immuno testing Flashcards
difference between polyclonal and monoclonal antibodies
polyclonal- recognize multiple epitopes, contains various antibodies of different affinities
monoclonal- detect only 1 epitope
steps in the production of monoclonal antibody (5)
- isolate spleen cells from mouse that was previously immunized against antigen
- fuse spleen cells with cell line to make hybridomas
- isolate clones
- screen supernatants for desired antibody
- expand positive clones
4 elements in the back bone for all ABO antigens
2 galactose
fructose
N-acetyl-galactosamine
extra sugar for type A
N-acetyl- galactosamine
extra sugar for type B
galactose
Anti-ABO antibody type, implications
IgM, means they will not cross placenta
acute intravascular hemolytic transfusion run is mediated by:
complement (classical pathway)
Rh antibodies type and implications
IgG, can cross placenta
testing for Rh antibodies
coombs test
direct coombs test uses ___from patient
RBCs
indirect coombs test uses ___ from patient
plasma
what is the “anti-human globulin” used in coombs testing?
mixture of anti-IgG and anti-complement, used to determine if there is IgG or complement present on RBC surface in direct test or in plasma in indirect test
direct ELISA tests for presence of-
antigen
indirect ELISA tests for presence of-
antibody
how can recent infections be confirmed using ELISA?
look at labs from acute and convalescing phases of illness, 4x or greater difference in IgG = acute infection
what is the advantage of western blot?
very specific, allows for determination of which viral proteins are reacting with antibody
2 types of “scatter” in flow cytometry
forward = size side = internal granularity/complexity
what is antibody staining in flow cytometry?
use antibodies that have been conjugated with fluorophores to detect cellular antigens (FACS)
why is “threshold” important in flow cytometry?
focuses the data on the cells of interest
2 mitogens
PHA- phytohemoagglutinin
PWM- pokeweek
3 substances that can be used to determine the extend of cell proliferation
BrdU
titrated thymidine
CFSE
how to interpret results when cells have been treated with CFSE?
if cells are actively proliferating, will lose fluorescence (diluted)
left shift indicates
increase in neutrophils
