LECTSTEPHEN
Lab 8 Notebook
BS08 Biochemical Assays for Bacterial Antigen Detection
Objective: To understand the assays available for detecting bacterial or viral antigens.
Antigen – antibody generating pathogen
Notes:
ELISA: Enzyme-Linked Immunosorbent Assay utilizes antibodies and colorimetric readout
to indicate and antigen of interest.
o Sandwich ELISA
Each well is labeled with an antibody (A)
Antibody= blue y-shaped structure, adhere to the bottom of the
well
To each well, you would then add a patient sample (i.e., serum or cultures
that you have grown)
If antigen is present and recognized by the antibody, it would be bound
shown in B.
Arrows indicate wash steps
Once antigen is bound, you wash to remove and antigen that is
unbound.
Part C: capture of the antigen to the antibody
Incubation with a secondary antigen that also recognizes the antigen.
Reason why it’s called a sandwich, because your antigen is now
sandwiched between 2 different antibodies.
Secondary antibody has a tag on it (D)
Tag either a enzyme or a substrate that will react once activated.
Wash performed to the wash away the unbound secondary antibody.
Well E: final step where everything is activated, and you get a colorimetric
change.
Well E can have a very dark color or a light-yellow color, or some
won’t change at all
From A to D = most likely to be a clear culture.
o Notes: (shown is a 96 well ELISA)
Color intensity = abundance of antigen
This study source was downloaded by 100000829684747 from CourseHero.com on 03-18-2022 22:08:02 GMT -05:00
https://www.coursehero.com/file/102330455/lab-8docx/
Lab 8 Notebook
BS08 Biochemical Assays for Bacterial Antigen Detection
Objective: To understand the assays available for detecting bacterial or viral antigens.
Antigen – antibody generating pathogen
Notes:
ELISA: Enzyme-Linked Immunosorbent Assay utilizes antibodies and colorimetric readout
to indicate and antigen of interest.
o Sandwich ELISA
Each well is labeled with an antibody (A)
Antibody= blue y-shaped structure, adhere to the bottom of the
well
To each well, you would then add a patient sample (i.e., serum or cultures
that you have grown)
If antigen is present and recognized by the antibody, it would be bound
shown in B.
Arrows indicate wash steps
Once antigen is bound, you wash to remove and antigen that is
unbound.
Part C: capture of the antigen to the antibody
Incubation with a secondary antigen that also recognizes the antigen.
Reason why it’s called a sandwich, because your antigen is now
sandwiched between 2 different antibodies.
Secondary antibody has a tag on it (D)
Tag either a enzyme or a substrate that will react once activated.
Wash performed to the wash away the unbound secondary antibody.
Well E: final step where everything is activated, and you get a colorimetric
change.
Well E can have a very dark color or a light-yellow color, or some
won’t change at all
From A to D = most likely to be a clear culture.
o Notes: (shown is a 96 well ELISA)
Color intensity = abundance of antigen
This study source was downloaded by 100000829684747 from CourseHero.com on 03-18-2022 22:08:02 GMT -05:00
https://www.coursehero.com/file/102330455/lab-8docx/
