters the laser light it is detected.
5. If cell bound by fluorescent Ab then signal emitted and recorded.