Antibody based immunoassays

Question 1

If you injected a goat with human IgM what would you call the resulting antibody made from the goat?

Answer 1

Goat anti-human IgM

Question 2

What is crosslinking?

Answer 2

1+ antibody bound to same antigen @ different epitopes

Question 3

What is the difference between agglutination and precipitation in relation to antigen-antibody reactions?

Answer 3

• Agglutination: crosslinking on large antigens (e.g. cells)

- Precipitation: crosslinking causes soluble antigens to precipitate

Question 4

Why would IgM be the best antibody isotype to crosslink?

Answer 4

Because it has 10 binding sites

Question 5

Explain the difference between haemagglutination assays and haemagglutination inhibition assays.

Answer 5

• Haemagglutionation assay: RBC agglutinating by serum Ab [to see specific Ab in serum]
• ” inhibition “: Ab is treated w/ Ag => neutralise antigen => when mixed w/ RBC ≠ haemaglutination [to see if Ab match Ag)

Question 6

How are latex particle agglutination assays used to detect antigens and how are they used to detect antibodies?

Answer 6

• detect Ag: latex particle coates w/ Ab => agglutination = presence of Ag = +ve for Ag
• detect Ab: latex particle coated w/ Ag => agglutination = presence of Ab = +ve for Ab

Question 7

What is the zone of equivalence?

Answer 7

proprotion/ [ ] of Ab = propor./ [ ] of Ag = crosslinking => precipitate
*too much/little of each ≠ precipitate

Question 8

Explain how single radial immunodiffusion is used to determine the concentration of a protein.

Answer 8

• Ag is put in the well of an agar containing Ab => precipitation forms
• Zone of precipitation = [Ag]

Question 9

Explain the basic principal behind an Ouchterlony gel.

Answer 9

(aka double immunodiffusion)

• 2 adjacent wells in agar: 1 filled w/ Ag & the other filled w/ Ab
• both diffuse through the gel
• as they come in contact => form antigen-antibody complexes => line of precipitation b/w wells

Question 10

What precipitation reactions you would see for an identical, non-identical and partially identical antigens in an Ouchterlony gel?

Answer 10

• identical: 2 lines would meet at a point = same Ag
• partially: 2 lines meet but one line exceeds the point = have 1 same Ag in both wells & another Ag type in one well
• non-identical: 2 lines cross over = diff. Ag in each well

Question 11

Describe an Immunofixation Electrophoresis method and what is it used for?

Answer 11

• Serum electrophorised -> antisera (anti-G, M, A, K & Mu) => Ag-Ab complexes form = precipitation
• detect paraprotein in serum (monoclonal Ig) bc gel will have single band of precipitate instead of large, smudged area (bc gamma/Ig fraction reduced)
• test for multiple myeloma

Question 12

Explain the Nephelometry method of determining protein concentrations.

Answer 12

• Ag mixed w/ specific Ab => precipitation forms = cloudy
• Light is passed & light scatter is measured
• rate of increase light scatter is proportional to [Ag]

Question 13

What is a “conjugate”?

Answer 13

aka conjugated Ab:

when Ab is labelled w/ fluorecent dyes, enzymes, radio isotope

Question 14

General structure of naming antibody (/serum)

Answer 14

Host - anti - antibody (/serum)