Serology Flashcards
serology
detection and measurement of antibodies or antigens
what is serology used for?
can confirm viral, bacterial, or fungal infections
what type of antibodies are used in serology tests?
monoclonal antibodies to many different antigens which help to rapidly detect disease
what does protein electrophoresis do? (EPH)
assesses cause of severe hyperproteinemia (usually from a rise in immunoglobulins)
measurement results in a graph
gamma = immunoglobulins
IEP
patient’s serum is placed in a well and antibody to whole serum in a trough
comparisons are done to normal serum which can determine if there are deficiencies in 1 or more serum components or if there is an overabundance of some serum components (based on thickness of line on graph)
PCR tests
polymerase chain reaction
DNA or RNA is amplified into millions of copies and can then be detected for analysis of hereditary diseases, forensic, paternity, or diagnosis of infectious disease
advantages of PCR tests
much more sensitive and specific, quick turn-around time
disadvantages of PCR tests
high cost, false positives from contamination, technical expertise needed
how are PCR tests done?
- 2 strands of DNA double helix are melted, separated, and then cooled
- DNA enzymes are added using DNA primers that are specific for the target DNA so they complement and are copied exponentially
electrophoresis
technique used to separate molecules of DNA, RNA, and protein based on size
larger fragments move slow, smaller fragments move fast
heavier fragments stop sooner, they move from negative to positive change
interpretation of electrophoresis
look for bands at specific markers
no bands = negative
indeterminate = bands don’t match criteria for specific disease
Coombs test
detects auto-antibodies
direct test for hemolytic disease (especially IMHA)
RBCs from patient are incubated with species-specific reagent: agglutination seen if positive
too expensive to stock in-house, usually send out
Rh factor
protein on RBC
can produce antibodies against fetus in human
associated with Coombs test
sensitivity
true positives
proportion of animals with a positive result actually having the disease
specificity
true negatives
proportion of animals with a negative test that don’t have the disease
ELISA tests
enzyme-linked immunosorbent assay
types: direct, indirect, sandwich, and competition/blocking
involves lots of pipetting and washing
some are available as kits
direct ELISA
quick
can be opposite: looking for antigen in sample instead of antibodies
how do you perform a direct ELISA?
- antigen is affixed to the well surface
- patient sample is added (already linked with enzyme)
- if antibody is in the sample it will bind and form a complex
- wash
- read color change
ELISA SNAP tests vs rapid tests
similar specificity and sensitivity
ELISA includes washing step
rapid tests have more potential for false positives due to not having a washing step
ELISA tests are gold standard
send out for confirmation when there is a positive rapid test
lateral flow tests
a type of rapid test
immunochromatography assay (ICT)
uses colloidal gold instead of enzymes
gold buildup as sample migrates which can lead to a positive result
indirect ELISA
antigen is added to wells and then patient sample is added (if the antibodies for the antigen are present it will bind and form a complex)
then specific antibody is added to bind to the antigen: this antibody is linked to an enzyme that will change color if the antigen antibody complex is formed, otherwise it’s washed away
reading color change in a indirect ELISA
lighter color = less antibody in sample
are indirect ELISA tests more sensitive or specific?
increased sensitivity because there is increased antibody in the patient sample which means there is more epitope availability for secondary antibody
indirect ELISA test result interpretation
in the example of FIV:
< 0.300 = negative, 0.300-0.499 = indeterminate (retest), > 0.500 = positive
how do you perform a sandwich ELISA test?
- capture antibody coats the wells
- antigen to be tested is added and will bind to capture antibody (antigen in patient sample needs epitopes for both capture and detection antibody)
which disease is the sandwich ELISA test typically used for?
common for feline leukemia
is the sandwich ELISA test more specific or sensitive?
highly specific and has increased sensitivity
competition/inhibition blocking ELISA test (bELISA)
- wells are coated with a capture antibody
- antigen is added which forms complex
if the sample has increased antibodies to the antigen, it will compete with the detection antibody and will wash away so there will be significant reduction in signal
is the bELISA test more sensitive or specific?
increased sensitivity because we are looking for a specific antibody to antigen
how is a bELISA test read?
color change is opposite: negative = darker color and positive = lighter color
immunodiffusion test
Coggins test
antigen and patient sample are added to the wells on an agar plate —> both will diffuse into the agar and form a visible band of precipitation as a reaction
a nonidentity reaction will occur when the Ag-Ab complexes are different (the resulting “X” or crossed reaction indicates that 2 unrelated complexes are present)
what are 2 other immunodiffusion tests?
fluorescent antibody and RIA tests
fluorescent antibody test
antibodies from the patient are added to a fluorescent dye-coated antigen slide which is viewed under a fluorescent mircoscope
RIA test
radioimmunoassay
determines antibody levels by attaching radioisotope to antigens and measuring radioactivity
antibody titer
measurement of how many antibodies an animal has produced against a specific antigen
done by making serial dilutions of the sample
antibody titer results
low titer = previous exposure
high titer = active infection
booster vaccine will elicit a secondary response (IgG) which will give you a higher titer result
seroconversion
negative antibody converts to positive antibody titer or low antibody titer converts to high level (via vaccine or exposure)
seroreversion
antibody decay
seroprevalence
proportion of seropositive animals in a population
