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20 Cards in this Set
- Front
- Back
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direct agglutination assays
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-soluble antibodies cause clumping of antigens that are on the surface of a particle or cell
-ex. agglutination of blood cells in blood typing (hemagglutination) or bacterial cells (stereotyping) |
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indirect agglutination assays
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-antigens or antibodies are chemically coupled to particles such as latex beads-latex agglutination assay
-ex: latex bead coated with antibodies to protein-A which is a protein found only on the surface of S. aureus cells -very specific and rapid assay for presence of this bacterium |
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fluorescent antibodies
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-purified antibodies that recognize specific antigens are chemically modified with fluorescent dyes
--often used to examine for a pathogen in a complex clinical sample ------antibodies are used that bind to certain specific bacterial cells or viruses -------samples are often visualize by fluorescence microscopy |
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direct immunofluorescence assay
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-antibacterial antibody, labeled with fluorescent dye
-the antibody that interacts with the surface antigen is itself covalently linked to the fluorescent dye |
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indirect immunofluorescence assay
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-antibacterial antibody (unlabeled) made in rabbit
-abti-rabbit lg, labeled with fluorescent dye -the presence of a nonfluorescent antibody on the surface of a cell is detected by the use of a fluorescent antibody directed against the nonfluorescent antibody |
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Direct Enzyme-Linked immunosorbent Assay (ELISA)
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-can be used to detect antigenic pathogen components or antigenic metabolites in blood, urine, and other specimens
-detects antigens |
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Indirect Enzyme-Linked Immunosorbent Assay
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-used i many application including the detection of antibodies to HIV
-detects antibodies |
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steps in direct ELISA
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-coat plate with antibody
-add clinical sample -add enzyme-antibody -wash with buffer -develop color reaction |
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steps in indirect ELISA
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-purified antigen is absorbed to microtiter plate
-patient's serum (containing antibodies to that antigen) is added -wash with buffer -then anti-bodies linked to an enzyme are added -wash with buffer -develop color reaction |
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what is detected in direct ELISA
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detects antigens such as virus particles from a blood or fecal sample
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what is detected in indirect ELISA
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to detect antibodies in human serum
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enzyme-linked antibody
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various enzymes that are chemically attached to antibodies. Enzyme catalyze reactions that lead to readily visible color changes to allow for rapid and sensitive detection of antigens
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gel electrophoresis to separate proteins
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1. denature proteins by boiling in detergent and subject to electrophoresis; proteins separate by molecular weight
2. blot the separated proteins from the gel to membrane 3. treat membrane containing blotted proteins with antibodies; each antibody recognizes and binds to a specific protein 4. add marker to bind to antigen-antibody complexes, either radioactive Staphylococcus protein A-125I, or antibody containing conjugated enzyme |
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immunoblots
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-use antibodies with chemical tags to detect a protein in a clinical sample that has previously been separated by gel electrophoresis. They require a thin, porous membrane that tightly binds all types of proteins. The term “blot” means to transfer the proteins from gel to the membrane. Immunoblots are most commonly performed after protein gel electrophoresis (called a Western Blot)
-bacterial cells --> separate proteins on SDS-polyacrymide gel --> transfer proteins to polymer sheet --> polymer sheet is exposed to radiolabled or enzyme-linked antibodies --> protein bands detected by specific antibodies are exposed to film |
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membrane filter assay
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clinical samples are treated to release genomic DNA, denature, and then hybridize with a probe tagged with a reported molecule
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probe
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single-stranded oligonucleotide with a sequence that is complementary to a pathogen-specific DNA sequence
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reporter molecule
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probes link to these molecules to allow some type of detection using enzymes, or fluorescent compounds
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dipstick assay
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probe has one end of the sequence that hybridizes with the target DNA of the pathogen, and the other end is used for subsequent capture and measurement
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how can PCR be used as a diagnostic method to test for the presence of DNA of a pathogen in a clinical sample
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-used to detect DNA sequences that are specific to a particular pathogen (viral, fungal, protozoan, bacterial)
-use synthetic oligonucleotides with a sequence that will amplify specific short regions of the genomes of pathogens -source of DNA for analysis can be blood, serum, sputum, pus, urine, appropriately treated tissue samples -PCR products can be analyzed by: gel electrophoresis and real-time OCR |
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what is real-time PCR
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is also called Quantitative real time PCR, qPCR. Fluorescent dye is incorporated into double-stranded PCR reaction products. Properly equipped thermocycling machines can monitor formation of reaction products by observing increase in fluorescence
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