Question

1. What does an ELISA test for?

2. What variables can affect the outcome? (Name 4)

3. What are the three important limitations of an ELISA? Explain each.

4. In the virtual lab, the ELISA plate has been pretreated to bind SLE antigen to each well.

WHY? How do you expect the assay setup to differ in our case where the antigen of interest is present in a heterogenous sample in small quantities?

5. Why are there both a positive and negative control done?

6. What is the purpose of incubating the ELISA plate?

7. Why is the HRP substrate added?

Answer 1

1 Enzyme - linked Immunosorbent assay, also called ELISA is a test that detects and measures antibodies in your blood. This test can be cosed to determine if you have antibodies related to certain infectious Conditions. Elisa cused to identify peptides, Proteins, antibodies and Hormones. ELISA has been used as a diagnostic tool in medicine Plant pathology and biotechnology. It is a useful tool for determining Serum antibody concentrations, such as with HIV Test or West Nile virus. ELISA can also be used in toxicology as a rapid presumptive Screen for Certain classes of drugs. Ол

© ELISA experiment is affected by a variety of factors. (a) specimen factor (b) Reagent factor () Operating Factor (d) Application Species (e) Test sample. Limitations of ELISA (a) Results may not not be absolute. (b) Antibody must be available. (c) Concentration may be an clear. (d) False positive possible (e) False Negative possible.

6 ELISA plate is pretreated with SLE antigen, so that if the patient to bas SLE their serum will contain antibody Anti-SL castibodies that will bind to the antigen. Adding a second antibody that binds to the anti-SLE antibodies helps produce a colour change that gives a visual result. 15 Running appropriate controls help you to accurately separate true positive results from potentially false results. Positive and Negative control will also be useful if you ever need to trouble shoot your protocol.

6 Incubating serum samoples in antigen coated sell wells helps ensure that the antibody present in the sample will interact correctly with the astigen. Because SLE is a disease of bumans, the reaction cessually occurs at the temperature of the human body, which is 37c.

CHRP) (HRP) Horseradish perocidase, is Commonly used in techniques such as ELISA and inmunohisto- - Chemistry due to its monomeric nature and the ease with which it produces coloured products.