Antibodies are Y-shaped proteins used by the immune system to identify and help remove foreign objects like bacteria and viruses. Their specific shape helps them to recognize many different antigens (the unique parts of a molecule that causes an immune response).
An ELISA (enzyme-linked immunosorbent assay) uses scientifically designed antibodies to detect if a particular substance, such as a viral antigen, hormone, or another specific antibody, is present in a sample.
In this lab, you will use an indirect ELISA protocol to identify if a patient or sample is positive or negative for a specific antibody in response to infection. By comparing your patient results to positive and negative controls, you will be able to determine if the samples were positive or negative for a particular infection.
Your medical lab has been given four blood serum samples from patients who suspect they may be at risk for the modified influenza virus from the episode of Containment.
Jake Riley (police officer) – multiple contacts with infected people including Sayid and infected doctors. Katie Frank (school teacher) – multiple interactions within the hospital. Went into secure areas with her mask down. Pete Walden (police officer) – Sayid coughed mucus on him, he got his mask down but did he get it down in time. Henry Carver (doctor) – worked with infected doctors and patients. Sneezed while talking to Katie and Jake.
Your group will be testing two of the four patient samples along with positive and negative controls. To ensure that your results are accurate, all your samples will be run in triplicate.
Part 1: Plan for Loading the ELISA Plate
Each group will have 2 of the 4 patient samples. You will run your samples in triplicate (3 wells per control and patient sample. That’s 12 wells total.)
Record the location of your controls and patient samples by circling the available row on the diagram to the right.
Part 2: Perform ELISA
Add 50 µl of the antigen (green tube) to each of the 12 wells. Discard pipet tip.
Incubate for 5 minutes at room temperature. This allows for the antigen to bind to the plastic wells.
Remove the unbound antigen.
Turn the 96-well plate upside down on a stack of paper towels.
Bang it forcefully several times to remove any unbound antigen.
Discard the top layers of paper towels (see Figure).
Wash Step:
Use a transfer pipet to fill each well with wash buffer. Be careful not to touch the sides of the wells with the tip. PLEASE KEEP THE TRANSFER PIPET AND USE FOR FUTURE WASHES.
Remove the buffer by turning the 96-well plate upside down on a stack of paper towels and banging Discard the top layer of paper towels and keep the transfer pipet for future wash steps.
4. Add controls and patient samples.Add 50 µl of the controls and patient samples the wells designated in your 96-well template. Be sure to use a fresh pipet tip after pipetting a sample in triplicate.
3 wells (+) control (pink tube)
3 wells (-) control (purple tube)
3 wells patient ______ sample (clear tube)
3 wells patient ______ sample (clear tube)
5. Incubate 5 minutes at room temperature. This allows for the antibodies, if present in the patient sample, to bind to antigens. 6. After the 5 minutes, remove the unbound antibodies. as you did in STEP 3.
Wash Step:
Use a transfer pipet to fill each well with wash buffer. Be careful not to touch the sides of the wells with the tip. PLEASE KEEP THE TRANSFER PIPET AND USE FOR FUTURE WASHES.
Remove the buffer by turning the 96-well plate upside down on a stack of paper towels and banging Discard the top layer of paper towels and keep the transfer pipet for future wash steps.
7. Add secondary antibodies.Add 50 µl of secondary antibodies(yellow tube) to all 12 wells. Change the pipet tip after each triplicate. The secondary antibody is bound to an enzyme. These enzyme-bound antibodies are called “conjugated”. This enzyme interacts with the substrate we will add next, allowing us to visualize whether the primary antibodies are present. 8. Incubate for 5 minutes at room temperature. 9. Remove the secondary antibodies. As you did in STEP 3.
Wash Step - repeat 3 times:
Use a transfer pipet to fill each well with wash buffer. Be careful not to touch the sides of the wells with the tip. PLEASE KEEP THE TRANSFER PIPET AND USE FOR FUTURE WASHES.
Remove the buffer by turning the 96-well plate upside down on a stack of paper towels and banging Discard the top layer of paper towels and keep the transfer pipet for future wash steps.
10. Add substrate. Add 50 µl of peroxidase substrate (brown tube) to each well. Be careful not to touch the sides of the well with the tip. 11. Incubate for 5 minutes then record your results.