10 Immunoassays and Flow Cytometry

Question 1

Western Blot and Gel based immunoassays are unable to tell what?

Answer 1

Concentration of antibodies in serum

Question 2

What is affinity?

Answer 2

Strength of binding on one site of a molecule on its ligand

Question 3

What is avidity?

Answer 3

Strength of binding of multiple binding sites of a molecule on its ligand

Question 4

What Ig has the highest avidity?

Answer 4

IgM

Question 5

What tests are non-quantitative analysises of antibody levels?

Answer 5

Immunodiffusion and Immunoelectrophoresis gels

Western Blot

Question 6

How do most titers show antibody levels?

Answer 6

By serial dilution

Question 7

What tests are used for blood matching?

Answer 7

Coomb’s test

Question 8

What does the presence of RBCs in hemmaglutination test indicate?

Answer 8

Negative antibody presence

Question 9

What does the presence of RBCs in complement fixation assay indicate?

Answer 9

Positive antibody presence, these cells do not get lysed

Question 10

What does a hemagluttination test look for?

Answer 10

Self antibodies

Question 11

What does a complement fication assay look for?

Answer 11

Antibody-antigen complexes, if it binds the RBCs are not lysed

Question 12

No RBC lysis is positive in what test?

Answer 12

Complement

Question 13

What does self-recognizing antibodies to RBCs indicate?

Answer 13

Autoimmune hemolytic anemia

Question 14

Direct Coombs test outline:

Answer 14

Patient blood taken
Antihuman antibodies added
Binding of antibodies means RBCs have antigens recognized by that antibody

Question 15

Indirect Coombs test outline:

Answer 15

Patient Serum obtained
Donor blood added
Antibody binding occurs
If antibody binds, cells clump
Positive means NO TRANSFUSION

Question 16

What is a monospot test for?

Answer 16

Epstein-Barr Virus

Question 17

What test is positive for antibodies if no lysis occurs?

Answer 17

Complement fixation assay

Question 18

How are monoclonal antibodies produced?

Answer 18

Fusion of B cells with Myelonas to make eternal antibody factory called a hybridoma

Question 19

How can we purify antibodies?

Answer 19

Affinity chromatography (Ig binding beads, then wash)

Question 20

How can we make antibodies easy to detect?

Answer 20

Radiolabeling
Enzyme addition
Fluorescent tags

Question 21

What is a primary antibody?

Answer 21

Test sample, may or may not bind to antigen

Question 22

What is a secondary antibody?

Answer 22

Pre-made antibody, known specificity, conjugated to indicator molecule

Question 23

What is serum that is drawn after infection and Ig’s have been made?

Answer 23

Convalescent Serum

may be of use in treating another person with the same infection

Question 24

What is serum that is drawn during an active infection?

Answer 24

Acute phase serum

Question 25

What does acute phase serum contain?

Answer 25

High IgM, Low IgG

High Mannose Binding Protein and C reactive protein

Question 26

What does convalescent serum contain?

Answer 26

Antibody specific Ig’s

Question 27

What test uses pre-bound known antibodies in a plate of wells?

Answer 27

ELISA

Question 28

What does foward scatter in flow cytometry show?

Answer 28

Cell size

Question 29

What does side scatter in flow cytometry show?

Answer 29

Cell granularity

Question 30

What is a “gated population” in flow cytometry?

Answer 30

A parameter of cell features that can allow you to focus in on a certain group of cell types within a “gate”

Question 31

What allows analysis of two types of cell reactions in flow cytometry?

Answer 31

Double staining by Fluorescence of two different colors

Question 32

What can be used to separate cells?

Answer 32

Fluorescent Activated Cell Sorting in a flow cytometery

Question 33

What color is FITC?

Answer 33

Green

Question 34

What color is PE?

Answer 34

Red

Question 35

Do you ever find monoclonal antibodies in patient serum

Answer 35

(resounding) NO, because of hypermutation