Micro I-3: Methods In Immunology

Front 1

What is ELISA?

Back 1

A solid phase immunoassay where one component absorbed to solid support (usu. a 96-well plate) and a second component in solution is added in a series of dilutions. Protein is absorbed to plastic and antibody-antigen interactions are qualitatively or quantitatively measured, based on light absorbance

Front 2

How does detection occur in ELISA?

Back 2

ELISA detection is generally via an enzyme-coupled antibody yielding a color reaction

Front 3

What is FACS (flow cytometry)?
What makes FACS (flow cytometry) powerful?

Back 3

- Most frequently used for the analysis of leukocytes, although any cell type that can be prepared as a suspension can be analyzed
- Ability to quantitatively analyze large numbers of cells, and to accurately identify complex or small populations. Also possible to separate specific populations of cells, based on specific phenotypes

Front 4

What is immunofluorescence?
When would immunofluorescence be used?

Back 4

- Similar to FACS, but fluorescence detection is by microscopy. Only a small number of cells can by analyzed
- To analyze or identify subcellular structures, to identify specific populations of cells, or to analyze a biopsy in which the available cell population is very small

Front 5

What is Radioimmunoassay (RIA)?
What is RIA useful for?

Back 5

- Essentially an ELISA that uses radioactivity in place of the enzyme to allow detection of the bound antibody
- RIA is highly sensitive, so it is particullarly useful for the quantification of low-abundance substances

Front 6

When RIA is performed in solution, which amounts (of radiolabeled antigen, unlabeled antigen, and specific antibody) are known?

Back 6

a known amount of radiolabeled antigen is mixed with an unknown amount of unlabeled antigen and a known amount of specific antibody

Front 7

Which assay fractionates proteins based on size using an SDS-polyacrylamide gel?

Back 7

Western blot

Front 8

Which assay fractionates DNA fragments based on size using an agarose gel?

Back 8

Southern blot

Front 9

What is the Coombs test used for?

Back 9

- Testing for anti-Rh antibodies and to diagnose immune-mediated hemolytic anemia. Specifically detects antibodies against red blood cells

Front 10

What is the direct Coombs test?

Back 10

Pt's red blood cells are recovered from a patient via blood draw, and mixed with anti-human Ig (Coombs reagent). Positive test is agglutination

Front 11

What is the indirect Coombs test

Back 11

Donor's red blood cells are mixed with the patient's and then with anit-human Ig (the Coombs reagent). Commonly used for pregancy

Front 12

Name three sources of antibodies

Back 12

- Human Serum (used to measure a patient's antibody response)
- Polyclonal antibodies (used as detection reagents)
- Monoclonal antibodies (hybridomas)

Front 13

How would one produce Polyclonal antibodies?

Back 13

IgG (or other isotypes) is purified from the serum of the immunized animal

Front 14

How would one produce Monoclonal antibodies?

Back 14

fusion of spleen cells with an immortalized B cell tumor resulting in hybridomas

Front 15

What are proliferation assays?

Back 15

assays used to measure the ability of B cells or T cells to proliferate in response to mitogenic stimuli?

Front 16

What is a functional assay used for?

Back 16

To measure specific effector functions of leukocytes

Front 17

Name two nucleic acid-based assays

Back 17

Southern blot and Microarrays

Front 18

What can a Southern blot be used to detect?

Back 18

restriction fragment length polymorphisms (RFLPs)

Front 19

Which assay permits the analysis of gene expression of thousands of genes?

Back 19

Microarrays