LECTURE 14 (ELISA)

Question 1

What is ELISA?

Answer 1

An enzyme immunoassay that employs enzyme-labeled immunoreactants and an immunoadsorbent to determine the presence and concentration of certain proteins in serum

EXPLANATION: the detection method is based on the highly specific immunologic interaction between an antibody and its antigen

Question 2

What happens in ELISA?

Answer 2

1) Antigen is fixed on a micrometer plate + bound by an enzyme-coupled antibody
2) Enzyme catalyses a reaction when incubated with its substrate which is chromogenic
3) Intensity of signal is proportional to the amount of captured antigen

Question 3

What are the two main types of ELISA tests?

Answer 3

• DIRECT ELISA = tests for antigen directly
• INDIRECT ELISA = tests for antibody, which indicated the presence of antigen (indirectly)

Question 4

What is a “Sandwich ELISA”?

Answer 4

An indirect ELISA that employs two antibodies that bind to different epitopes on the antigen of interest

Question 5

What happens in Direct ELISA?

Answer 5

1) The patient’s sample supposedly containing the antigen is added to a well of micrometer plates with a buffered solution
2) The specific antibody-enzyme conjugate is added to the solution + a substrate for the enzyme is added
3) Spectrometry is used to detect the generated chromophore
[Higher conc of antibodies binding to antigen -> stronger signal, Lowe conc of antibodies binding to antigen -> weaker signal]

Question 6

What happens in Indirect ELISA?

Answer 6

Same procedure as Direct ELISA with the following exceptions:
- antibody specific for antigen of interest isn’t labeled itself + is called “primary antibody”
- primary antibody is detected by a secondary, labeled antibody

Question 7

What happens in Sandwich ELISA?

Answer 7

1) A surface plate is coated with capture antibodies + sample is added to coated plate where captured antibodies bind the antigen of interest
2) Specific labeled antibodies for antigen are added -> if antigen present, antibody binds to antigen
3) Substrate for enzyme is added + spectrometry, fluorescence or electrochemical studies are performed to assess for amount of antigens present

Question 8

What are the uses for ELISA?

Answer 8

• Screening for HIV antibodies (high sensitivity, low specificity)
• Testing for West Nile virus antibodies
• Detection of: Mycobacterium tuberculosis, Rotavirus (in faeces), Hepatitis B virus, E. coli enterotoxin (in faeces)

Question 9

What is Direct Fluorescent Antibody test (DFA)?

Answer 9

DFA is used for antigen localisation and detection via a fluorescently labeled antibody that binds to the antigen of interest

USED TO HELP IN DIAGNOSING:
- Varicella zoster virus (VZV) infection
- Legionella infection
- Herpes simples virus (HSV) infection

Question 10

What is Indirect Fluorescent Antibody Test (IFA)?

Answer 10

IFA is used for pathogen-specific antibody detection via a fluorescently labeled anti-antibody mixed with the patient’s serum that contains the produced antibody and the lab-generated infected tissue sample

USED TO DETECT PRESENCE OF:
- Anti-nuclear antibodies
- Anti-dsDNA antibodied
- Anti-glomerular basement membrane antibodies