immunodiagnostic Testing

Question 1

Serology

Answer 1

the amount and specificity of antibody are measured by direct binding to antigen

Question 2

purpose of immunodiagnostic tests

Answer 2

identify infections, autoimmune diseases, allergies, and vaccine repsonses

Question 3

Primary interaction?

Answer 3

measuring direct binding of the antibody to its antigen

Question 4

secondary interaction?

Answer 4

measuring the changes antibodies are inducing in the physical state of the antigen

Question 5

in vitro

Answer 5

done outside of a living organism

Question 6

in vivo

Answer 6

done with living organism

Question 7

Types of reagents in primary interactions

Answer 7

serum, complement, antiglobulins, monoclonal antibodies

Question 8

radioimmunoassay (RIA) measures..

Answer 8

the concentration of antigens or hormones using radioactively labeled antibodies

Question 9

two types of RIA

Answer 9

antigen method and antibody method

Question 10

steps of antigen method of RIA

Answer 10

1. Ag* and Ag are added into solution with fixed amount of antibodies
2. they compete for binding sites

Question 11

For RIA, if [Ag] is high,

Answer 11

less bound Ag* and more free Ag*

radioactivity of free Ag* ↑

radioactivity of bound Ag* ↓

positive result

Question 12

For RIA, if [Ag] is low,

Answer 12

more bound Ag* and less free Ag*

radioactivity of free Ag* ↓

radioactivity of bound Ag* ↑

negative result

Question 13

ELISA detects …

Answer 13

Detects antibodies or antigens in serum or other fluids

Question 14

steps of direct ELISA

Answer 14

1. fixed antigens in the sample
2. add enzyme labeled antibodies → antigen-antibody complexes
3. add chromogenic substrate → change color
4. measure absorbance

Question 15

steps of indirect ELISA

Answer 15

1. fixed antigens in the solution
2. add unlabeled Ag (primary) from patient → antigen-antibody complexes
3. add enzyme-labeled antibodies (secondary)
4. add chromogenic substrate → color change
5. measure absorbance

Question 16

steps in SNAP test

Answer 16

1. mix sample with enzyme-labeled antibodies
2. fixed antigen-specific antibodies on the snap test
3. antigen-antibody complexes bind to those fixed ones → sandwich forms

Question 17

secondary antibodies bind to primary antibodies via which region?

Answer 17

Fc region of primary

Question 18

two types of IFA

Answer 18

direct (competitive too)

indirect

Question 19

steps of direct IFA

Answer 19

1. antigen on a slide
2. fluorophore-labeled primary antibody is added
   → binds to target protein
3. wash, slide examined under fluorescence microscopy

Question 20

steps of indirect IFA

Answer 20

1. antigen on a slide
2. unlabeled antibodies from the patient are added
3. Ag-Ab complexes
4. add fluorophore-labeled antibodies → secondary complexes

Question 21

Flow cytometry sorts …

Answer 21

luorescence-activated cells

Question 22

steps of flow cytometry

Answer 22

1. incubate the cells with surface antigens with fluorescent monoclonal antibodies
2. cells are washed and passed across a laser beam
3. Two scatters: forward and side scatters

Question 23

forward scatter of flow cytometry measures …

Answer 23

size, granularity of cells

Question 24

side scatter of flow cytometry measures…

Answer 24

fluorescence

Question 25

western blotting is mainly sued to identify antigens in

Answer 25

micro-organisms or parasites

Question 26

steps in western blotting

Answer 26

1. gel electrophoresis 2. gel is placed against nitrocellulose paper; sponge in the middle 3. electrical current is passed between sponges → antigens are transferred onto the membrane 4. primary antibodies from the patient are added 5. can be visualized by RIA and ELISA EX: soy extracts for allergic dogs!

Question 27

Immunohistochemistry helps diagnosing ...

