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Immunology lecture 03


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What is ELISA?
A solid phase immunoassay in which protein is absorbed to plastic and antibody-antigen interactions are qualitatively or quantitatively measured, based on light absorbance
What is Radioimmunoassay (RIA)
Essentially an ELISA that uses radioactivity in place of the enzyme to allow detection of the bound antibody
What is RIA useful for?
RIA is highly sensitive, so it is particullarly useful for the quantification of low-abundance substances
What is FACS (flow cytometry)
Most frequently used for the analysis of leukocytes, although any cell type that can be prepared as a suspension can be analyzed
What makes FACS (flow cytometry) powerful?
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
What is immunofluorescence?
Similar to FACS, but fluorescence detection is by microscopy. Only a small number of cells can by analyzed
When would immunofluorescence be used?
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
What does Western blot do?
Fractionates proteins based on size
What is the Coombs test used for?
Testing for anti-Rh antibodies and to diagnose immune-mediated hemolytic anemia. Specifically detects antibodies against red blood cells
What is the direct Coombs test
Red blood cells are recovered from a patient via blood draw, and mixed with the reagent. Positive test is agglutination
What is the indirect Coombs test
Red blood cells are mixed with the patient and then with the Coombs reagent. Commonly used for pregancy
Name three sources of antibodies
Human Serum (used to measure a patient's antibody response)
Polyclonal antibodies (used as detection reagents)
Monoclonal antibodies (hybridomas)
What is a proliferation assay used for
To measure the ability of B cells or T cells to proliferate in response to mitogenic stimuli
What is a functional assay used for
To measure specific effector functions of leukocytes
Name two nucleic acid-based assays
Southern blot and Microarrays

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