Assays- Antibody and Cell

Question 1

The affinity with which antibody binds antigen results from a balance between what forces?

Answer 1

Attractive and repulsive forces

Good fit: high attraction, low repulsion

Question 2

Avidity

Answer 2

Combined affinity of many antibody-antigen interactions

Affinity + valence (ex- IgM has high valence due to multiple binding regions)

Question 3

Sensitivity

Answer 3

Ability of test to correctly identify true positives

Question 4

Specificity

Answer 4

Ability of test to correctly identify true negatives

Question 5

Most commonly used antibody-based assay

Answer 5

ELISA (enzyme-linked immunosorbent assay)

Question 6

Precipitation reaction

Answer 6

Antibody and antigen bind in aqueous solution, forming precipitate
Both “arms” of immunoglobulin must bind

Question 7

Radial and double immunodiffusion

Answer 7

Immunoprecipitation reaction in gel
Ag and Ab diffuse towards each other
Line of precipitation occurs at the line of equilibrium

Question 8

Is precipitation a quantitative or qualitative test?

Answer 8

Qualitative

Question 9

Downsides to precipitation

Answer 9

Slow- often takes a day or two

Requires large amount of Ag and Ab

Question 10

Immunoelectrophoresis

Answer 10

Combination of electrophoresis and double immunodiffusion
Antigen mixture is electrophorised to separate components by charge
Antigen and antibody will diffuse and produce line of precipitation

Question 11

Is precipitation still commonly used?

Answer 11

No- faster methods have replaced

Question 12

Agglutination

Answer 12

Visible clumping formed by interaction between insoluble antigen and antibody

Question 13

Example of agglutination assay

Answer 13

Blood type: red blood cells are mixed with anti sera to A or B antigen

Question 14

Radioimmunoassay

Answer 14

Rarely used
Radioactively labeled and unlabeled antigen compete for antibody binding site
Displaced radioactive isotope is measured

Question 15

ELISA (enzyme-linked immunosorbent assay)

Answer 15

Enzyme conjugated with antibody reacts with a colorless substrate to generate a colored reaction product
Can use either known antigen or known antibody, which is coated to plate

Question 16

Immunofluorescence

Answer 16

Antibodies or proteins bound to antibodies are fluorescently labeled
When antibodies bind to antigen on cell membranes, emit wavelength of light that can be viewed with fluorescence microscope

Question 17

Flow cytometry

Answer 17

Used to separate antibody-bound cells stained with fluorescent dye
Enables counting of cells in suspension

Question 18

Which antibody assay methods are quantitative?

Answer 18

Radioimmunoassay, ELISA, immunofluorescence with flow cytometry

Question 19

Cell-mediated immune assays: panning

Answer 19

Cells are washed over antibody-bound plate: cells with antigen that is specific to antibodies bind and other cells are washed away

Question 20

ELISPOT

Answer 20

Similar to ELISA, but using panning techniques
Used to determine number of cells (B or T cells) that are reactive against a certain antigen
B cells release antibodies that bind with antigen on plate
T cells release cytokines that bind with anti-cytokines on plate
Enzyme-conjugated anti-cytokines or anti-antibodies bind to cytokines or antibodies
Addition of substrate produces color

Question 21

Lymphocyte stimulation

Answer 21

Most common test for T cell activation
Lymphocytes are separated from blood, then put into test tubes with antigen
Radioactive thymidine is added and amounts of radioactivity are measured (activated T cells proliferate and incorporate thymidine)

Question 22

Cell-mediated cyototoxicity

Answer 22

Measures activity of T cytotoxic cells
Target cells are incubated with 51Cr, which is taken up into cells and binds to protein
Cells are plated out and are co-cultured with T cytotoxic cells
Cells that lyse release chromium, which can be measured

Question 23

Quantitative tests focus on specificity or sensitivity?

Answer 23

Specificity (ability to identify true negatives and prevent false positives)

Question 24

Qualitative tests focus on specificity or sensitivity?

Answer 24

Sensitivity (ability to identify true positives and prevent false negatives)