Lab 9 - ELISA

Question 1

Know the types of ELISA procedures, and key differences between the three

Answer 1

Direct ELISA: 1° Ab conjugated to an enzyme
Indirect ELISA: 1° Ab and 2° Ab conjugated to an enzyme
Sandwich ELISA: capture Ab, 1° Ab and 2° Ab conjugated to an enzyme (antigen is sandwiched between the capture Ab, 1° Ab and 2° Ab)

Question 2

Know the applications of this procedure (ELISA), and what type of macromolecule it identifies

Answer 2

Applications: clinical diagnostics, microbiology, virology, etc.
It identifies a pathogen or protein

Question 3

Understand and memorize each step of the ELISA procedure

Answer 3

1) Coat wells with sample: antigen or antibody absorbed into well so we can detect it
2) Wash: removes unbound sample
3) Block: prevents nonspecific binding
4) Primary antibody incubation: binds protein if present; also serves as a binding site for the 2° Ab if the protein is present
5) Wash: removes unbound antibodies; prevents false positives
6) Secondary antibody incubation: binds 1° Ab; has HRP conjugated to it so we can detect it later
7) Wash: removes unbound antibodies; prevents false positives
8) Develop: allows us to visualize the concentration of antigen-antibody binding using a microtiter plate reader

Question 4

Know the purpose of each step of the ELISA, and what could happen if certain steps are omitted/performed incorrect

Answer 4

• Forgetting to block would cause a nonspecific binding to occur
• Forgetting to wash may cause a false positive since the wells were not cleaned out
• Forgetting to use Ab with enzyme conjugated to it would mean we cannot visualize the results since the TMB substrate needs to react with the enzyme to produce a result

Question 5

Know the important solutions used in an ELISA, and the purpose of each component

Answer 5

The wash buffer, primary antibody, secondary antibody, some kind of dye, and then the antigen of what pathogen you’re testing for

Question 6

Understand what results of an ELISA can tell you about a sample, how this can be used to diagnose a patient, and how mistakes in the procedure can influence the final result

Answer 6

The results of an ELISA tell us if out specific protein in our sample is present. It can be used to diagnose a patient because if the test results in a positive for a certain disease-causing antigen or antibody, then we know the patient is also positive for the associated disease. Some mistakes can include: forgetting to block which would cause a nonspecific binding to occur, forgetting to wash which may cause a false positive since the well were not cleaned out, forgetting to use Ab with enzyme conjugated to it which would mean we cannot visualize the result since the TMB substrate needs to react with enzyme to product a result

Question 7

What does ELISA stand for?

Answer 7

Enzyme Linked Immunosorbent Assay