Immunodiagnosis Flashcards
how can serological applications be preformed? (mechanisms)
using antibodies to detect antigens (see if someone has infection) or antigens to detect antibodies (to see if someone HAS had that infection)
what is the difference between agglutination and precipitation
A- particles
P- soluble proteins
what is an agglutination based assay on cards?
used to identify antigens that are particles (like a bacteria or Red cells)
how does agglutination based assays work?
cross linking aggregation (usually uses latex particles coated in antigen to detect)
what is passive agglutination?
soluble molecule (bacterial antigen or antibody) attached to carrier like latex
what is the difference between indirect/passive agglutination ad reverse passive agglutination?
indirect: antigen on latex used to DETECT ANTIBODIES
reverse: antibody on latex used to detect antigen in patients serum
what is a titer on agglutination based assay?
last dilution to still show agglutination
what is prozone?
antigen is completely covered with antibody molecules so no cross linking can occur
what is used to dest for RF?
human IgG coated latex particles and RF (IgM in rheumatoid arthritis patients that reacts with human IgG)
what does secondary antibody do for agglutination reactions?
helps to avoid false negatives in the prozone (binds to primary antibody which is already attached to antigen, bridging antigen bound primary antibodies together causing visible aggregates making test more sensitive)
what are the limitations of the agglutination test?
positive result does not necessarily mean pt is sick, person may have recovered
what could cause a false negative test result?
poor producer of antibody
what can cause a false positive test result?
if an unrelated antibody reacts with the antigen nonspecifically
what is a quantitative precipitation curve?
using a constant amount of antibody you titrate variable amounts of antigen to get to the equivalence zone (marked by precipitation)
In a quantitative precipitation curve, what is the antibody excess zone?
not enough antigen molecules for cross link to form stable complex, no precipitation
In a quantitative precipitation curve what is the equivalent zone?
antigen-antibody ratio is in good proportion for cross linking to form large aggregates- precipitation
In a quantitative precipitation curve what is the equivalence point?
no antigen or antibody in solution, they are completely precipitated out
In a quantitative precipitation curve what is the antigen excess zone?
each molecule of antibody is saturated with antigen, insufficient cross linking, o precipitation
what is a precipitation based assay?
add antigen to one well and antibody to another well, solutions diffuse out and bind where they meet precipitation
line forms at the equivalence point
in an immunodiffusion test what is a line of identity?
antibody prep recognizes identical antigenic epitopes (lines equal on both sides)
what are lines of non-identify in immunodiffusion?
lines extend to both sides of the wells and diffuse past each other, antibody prep recognizes different antigens or antigen epitopes(lines cross each other making a # shape)
what are lines of partial identity or spurs?
one antigen has two epitopes and other only has one causing one side to cross but not other leaving a spur)
What does radial immunodiffusion test for?
normal levels if IgG
what are monoclonal antibodies?
(superior reagents)
all the antibody molecules have the same specificity and recognize the same epitope
what is hybridoma tecnology?
allows you to take cells from the spleen and make a lot of a specific antibody
what is immunohistochemistry?
antibody conjugated with fluorescent tag that can be seen as it tags appropriate antigen
what is FACS?
laser equipment detects fluorescence on individual cells one by one and can sort them
what is ELISA?
enzyme is conjugated to secondary antibody by tagging antibody with enzyme, add substrate, and see if a color develops which means positive test
what is western blot?
taking antigen and dissociating the proteins on agar gel then blot on membrane with sample and sort
what is radioimmunoassay?
label antigen with radioactive compound (quantitative and vey sensitive) uses competition for binding
what is complement fixation/inhibition assay?
can be used to detect the presence of a specific antibody or antigen in serum