Answer 27

diff types of cancer

Question 28

two types of Immunohistochemistry

Answer 28

direct and indirect

Question 29

steps of direct immunohistochemistry

Answer 29

1. tissue section is incubated with enzyme-labeled antibodies → antigen-antibody complexes 2. wash → add chromogenic substrate → produces a colored precipitate 3. examine under light microscopy

Question 30

steps of indirect immunohistochemistry

Answer 30

1. tissue section is incubated with unlabeled antibodies → antigen-antibody complexes 2. add enzyme-labeled antibodies → secondary complexes 2. wash → add chromogenic substrate → produces a colored precipitate 3. examine under light microscopy

Question 31

similarities between immunohistochemistry and IFA?

Answer 31

1. use slides 2. seen under microscopy

Question 32

similarities between immunohistochemistry and ELISA?

Answer 32

use enzyme-labeled

Question 33

Antiglobulin test measures for ...

Answer 33

antibodies that cause autoimmunity

Question 34

type of antiglobulin test

Answer 34

coombs test (direct and indirect)

Question 35

direct coombs test detects ...

Answer 35

antibodies/complement already bound to RBCs

Question 36

indirect coombs test detects ...

Answer 36

free antibodies that could bind to donor's RBCs

Question 37

steps in direct coombs test

Answer 37

1. collect p's blood and wash 2. incubate with coombs reagent 3. examine for agglutination

Question 38

indirect steps of coombs

Answer 38

1. p's serum with free Ig is incubated with donor's RBCs → Ig-RBC complexes 2. wash any unbound ones → add coombs reagent 3. agglutination = positive

Question 39

agglutination in indirect coombs test means

Answer 39

circulating antibodies that can cause hemolysis of specific RBC type are present

Question 40

agglutination in direct coombs test means

Answer 40

RBCs are coated with Ig or complement (signs of hemolytic activity)

Question 41

EX of monoclonal antibody testing

Answer 41

ELISA, flow cytometry, IHC, western blotting, RIA

Question 42

how is monoclonal antibody produced?

Answer 42

1. antigen is added to mice 2 mice build up plasma cells 3. extract those plasma cells and mix with tumor cells (myeloma cells) 4. form hybridoma → mass producing Igs

Question 43

monoclonal antibodies?

Answer 43

Ig from one B-cell clone → antigen-specific plasma cells

Question 44

What is secondary antigen–antibody binding?

Answer 44

When multiple antigens and antibodies interact, cross-linking into lattices

Question 45

What type of antigen does precipitation detect?

Answer 45

Soluble antigens.

Question 46

What is required for precipitation lattice formation?

Answer 46

Multivalent antigens and bivalent antibodies

Question 47

What are common types of precipitation tests?

Answer 47

Immunodiffusion, immunoelectrophoresis, rocket electrophoresis

Question 48

which precipitation tests are qualitative or which ones are quantitative?

Answer 48

immunodiffusion, immunoelectrophoresis - qualitative rocket electrophoresis, radial immunodiffusoin - quantitative

Question 49

How does immunodiffusion work?

Answer 49

1. antigen and antibody are placed on a gel → they diffuse thru it 2. when reach zone of equivalence → precipitin lines form

Question 50

How do you interpret immunodiffusion lines? Lines that fuse smoothly ? Lines that cross ?

Answer 50

Lines that fuse smoothly → identical antigens. Lines that cross → different antigens

Question 51

Example of an immunodiffusion test?

Answer 51

Coggins test (for Equine Infectious Anemia)

Question 52

What does the gel contain in radial immunodiffusion?

Answer 52

A specific antibody distributed uniformly

Question 53

How does antigen form a visible pattern in radial immunodiffusion?

Answer 53

Antigen diffuses outward from a well → precipitation ring forms

Question 54

What does the ring area represent in radial immunodiffusion?

Answer 54

proportional to antigen concentration, compared against a standard curve big ring - more antibody-antigen complexes

Question 55

What is immunoelectrophoresis used for?

Answer 55

Mainly to find the presence of immunoglobulin levels in serum but can also be quantitative if radial is used

Question 56

What two methods are combined in immunoelectrophoresis?

Answer 56

Electrophoresis (separates serum proteins) + immunodiffusion (antibody interaction)

Question 57

How are precipitin arcs formed in immunoelectrophoresis?

Answer 57

Antibodies diffuse sideways into separated proteins → each arc represents an antigen–antibody complex