Final Exam- lab techniques

Question 1

best labeling technique

Answer 1

radioactive, but presents health hazards

Question 2

second best labeling technique

Answer 2

fluorescent (less sensitive and hazardous than radioactive)

ex- FITC and PE

Question 3

fluorescein isothiocyanate

Answer 3

fluorescent label: FITC

green light

Question 4

phycoerythrin

Answer 4

fluorescent label: PE

red protein from cyanobacteria

Question 5

indirect immunostaining

Answer 5

tissue section on slide
primary ab binds antigen on tissue w/Fab
secondary binds primary Fc w/Fab
secondary is tagged with fluorescent label

Question 6

Agglutination

Answer 6

reaction b/w Ag-Ab that may lead to precipitation (due to colloidal instability)
Formation of complexes manifests as inc light scattering (more lattices=less transparent)
Requires MULTIPLE INTERACTION SITES b/w antigen and antibody (multiple Ag epitopes-> cross linked lattice)

Question 7

colloidal instability

Answer 7

causes precipitation in agglutination rxn b/w antigen and antibody

Question 8

turbidimetric detection

Answer 8

photometric monitor of light scattering that does not involve staining
helps determine soluble antigens in soluble tissues

Question 9

nephelometric detection

Answer 9

photometric monitor of light scattering that does not involve staining
helps determine soluble antigens in soluble tissues

Question 10

agglutination binding

Answer 10

Abs: divalent (2 Fab regions)
Ags: polyvalent
Crosslinking b/w polyvalent Ag
More crosslinking-> less stable complex-> agglutination-> complex precipitation

Question 11

Heidelberger-Kendall Curve (agglutination)

Answer 11

3 areas:

1. [Ab] > [Ag]
2. [Ab]=[Ag] OPTIMUM RATION
3. [Ab] inc rxn rate

Question 12

Ab used for agglutination

Answer 12

polyclonal antiserum

monoclonal doesn’t work unless it has several identical epitopes

Question 13

maximum specific agglutination rate

Answer 13

around neutral pH
below pH6 (or the isoelectric point, pI)-> many proteins self-agglutinate

Question 14

direct agglutination assay

Answer 14

IgM antibodies cross-link epitopes on particulate antigens
potential for error at high [Ag] or [Ab][Ag]
Add diff dilutions of Ab to identical conc of Ag
lowest [Ab] to cause agglutination=titer

Question 15

indirect agglutination assay

Answer 15

Detects non-IgM ab binding to particulate antigens or to detect low levels of Ab binding
Mechanism:
1. particulate Ag incubated w/primary ab
2. secondary Ab (anti-Ig) added to react w/primary-> cross-linking/agglutination
Sensitivity enhanced by adding anti-Ig (second-step Ab)-> inc binding
competitive assay based on polyvalent Ag/Ab reagents in provided kit
solution has equimolar [Ag] and [Ab]

Question 16

titer

Answer 16

lowest concentration of Ab that causes agglutination in a direct agglut assay
relative measure of Ab activity

Question 17

Coombs test

Answer 17

detects presence of autoantibodies to RBC self-antigens
Autoimmune Hemolytic Anemia: autoabs do not directly agglutinate RBCs due to isotype or concentration
direct and indirect

Question 18

direct Coombs test

Answer 18

Autoabs detected by addition of anti-human Ig (secondary Ab)

helps diagnosis autoimmune hemolytic anemia

Question 19

indirect Coombs test

Answer 19

Autoabs detected by incubating RBCs in serum, washing, then adding anti-human Ig (secondary Ab)
helps diagnosis autoimmune hemolytic anemia