Assays- Antibody and Cell Flashcards
The affinity with which antibody binds antigen results from a balance between what forces?
Attractive and repulsive forces
Good fit: high attraction, low repulsion
Avidity
Combined affinity of many antibody-antigen interactions
Affinity + valence (ex- IgM has high valence due to multiple binding regions)
Sensitivity
Ability of test to correctly identify true positives
Specificity
Ability of test to correctly identify true negatives
Most commonly used antibody-based assay
ELISA (enzyme-linked immunosorbent assay)
Precipitation reaction
Antibody and antigen bind in aqueous solution, forming precipitate
Both “arms” of immunoglobulin must bind
Radial and double immunodiffusion
Immunoprecipitation reaction in gel
Ag and Ab diffuse towards each other
Line of precipitation occurs at the line of equilibrium
Is precipitation a quantitative or qualitative test?
Qualitative
Downsides to precipitation
Slow- often takes a day or two
Requires large amount of Ag and Ab
Immunoelectrophoresis
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
Is precipitation still commonly used?
No- faster methods have replaced
Agglutination
Visible clumping formed by interaction between insoluble antigen and antibody
Example of agglutination assay
Blood type: red blood cells are mixed with anti sera to A or B antigen
Radioimmunoassay
Rarely used
Radioactively labeled and unlabeled antigen compete for antibody binding site
Displaced radioactive isotope is measured
ELISA (enzyme-linked immunosorbent assay)
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
Immunofluorescence
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
Flow cytometry
Used to separate antibody-bound cells stained with fluorescent dye
Enables counting of cells in suspension
Which antibody assay methods are quantitative?
Radioimmunoassay, ELISA, immunofluorescence with flow cytometry
Cell-mediated immune assays: panning
Cells are washed over antibody-bound plate: cells with antigen that is specific to antibodies bind and other cells are washed away
ELISPOT
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
Lymphocyte stimulation
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)
Cell-mediated cyototoxicity
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
Quantitative tests focus on specificity or sensitivity?
Specificity (ability to identify true negatives and prevent false positives)
Qualitative tests focus on specificity or sensitivity?
Sensitivity (ability to identify true positives and prevent false negatives)
