Serology Flashcards
serology
the measurement and characterization of antibodies or antigens in body fluids
mostly done on serum or plasma
is it more useful to detect antigen or antibody
antigen because it indicates active or current infection
ex. heart worm, FeLV, parvovirus, some fungi
do the majority of serologic tests detect antigen or antibody
antibody - useful for pathogens that may not be in the blood at the time of the test
antibody characteristics
- produced by plasma cells
- have an Fab and Fc region
(Fab = variable, binds antigen; Fc = constant) - can be monomeric (IgG), dimeric (IgA), or pentameric (IgM)
what is an antigen
the entire molecule that is recognized by antibody
usually a protein
what is an epitope
the portion of a molecule that is recognized by an antibody
antigens usually have >1 epitope
monoclonal antibodies
reagents that are produced to be used in serologic tests
the exogenous antibody required to detect endogenous (patient) antibody
NOT the same as the antibody produced by the patient
how are monoclonal antibodies formed
- mouse vaccinated with antigen of interest
- splenocytes that contain antigen specific plasma cells are harvested and fused with myeloma cells to form a hybridoma
- antigen specific hybridomas are selected and propagated
- antibodies are harvested from hybridoma supernatant
what are the main serologic tests
immunodiffusion
agglutination
ELISA
immunofluorescence
primary binding tests
measure immune complexes that form after combining antigen and antibody
requires labeling of a reactant in order to detect the immune complexes
ex: ELISA, immunochromatography
lateral flow assays
paper based platforms for the detection and quantification of antigens + antibody
point of care tests done in clinic
immunochromatography
lateral flow assay in which antigen solution flows unilaterally through a porous strip
- sample placed on sample pad
- flows to the conjugate pad containing labeled antibody
- if antigen present in sample: forms immune complex - flows to the detection zone (test line) where immune complexes are captured by immobilized detection antibody
- if immune complexes present: color change occurs - flows to the control line where non-antigen related antibodies are captured to induce color change
ex. at home covid tests
ELISA
enzyme linked immunosorbent assay
can detect antigen or antibodies
antibody ELISA
- antigen immobilized
- add sample
- if antibody present: will bind antigen and form immune complex - add conjugate containing detection antibody and enzyme
- wash and add substrate
- if immune complexes formed: color change
antigen ELISA
- capture antibody immobilized
- add sample
- if antigen present: will bind antibody and form immune complex - add conjugate containing detection antibody and enzyme
- wash and add substrate
- if immune complexes formed: color change
SNAP tests
ELISA based test where the flow matrix is connected to reagent reservoirs that are released when “snapped”
solution flows bidirectionally through strip
- sample is mixed with labeled detection antibody and placed on sample pad
- flows (left to right) to the antigen window containing capture antibodies
- if antigen present: forms immune complexes on the “dots” in the window - test is snapped to initiate flow of wash and substrate reagent (right to left)
- detection antibodies bind immobilized immune complexes to cause color change
secondary binding tests
measure the downstream effects of immune complex formation after combining antigen and antibody
ex: precipitation or agglutination
immunodiffusion
precipitation test
detects antibodies in blood
antigen: small and soluble –> forms a lattice and precipitates
antibody amount is constant (based on patient sample)
antigen amount if variable (added to sample to reach optimal proportion)
ex. Coggins test
what happens if too much or not enough antigen is added to the patient sample
no precipitate will form
Coggins test
immunodiffusion assay
antigen-antibody complex formation depends on proportions of each (must be in the equivalent zone)
used to diagnose EIA (equine infectious anemia)
produces a QUALITATIVE result (positive vs negative)
radial immunodiffusion assay
antibody is located in agar and antigen is located in a well –> ring of precipitation indicates a positive result and the area is proportional to the amount of antigen in the well
produces a QUANTITATIVE result using standardized curve
ex. measuring serum ig levels in foals
agglutination tests
detect antibodies on RBCs in IMHA
antigen: large and particulate –> clumps and agglutinates
ex. Coombs test
Coombs test
detects autoantibodies on RBCs to test for IMHA
direct: Coombs reagent added to blood and check for agglutination
indirect: Coombs reagent added to donor blood mixed with patient serum and check for agglutination
what does a positive vs negative Coombs test indicate
positive: confirms there is an immune mediated component to anemia BUT that main mechanism may or may not be immune mediated
negative: does not rule out IMHA
functional tests
measure the protective effect of antibodies in animal or in vitro
ex. neutralization tests
neutralization tests
rely on the ability of antibody to neutralize biological activity of antigen
qualitative results
determine the presence or absence of antibodies
result is positive or negative
semiquantitative results
determines the levels of antibody or antigen present
results is low vs high levels
quantitative results
determines the amount of antibody (titer)
achieves an actual measurement by doing serial dilutions
titer: highest dilution that produces a positive result
sensitivity
ability to correctly identify patients with a disease (true positives)
Sensitivity = TP / (TP + FN)
percentage of true positives out of all disease positives
specificity
ability to correctly identify patients without a disease (true negatives)
Specificity = TN / (TN + FP)
percentage of true negatives out of all disease negatives
why is it a challenge to determine a specific titer cutoff point
some disease + animals have low titers and some disease - animals have high titers
there is always going to be overlap, will cause there to always be some false positive and negatives
highly specific tests = insensitive
highly sensitive tests = non specific
what are paired titers used for
identifying acute infection
acute sample: first sample obtained at presentation
convalescent sample: second titer obtained 3 weeks layer
what is the criteria for a positive paired titer
convalescent sample has 4x increase in IgG or total Ig compared to acute sample
OR
there is IgM present
