Immunologic Tests

Question 1

how to make hydridomas

Answer 1

Put purified antigen into an animal
get antibody-producing B cells from spleen
fuse w/ myeloma cells
grow in vitro in HAT medium to select for hybridomas
continually making monoclonal Ab

Question 2

HAT medium

Answer 2

aminopterin block DHRF making TTP and GTP
Add in thymidine (to make TTP)
Add in hypoxanthine - need HGPRT to make GTP

Myeloma does not have HGPRT
B cell do but will die anyway

hybridomas left

Question 3

equivalence zone of antigen/antibody

Answer 3

if too much antigen – no precipitate
if too little – no precipitate

need just the right amount for equivalence

tested in immunoprecipitation assays

Question 4

Ouchterlony radial immunodiffusion test

Answer 4

S = serum
3 different antigens in A, B, C

Agarose on glass side – punch out – diffuse out of well into cell – diffusing out
Get lattice formation where ag-ab bind

Close to ag well – too much ag, close to ab – too much ab
Tells you what serum binds with

Question 5

spur in ORI test

Answer 5

spur points away from the one that is uniquely reactant

Question 6

direct agglutination assays

Answer 6

what we are testing is big enough to see (ie ABO)

see clumping = positive

Question 7

passive agglutination assays

Answer 7

if too small to see, use latex beads as markers that have antibodies on them that recognize the antigen
see latex beans clumping

Question 8

hemagglutination

Answer 8

Viruses that interact w/ RBC and induce agglutination (so clump w/ virus)

add in serum at diluted levels

if ab - no clumping because viruses are block = positive result (no clumping)

Question 9

neutralization assays

Answer 9

to see if a person’s serum has abs to neutralize a toxin
(serum + toxin) + cells
if cells survive - have ab

Question 10

complement fixation assays

Answer 10

control: RBCs + hemolysin + complement –> complemnet cascade and lysis

pos result: pts abs + complement + RBCs + Hemolysin
all complement bound up in ag-ab rexn –> no lysis

Question 11

immunohistochemical microscopy

Answer 11

can stain w/ antibody to certain markers

Question 12

fluorescent microscopy

Answer 12

can mark ab w/ different specific wavelength tags

can detect multiple things on one slide at once

Question 13

direct immunofluorescence

Answer 13

fluorescently labeled antibody

Question 14

indirect immunofluorescence

Answer 14

unlabeled antibody for specific antigen + fluorescently labeled generic (anti-organism) antibody

Question 15

direct elisa

Answer 15

antibodies attached to wells
add patient sample
add antivirus antibody containing conjugated enzyme
add substrate for enzyme and measure change in color
positive results = colored wells

if no antigen, does not bind to ab on well. No antigen for second antibody to bind to.

Question 16

indirect elisa

Answer 16

wells / antigen prep from certain virus
add pt serum (suspected to have antibodies)
add anti-Ab abs w. conjugated enzyme
add substrate for enzyme and measure color change = + result

if no antibodies in serum - no ab binds to ag in well – washed away – no ab for indirect ab to bind to

Question 17

special pro to indirect elisa

Answer 17

can determine which class of ab patient has (IgG or IgM etc)

Question 18

competitive elisa test

Answer 18

mix pt specimen containing antigen w/ known amount of antigen-specific antibody
add to antigen-coated microtiter wall
wash
add new ab w/ enzyme - substrate to change color

NO color change is a positive result - means that the pt had more antigen
(color inverse to amt antigen)

Question 19

ELISPOT assays

Answer 19

take cells - put on plate and grow - put labed ab against cytokines or general things - if looking for cells producing special things, use that ab and see spots

cells you took were actually able to produce certain things

Question 20

western blotting

Answer 20

antigens denatured and run through gel - electrotransfer to membrane
bind antigens on membrane to enzyme-linked antibodies

see based on size and reaction

Question 21

flow cytometry

Answer 21

fluorescence emited from stained cells detects and forward/side scattered light from all cells

get size and granulation