Diagnostic Testing (Lec. 22 and 23) Flashcards
Discuss morphological identification test methods
Microscopic morphology and colony morphology.
Discuss biochemical identification test methods
Determine the presence of enzymes through identification trees.
Discuss immunological identification test methods
Involve interactions of humoral antibodies with antigens; a known antibody can identify an unknown pathogen (antigen), and a known antigen can determine the presence of an unknown antibody. Require commercial kits and are useful for bacteria, fungi, protozoa, and viruses. Advantages: inexpensive, low tech, easy to use, fast (TAT = 5 min to a few hours). Disadvantages: lower sensitivity/specificity than culture; cannot always distinguish between current and past infection.
Discuss molecular profiling
A lab method that uses a sample of tissue, blood, or other body fluid to check for certain genes, proteins, or other molecules that may be a sign of a disease or condition.
Discuss nucleic acid identification test methods
Detection of DNA/RNA. Require commercial kits and are useful for bacteria, fungi, protozoa, and viruses. Advantages: fast: TAT (Turn Around Time) is 30 min to a few hours; highly sensitive and highly specific. Disadvantages: high tech and expensive.
Differentiate sensitivity from specificity in a diagnostic test
Both are measures of a test’s ability to correctly classify a person as having a disease or not having a disease. Sensitivity refers to a test’s ability to designate an individual with disease as positive (highly sensitive test = few false negatives). Specificity refers to a test’s ability to designate an individual who does not have a disease as negative (highly specific test = few false positives).
Describe PCR tests
NAAT is a more general term (does not specify whether you’re starting with RNA or DNA). PCR is specifically having a DNA template. (RT (reverse transcriptase) PCR is an RNA template). Polymerase chain reaction: multiple rounds of DNA replication; sensitive enough to detect DNA of a single cell/virion in a sample; highly specific: only DNA that is an exact match to primers will be amplified.
Describe DNA/gene microarray
Based on ability of ssDNA to hybridize to complementary ssDNA. Short ssDNA molecules (oligos) are bound to a glass slide in a microscope array. If complementary DNA molecules are in the sample, they’ll bind and can be visualized by a detector. Requires commercial kits and specialized equipment. Useful for blood culture panels and respiratory panels.
Define antibody
a protein produced in response to and counteracting a specific antigen
Define antigen
a toxin or other foreign substance which induces an immune response in the body
Define epitope
Specific 3D portion of a protein that is recognized by an Ab. Every protein has multiple epitopes.
Differentiate monoclonal from polyclonal antibodies
Monoclonal Ab (mAb): An Ab produced by a single clonal B cell from an animal that can only recognize one specific epitope on a protein of interest.
Polyclonal Abs: from an animal that can recognize multiple epitopes of a foreign protein.
Differentiate primary from secondary antibodies
A primary antibody binds directly to a particular antigen (used to detect, purify, or measure antigens). Secondary antibodies bind to the primary antibody.
Describe the advantages/disadvantages of immunological diagnostic methods over other identification methods (culture/biochemical methods or nucleic acid methods)
Advantages of immunological diagnostic methods: rapid, relatively inexpensive, can detect either antigens or antibodies (useful for monitoring disease progression. DisadvantageL may not be as sensitive as nucleic acid-based methods, and can lead to false positive results.
Describe precipitation tests
Use the formation of a precipitate as an indicative result of the presence or absence of certain elements (antibodies and/or antigens) in a test sample.
Describe agglutination tests
Based on the principle that antibodies can cross-link particulate antigens, causing them to clump together. Used to detect the presence of antibodies or antigens in bodily fluids.
Describe neutralization tests
Serum and virus are reacted together in equal volumes and inoculated into a susceptible animal host or cell culture. If antibodies to the virus are present then CPE will not be observed.
Describe complement-fixation tests
A blood test in which a sample of serum is exposed to a particular antigen and complement in order to determine whether or not antibodies to that particular antigen are present.
Describe EIA/ELISA
Enzyme immunoassay (EIA) aka enzyme-linked immunosorbent assay (ELISA) use enzyme-bound antibodies to detect the presence of antigens. The antigens bind to a surface, the antibody-enzyme conjugate attaches to the antigen, and this substrate and enzyme interaction creates a color change. Requires a microtiter plate, a patient sample, a specific antibody, an enzyme, and a plate reader.
Describe immunofluorescence assays
A microscopy-based technique that use antibodies and fluorescent dyes to detect and quantify antigens or antibodies in biological samples (requires use of a fluorescence microscope).
Direct: fluorescent dye-labeled antibodies bind to antigens in a patient sample.
Indirect: normal primary antibodies bind to the antigen in a patient sample, then fluorescent dye-labeled secondary antibodies bind to the primary antibodies.
Describe immunochromatography assays (ICAs)
Also known as lateral flow assays (LFAs): simple device to detect the presence/absence of antigens or antibodies. Is a combination of chromatography (separation of components of a sample based on differences in their movement through a sorbent) and immunologic reactions.
Explain how fluorescent-tagged antibodies may be used in diagnostic testing
The fluorescent antibodies bind to the antigens on a microscope slide, allowing ready detection of the antigens using a fluorescence microscope.
Explain how Western blotting works
Identifies proteins via electrophoresis and a blot. Proteins in a mixture are separated by electrophoresis in a gel, and are then transferred to a protein-binding sheet (blot), which is then flooded with specific enzyme-linked antibodies. A substrate is added, and color changes allow visualization of location of antibody-antigen reaction.
