MBIO 4310 FINAL

MBIO 4310 FINAL - MBIO 4310: INTRO TO VIROLOGY (FINAL EXAM...

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MBIO 4310: INTRO TO VIROLOGY (FINAL EXAM REVIEW) 10/27/2009 Physio-chemical assays: Hemagglutination assay: -RBC in test tube roll to center -Many viruses adhere to RBC -RBCs form sheet in test tube -Determine highest dilution of virus that causes agglutination  -Relative titer only (must compare to stock with absolute titer known) Immunoassays: -"tagged" antibodies -immunostaining -radioimmunoassay (RIA) -Enzyme-Linked ImmunoSorbant Assay (ELISA) -Immunoblotting (western) Tagged Antibodies: -All antibodies from same line of clonal B-cells -Antibody "tagged" with fluorescent dye Fluorescent Antibody Assay: -1st antibody reacts with virus -2nd antibody reacts with 1st antibody -2nd antibody contains fluorescent tag (e.g. FITC) Radioimmunoassay: -Basically same as tagged antibodies, but tag is radioactive isotope ELISA: -Same as tagged antibodies; tag is enzyme (e.g. horseradish radish peroxidase) -Substrate changes color; measure absorbance
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Immunoblotting: -Fractionate viral polypeptide (antigen) on SDS-PAGE gel -Blot to membrane (NC or Nylon) -Probe with phosphatase-tagged Ab then wash -Develop in substrate mix -Substrate reacts with antibody causing visible marker of antigen 10/29/2009 Competitive Immunoassay: 1) Mix antigen with patient serum 2) Wash 3) Mix with known antibody that binds the specific antigen (tagged to allow measuring) 4) Measure amount of binding of second antigen (100% means no patient antibody, 0%  means all patient antibody) HIV Testing: ELISA is performed first (95% accurate), some false positives. IF positive THEN Western Blot to confirm If truly positive, HIV viral load and CD4 count measured every 3 months New Methods of Viral Detection: Nucleic Acid Hybridization PCR Physical Assays: UV ABsorbance: Viruses absorb strongly at 260nm (peak for nucleic acids) Electron Microscopy: particle count Protein assays: Bradford etc. None of these physical assays are specific for viruses -------------------------------------------------------------------------- Phage-Bacterium Interaction --------------------------- Phases of virus replication:
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1) Adsorption 2) Entry (penetration) 3) Synthesis 4) Assembly 5) Release Adsorption: Reversible stage: non-covalent bonds Irreversible stage: covalent bonds Phage T4: Reversible stage: -All 6 tail fibers must bind before advancing to next step of adsorption -T4 receptor is LPS core polysaccharide Irreversible stage: -covalent cross-linking between spikes and p12 -Base plate undergoes conformational change when all 6 tail fibers bind -This in turn causes compression of tail sheath, exposing tail tube Fiber tips attach to receptors Triggers base plate Tail rearranged - shorter and thicker Base plate flattens tail core penetrates LPS and wall - touches cell membrane
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This note was uploaded on 04/29/2010 for the course MBIO 4404 taught by Professor Moss during the Fall '10 term at Texas Tech.

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MBIO 4310 FINAL - MBIO 4310: INTRO TO VIROLOGY (FINAL EXAM...

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