Lab 8 Notebook
Title: AB08 Biochemical Assays for Bacterial Antigen Detection
Objective:
To understand the assays available for detecting bacterial or viral antigens
Procedure:
ELISA
1. A- in a 96 well plate, each well is labels 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
Western Blot
1. Loading the gel
2. Running the gel
a. 35 mAmps/gel
3. Transferring the Gel
a. 25V for ~75 min
4. Block the gel
a. 2% BSA for ~45 min at RT
b. Place on rocker
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 Development
a. BCIP/NBT at RT, covered
b. Place on rocker
c. Closely monitor
Agglutination tests
- Commercial kits
- Each card is for a different sample
- Sell kits based on specific pathogens
- Kit we have today is specific for Staph.
- Mix your vial by inverting it a few times
- Place a drop in the circle of each card
o This is you test your reaction
- Grab a sterile wooden pick
- Going to grab a culture of known E Coli, so we know there is no staph, so we do not
expect it to react with a reagent.
o Mix the in well 2 and discard stick
- Quadrant 1 is empty bc it is our control and want to make sure no clumping occurs
- Pick our second colony, mix in well 3
o Immediately see dark blue clumps
o Discard stick
- Test 3rd colony in well 4
o Mix for about 20 seconds
o Discard stick
o Can see a positive agglutination signal
Notes:
Antigen= short for antibody generating pathogen
- Means that there is something foreign that is in your system that is going to require
antibodies to detect it
- ELISA
o Enzyme-Linked immunosorbent Assay
o Utilizes antibodies and a colorimetric readout to indicate an antigen of interest
▪ If it is present in your system or not
o Different types, but we will be using the Sandwich ELIZA, which uses a 96 well
plate
Title: AB08 Biochemical Assays for Bacterial Antigen Detection
Objective:
To understand the assays available for detecting bacterial or viral antigens
Procedure:
ELISA
1. A- in a 96 well plate, each well is labels 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
Western Blot
1. Loading the gel
2. Running the gel
a. 35 mAmps/gel
3. Transferring the Gel
a. 25V for ~75 min
4. Block the gel
a. 2% BSA for ~45 min at RT
b. Place on rocker
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 Development
a. BCIP/NBT at RT, covered
b. Place on rocker
c. Closely monitor
Agglutination tests
- Commercial kits
- Each card is for a different sample
- Sell kits based on specific pathogens
- Kit we have today is specific for Staph.
- Mix your vial by inverting it a few times
- Place a drop in the circle of each card
o This is you test your reaction
- Grab a sterile wooden pick
- Going to grab a culture of known E Coli, so we know there is no staph, so we do not
expect it to react with a reagent.
o Mix the in well 2 and discard stick
- Quadrant 1 is empty bc it is our control and want to make sure no clumping occurs
- Pick our second colony, mix in well 3
o Immediately see dark blue clumps
o Discard stick
- Test 3rd colony in well 4
o Mix for about 20 seconds
o Discard stick
o Can see a positive agglutination signal
Notes:
Antigen= short for antibody generating pathogen
- Means that there is something foreign that is in your system that is going to require
antibodies to detect it
- ELISA
o Enzyme-Linked immunosorbent Assay
o Utilizes antibodies and a colorimetric readout to indicate an antigen of interest
▪ If it is present in your system or not
o Different types, but we will be using the Sandwich ELIZA, which uses a 96 well
plate
