Immunoassays/ Flow Cytometry

Question 1

• Tests for precipitation of antibody binding to an antigen

- Used with simple antigen mixtures

Answer 1

Agarose Gel-Based Assays

Question 2

• Poor Sensitivity
• Non-Quantitative
• Antigen and Antibody placed in separate wells
  When they meet, they will form a line

Answer 2

Immuno- double diffusion (Ouchterlony double immunodiffusion assay)

Question 3

• Analyzes a complex mixture of antigens
• Can evaluate patient sera for immunoglobulin content
• Antigens separated by applying an electric charge across gel
• Qualitative, not quantitative

Answer 3

Immunoelectrophoresis

Question 4

• Used to determine levels of IgM, IgG, and IgA from serum/plasma sample
• Tests serum for Ig content using light scatter

Answer 4

Nephelometry

Question 5

• More sensitive than gel-based assays
• Can detect and measure antibodies specific for a particular antigen
• RBCs are used as an indicator
• If antigens bind to RBCs-
• If insufficient antigen-specific antibody is present (negative test) then-
• Can test for autoimmune hemolytic anemia and systemic lupus erythematosus

Answer 5

Hemagglutination

Question 6

• Detects antibodies or complement proteins bound to the surface of RBCs
• Positive test if RBCs agglutinates
• Used to detect antibodies or complement system factors that have bound to RBC surface antigens in vivo
• Only detects antibodies/complement that has already bound to a patient’s RBC in circulation

Answer 6

Direct Coomb’s Test

Question 7

• Primarily used for detection of antibodies within a patient’s sera that binds to “self” antigens following blood transfusions or during pregnancy
• Used to determine the presence of antibodies that could potentially bind to specific RBCs
• Screens pregnant women for antibodies that may cause hemolytic disease of newborn

Answer 7

Indirect Coomb’s Test

Question 8

• Diagnostic test for Epstein-Barr Virus (infectious mononucleosis)
• Simliar to indirect Coomb’s

Answer 8

Monospot Test

Question 9

• Used to ID the presence of antibodies specific for a variety of common human pathogens

Answer 9

Complement Fixation Assay

Question 10

• Quantitative method of measuring antigen-specific antibody
• Wells are coated with purified antigen and the unbound antigen is washed away
• Very sensitive, high throughput, and inexpensive
• Must have purified antigen or monoclonal Ab

Answer 10

ELISA

Question 11

• Not Quantitative
• Highly sensitive, purified antigen or antibody not needed
• Can deter me whether Ab sample can bind to protein antigen

Answer 11

Western Blot Analysis

Question 12

• Antibodies that are conjugated to visible particles
• Or enzymes that can metabolize a chromogenic substrate
• Can diagnose autoimmune disorders of the CT system

Answer 12

Immunohistochemistry

Question 13

• Involves use of antibodies that are conjugated to fluorochromes
• Can diagnose autoimmune disorders of the CT system

Answer 13

Immunofluroescence

Question 14

• Analysis of cell preparations used to rapidly analyze individual cells based on size and granularity and surface marker expression
• Measures light scattering
  
  1. Forward scatter: size and refractive index of particle
  2. Side scatter: rougher and irregular particles

Answer 14

Flow Cytometer

Question 15

Modification of Paul-Bunnel Test that differentiates among 3 types of sheep erythrocyte agglutinins

• Infectious mononucleosis
• Serum Sickness
• Natural Antibodies against Forssman Antigen

Answer 15

Paul-Bunnell-Davidsohn Test

Question 16

• Popular way of purifying antibodies

- Protein A will bind to Fc regions of IgG1, IgG2, and IgG4

Answer 16

Affinity Chromatography