31. ELISA

Question 1

What does ELISA stand for?

Answer 1

Enzyme Linked Immunosorbent Assay

Question 2

What are the main functions of the immune system? (2)

Answer 2

1) recognition of invading pathogens

2) destruction and removal of those pathogens

Question 3

What are antigens?

Examples?

Answer 3

• any molecule or structure that can elicit an adaptive immune response
  
  • Ex. Protein and polysaccharides

Question 4

What are linked enzymes also called?

Answer 4

-reporter enzymes

Question 5

How are antibodies produced in a lab setting?

Answer 5

-repeated injections of mammals or birds with antigen molecules and adjuvant > production of antibodies that bind the antigen

Question 6

What is the function of adjuvants?

Answer 6

-stimulate inflammation, stimulating an infection;inflammation is necessary to provoke a strong immune response

Question 7

Other names for antibodies

Answer 7

• immunoglobulin

- gamma globulin

Question 8

Common lab antibody sources

Answer 8

-mice, rats, guinea pigs, rabbits, goats, chickens

Question 9

How are antibodies named?
Ex.1) Rabbit with E.Coli O antigen 157
2) goat with mixture of antibody proteins from rabbits

Answer 9

1) rabbit anti-O157

2) goat anti-rabbit

Question 10

What are the roles of primary antibodies?

Answer 10

-directly bind the antigen you wish to detect

Question 11

What is the role of secondary antibodies?

Answer 11

-bind primary antibodies of the first species (antibodies can also be antigens)

Question 12

What is the difference between direct and indirect ELISA?

Answer 12

• Direct: antigen attached to plastic dish; enzyme-linked antibody binds only if the antigen is present
• Indirect: Antigen attached to dish; primary antibody binds to antigen; multiple secondary antibodies bind to primary antibody, allowing more amplification (more sensitive test)

Question 13

How can ELISA detect antigens using antibodies?

Answer 13

-the patient sample supplies the antigen; if it is absent, the antibodies don’t bind -can be direct or indirect ELISA

Question 14

How can ELISA detect antibodies using only antigens?

Answer 14

-use the patient as the source of the primary antibodies in an indirect ELISA

Question 15

Describe a Sandwich/Antigen capture ELISA

Answer 15

• use when antigen does not naturally bind to the plastic dish well
• used to eliminate antigen purification steps that are typically needed before a direct or indirect ELISA
• coat the dish with antibodies that capture the antigen
• similar to indirect ELISA, but uses 3 antibodies in total

Question 16

What are polyclonal antibodies?

Answer 16

• antibodies that bind the same antigen at multiple epitopes

- Many B ells recognize the same antigen, but at different epitopes; this leads to many different plasma cell clones

Question 17

What are monoclonal antibodies?

Answer 17

• antibodies that all bind exactly the same epitope

- single B cell leads to a single clone of plasma cells

Question 18

How does the standard urine pregnancy test work?

Answer 18

• early in pregnancy, the placenta produces human chorionic gonadotropin (hCG)
  
  • urine pregnancy tests detect hCG
• absorbent material (sponge) exposed to urine
  
  • urine travels down sponge, passes through enzyme-linked mouse monoclonal anti- hCG antibodies
  • If hCG is in urine, mouse anti-hCH antibodies bind hCG
• urine continues to move through absorbent material, drawing antibodies to second zone containing immobilized polyclonal anti hCH antibodies and substrate
  
  • immobilized anti-hCG antibodies capture enzyme-linked mouse antibodies by also binding the hCG antigen, at different epitopes
• if captured, the substrate and the enzymes now react to produce colorful product
• mouse antibodies without hCH continue down absorbent material to 3rd (control) zone containing substrate and immobilized anti- mouse antibodies

Question 19

What are the 2 statistics that determine how reliable a clinical test is?

Answer 19

1) sensitivity: probability that the test is reactive if the specimen is a true positive
- generally use samples that stimulate positive samples

2) specificity: probability that a test will not be reactive if a specimen is a true negative
- generally use samples that stimulate negative samples