BIOD 171:BIOD 171 Lab 8 Notebook:Lab 8 Notebook: Latest Updated A+ Score Solution

Biochemical Assays for Bacterial Antigen Detection Objective: To understand the assays available for detecting bacterial or viral antigens. Procedure: Elisa: enzyme-linked immunosorbent assay utilizes antibodies and a colorimetric readout to indicate an antigen of interest. 1. A- in a 96 well plate, each well is labeled with an antibody a. Antibody is the blue Y 2. B- add patient sample to each well 3. Wash- whatever is not bound will be washed away 4. C- where you have capture of the antigen to the antibody 5. Wash any antigen that isn’t bound 6. Incubation 7. D- secondary antibody (has a tag), binds 8. Wash all unbound secondary antibodies 9. E -everything is activated, and you get a colorimetric change Wester Blot 1. Loading the gel 2. Running the gel a. 35 mAmps/gel (called dye front) 3. Transferring the Gel a. 25V for ~75 min 4. Block the gel 5. Primary Antibody a. TBST/BSA for ~45 min at RT b. Place on Rocker 6. Secondary Antibody a. TBST/BSA for ~45 min b. Place on rocker 7. Membrane DevelopmentAgglutination Test: A rapid assay to determine if antibodies to a specific pathogen are present. Most commonly tested against patient serum or blood. 1. Commercial kits a. Each card is for a different sample b. Sell kits based on specific pathogens c. Kit we have today is specific for Staph. 2. Mix your vial by inverting it a few times 3. Place a drop in the circle of each card a. This is how you test your reaction 4. Grab a sterile wooden pick 5. Getting E Coli culture, so we know there is no staph, so we do not expect it to react with a reagent. a. Mix in well 2 and discard stick 6. Quadrant 1 is empty since it is our control. Make sure no clumping occurs 7. Pick our second colony, mix in well 3 a. Immediately see dark blue clumps b. Discard stick 8. Test 3rd colony in well 4 a. Mix for about 20 seconds b. Discard stick c. Can see a positive agglutination sig