Immunoassays/Flow Cytometry

Question 1

Describe immunodiffusion assay.

Answer 1

• Aka, Ouchterlony double immunodiffusion assay
• NOT sensitive, CANNOT be used to determine Ab concentration or be used with complex mixes of antigen
• Gel plate cut to form series of wells
  1. Sample extract placed in one well; sera or purified antibodies placed in other well (48 hrs)
  2. Sample antigens and sera antibodies diffuse out of their respective wells, immune complexes formed when they meet if Abs recognize antigens
  3. Precipitate and white line indicate antigen recognition

Question 2

Describe immunoelectrophoresis.

Answer 2

• NOT sensitive, and CANNOT be used to determine Ab concentration
• Serum samples (pt. w/recurrent inf & healthy) added to opposite sides of agar plate (trough in middle)
  1. Proteins in both samples separated by electrophoresis (+ to - pole, - to + pole)
  2. Rabbit anti-human antiserum added to central trough; serum proteins allowed to diffuse into gel
  3. As anti-Ig Abs from rabbit serum bind cognate antigens, precipitin lines form

Question 3

Describe nephelometry.

Answer 3

• Used to DETERMINE CONCENTRATIONS of IgM, IgG, and IgA Abs from a serum/plasma sample
• Performed by measuring scattering of light (laser) passed through diluted serum sample at an angle (70-75 degrees)
• Amount of light scatter measured and compared to amount of scatter from a known normal sample (positive control), then extrapolated from a standard curve

Question 4

What is an antibody (Ab) titer?

Answer 4

• The measurement of how much antigen-specific antibody is contained within an antibody sample.
• Titer is typically expressed as an endpoint titer which reflects the inverse of the last tested dilution that yields a positive result for antibody presence (i.e., 1/256 = 256)

Question 5

What is an agglutination titer?

Answer 5

The highest dilution of a serum sample that causes
clumping of microorganisms or other particulate antigens (often RBCs; visible if so b/c red)

Question 6

What is a serial dilution?

Answer 6

• A series of repeated dilutions (using the same dilution factor)
• Typically carried out on a microtiter plate