Test 1: lecture 12 and 13 testing Flashcards
antibody test shows evidence of ___
PIE (protection (vaccine), infection or exposure)
two main ways to make definitive diagnosis of an infection with an antibody test
4-fold increase in titer of non-class specific Ab
or
Detection of IgM antibodies (ideally paired with IgG)
___ is the highest dilution of an assay to give a positive reaction
titer
•Detecting a four-fold rise in titer requires___
acute and convalescent titers
2 weeks apart
(titer is the highest dilution that gives a positive reaction→ at lower dilutions they clump together and fall to bottom, at higher dilutions there are so little that they are all spread out, not clumping)
are titers absolute or relative value
relative (has no unit)
elevated liver and kidney values are common with ___
lepto
run agglutination antibody titer assay and again 2 weeks later
4x increase is positive for lepto
___ indicated recent exposure or infection
IgM
___ represents previous exposure
IgG
(secondary response)
antigen test
- Detects a SPECIFIC antigen of a pathogen.
- In the presence of clinical disease detection of an antigen is considered diagnostic.
- Can cross react with similar targets (from closely related pathogens for example).
antigen titers can increase with treatment
what is one down fall of the antigen test
pathogens can be very closely related
therefore antigen test can have cross react → react to wrong antigen
weird shape of nose, run cryptococcus ___ test and it is + at 1:8. Treat. Rerun test and it is + 1:16. why the increase after treatment?
antigen test
by treating the fungus, it is broken down leading to more antigen and more response by body = swelling and higher antigen test results
antigen titers can rise with treatment
treat until you get 0 on titer
what does a negative antigen test show
pathogen not there
antigen present, but not enough to detect
antigen present but bonded to antibody
technical error
what does a + antigen test show
pathogen present
antigen present but viable microbe absent
cross reactive antigen present from another pathogen
technical error
poor assay specificity (false +)
negative antibody test →
exposure to pathogen did not occur
too early in course of infection
severe immunosuppression
technical error
poor assay sensitivity (false negative)
positive antibody test _>
previous natural exposure to pathogen
previous vaccination
cross- reactive
technical error
poor assay specificity → false +
seronegative
used to screen blood donor
immunoassay studies are 0
antibody test → dog was never exposed to this pathogen and carries no antibodies for these diseases
what kind of test do you use for blood donors and why?
antibody→ cast broad net
will show if every exposed, previous immunization
don’t use antigen→ cause antigens at very low levels can’t be detected and some diseases can lead to long term effects that would exclude the patient from being a blood donor
why use titer for vaccination status
- Assess status prior to booster
- For Animals with Concurrent disease
- Assess immunity at > 24 weeks
- Unknown vax hx
- Assess prior to breeding
- To determine if animals are genetic “non-responders”
- Test in face of outbreak for quarantine purposes
IFA (immunoflourescent Ab)
slide with antigen are covered with antibodies that have been linked to specific fluorescent, if the smear contains the antigen the corresponding antibody will bind, wash away anything unbound, use dark field microscopy to see what sticks
- Used to detect presence of a specific antigen directly in a sample (tissue or smear).
- Slide is incubated with fluorescently labeled Ab and the unbound Ab washed away.
- Darkfield microscopy with ultraviolet light is use to detect bound antibody.
- Can also be used for tumors, cell types etc.
___ is a test where a slide with antigen are covered with antibodies that have been linked to specific fluorescent, if the smear contains the antigen the corresponding antibody will bind, wash away anything unbound, use dark field microscopy to see what sticks
IFA (immunofluorescent antibody)
ELISA
enzyme linked immunosorbent assay
•Either Bidirectional Flow ELISA or Immunochromatography for in-house assay.
indirect elisa
antigen bound to bottom of well
add serum from patient (may contain antibody)
wash away extra stuff
enzyme linked secondary antibody (or antiglobulin→ antibody that binds to antibody)
wash away
add chromogenic substrate that binds to secondary antibody
color change→ antibody was in serum = +
sandwich ELISA
•Capture Ab is coated on plate and Ag in sample is bound.••Unbound antigen is washed a detection Ab is added.••Finally a labeled antiglobulin is added for detection.
sandwich looks for antigen
capture antibody is coated on bottom of plate
add serum that might have the antigen in it
wash away every thing else
add enzyme linked antibody to that antigen we are looking for
wash everything else away
add labeled anti-globulin ( antiglobulin→ antibody that binds to antibody) that reacts to 2nd antibody(changes color)
what type of ELISA is for antigen identification
sandwich
•Capture Ab is coated on plate and Ag in sample is bound.••Unbound antigen is washed a detection Ab is added.•Finally a labeled antiglobulin is added for detection.
•Capture Ab is coated on plate and Ag in sample is bound.••Unbound antigen is washed a detection Ab is added.••Finally a labeled antiglobulin is added for detection.
sandwich elisa
looking for a specific antigen
- Ab is detected in sample when it is bound to antigen that is coated on the plate.
- Plates are washed of unbound Ab.
- Secondary Ab (or antiglobulin) with reporter is bound to sample Ab.
indirect ELISA
(looking for antibodies)
Bidirectional flow ELISA
snap test
matrix is pre-coated with specific antibodies
add sample, that has enzyme linked antibodies added to it
if antigen is present in sample will bind to the antibodies on the matrix and the enzyme linked antibodies will keep it from getting swept away
the substrate then moved across the cleaned matrix and blue dot = +
competitive ELISA
color change is inversely proportional to AB or Ag level
no color change = +
well is bound with antigen, add sample, if sample has antibodies will bind to the bound antigen, then add lab derived enzyme linked antibodies,
if there are no antibodies in the original sample lab Ab will bind and if there was antibodies in the original sample lab Ab will not bind
if lab sample Ab bind there will be a color change = negative
___ has color change is inversely proportional to Ab level in serum
competitive elisa
negative result = color change
positive result = no color change
why use competitive elisa
not species specific
other antibody ELISA need antiglobulins that are against the species Ig
where competitive just needs lab derived antibody
AGID
- Based on the passive diffusion of soluble Ab and Ag through agar gel.
- When “positive” precipitation occurs.
- Inexpensive but subjective.
4= negative
put lab derived antigen in the center, in wells around put +/- control and serum
precipitation line = +
serum neutralization
- Typically used for viral diagnosis.
- Observation of virus challenged cell cultures in the presence of test serum.
- If cells do not become infected then there is antibody in the serum.
- Results can be titered.
have specific cell line, add in virus, if the cells become infected → no antibodies in cell line. If cells do not become infected → + antibodies
have specific cell line, add in virus, if the cells become infected → no antibodies in cell line. If cells do not become infected → + antibodies
serum neutralization
used for viral diagnosis
complement fixation
if serum has antibodies, RBC will pellet and no lysis
have serum, heat it up to kill complement
add lab derived antigen, if serum has antibodies already present will bind
then will add lab derived complement
if Ab:Ag present, complement will bind
then add in sheep RBC and antibodies to sheep RBC
if complement is bound because antibodies are present in original sample→ RBC will settle into a pellet, no lysis (complement was used up → nothing happens to added RBC)
have serum, heat it up to kill complement
add lab derived antigen, if serum has antibodies already present will bind
then will add lab derived complement
if Ab:Ag present, complement will bind
then add in sheep RBC and antibodies to sheep RBC
if complement is bound because antibodies are present in original sample→ RBC will settle into a pellet, no lysis (complement was used up → nothing happens to added RBC)
complement fixation test
