Serology/Immunodiagnostics Flashcards
What is the difference between a molecular versus a serologic test?
Molecular: Direct (DNA or RNA) - PCR
Serologic: Indirect - antigen or antibody
What is Polymerase chain reaction (PCR)?
Synthesize and amplify specific target sequence of pathogens
-Nucleic Acid Extraction
-PCR Amplification (denature, anneal and extension)
-Fluorescence probes
What is Serology?
-ID antibody or antigen (IgG or IgM)
-Serum commonly used (plasma, feces, urine, saliva, CSF)
-Diagnose infection - blood type, host type or immunology
What is the lag phase?
Time post exposure to a pathogen before the body starts its primary response
Is IgM specific? What about IgG?
IgM is a general antibody that stalls the pathogen until the specific IgG can come in and clean up
What is different when it comes to the immune systems response between a primary and secondary exposure to a pathogen?
Primary: takes a bit longer to recognize and for IgG to kick in
Secondary: much faster response and IgG is prepped and already read to go
What does serostatus mean?
-Seropositive = sample contain antibodies and antigen (can report a titer)
-Seronegative = sample does not contain antibodies (not infected)
What is seroconversion?
Change from seronegative to seropositive - must be at least a 4 fold increase in titer between acute and convalescent sample- paired sample
How far apart should convalescent samples be taken for serology?
2-4 weeks apart
What kinds of serological tests are available?
ELISA, IFA, IHC, Agglutination, AGID, Virus neutralization, Complement Fixation
What should you remember about an Elisa test?
- send out or in house (snap, out give microtiter)
-can detect antibodies or antigens (can no distinguish vaccination versus exposure)
-detection (Antigen capture, capture antibody, add sample captured and second antibody with color indicator dropped in) - opposite for antibody test
What is an example of an Elisa test?
Snap - tick born diseases
What is Direct Immunofluorescence Assay (IFA)?
Direct FA
-Detect antigen
-Fluorescent labeled antibody conjugated
-Poor sensitivity - no individual viral particles, accumulated antigen, need to permeabilize cells before staining
What kinds of tests are Direct IFA?
Giardia and Cryptosporidium (feces) and Rabies (brain tissue)
What is Indirect IFA?
IFA or IFAT
-Test antibody (tell serum antibody titer)
-More sensitive than DFA
-Similar to Elisa - serum diluted and antibody isfluorescent, highest dilution of serum that results in specific fluorescence repoted as antibody tier to infectious agent of interest
What can you detect with IFA?
Anaplasma and Ehrlicihia, lyme
What makes immunohistochemistry different than IFA?
Conjugated with horseradish peroxidase
-Visualize with light microscope
How does agglutination work?
Detect antibody or antigen
-clumping of bacteria, RBC or antigen/antibody coated latex particles
-bivalence antibodies - cross linkage
-Determine titer like IFA
What can you test for with agglutination?
Brucellosis, leptospirosis, cryptococcus, blood typing, IMHA
What is AGID?
Agar Gel Immunodiffusion
-Round wells in agar - middle filled with patient serum, positive control included, reagent diffuse through agar, meet a parciptate forms
What do you run AGID to detect?
Coggins, Fungal (Aspergillus)
Why do we use a viral antibody titer?
Determine viral antibody titer
-serial dilution to plasma
-incubate
add cell culture monolayer
-higherst dilution of serum that prevetns cytopathic efect is the antibody titer
What may you run a virus neutralization test on?
DHPP, BVD
How do you determine the antibody titer?
Highest dilution of antibody that will detectably interact with the antigen
-lowest concentration of antibody
-presented as inverse concentration
1:1000>1:10 (Think of it as dilution)
How do you pick a diagnostic test?
Availability, type of pathogen, symptomatic or asymptomatic, time form exposure, treatment
-molecular more sensitive than serologic
What are some testing limitations?
High sensitivity - if negative test you can rule out disease, if positive may or may not have disease (false positives)
High specificity- negative may or may not have disease, positive you rule in (false negatives)
When you select a test remember there are tradeoffs for sensitivity and specififty. Cut off may have false results
What is a protective titer?
Animals with an antibody titer that did not get clinically ill when exposed to disease (core vaccines have one)
What are titers measuring and missing?
Measuring: Humoral Immunity
Missing: Cell Mediated Immunity
Why can you not offer to vaccinate pets based on titers?
-Cell mediated immunity is critical too
-Limited to diseases where immunity challenge studies have occurred
-Legal requirements (rabies)
-Not all labs test for them and may not be accredited
-More expensive than vaccination
What are the risks of using serologic assays in young animals?
-Maternal antibody
-length of time young animals retain maternal antibodies is species specific
-know when to recommend repeat testing
-Helpful for Lenti virus
Why do you test for many serovars of lepto?
Cross reacts, largest one generally the culprit
What is MAT Assay?
Microagglutination Test
Describe how each pathogen on the 4dx test is identified:
E. Canis/E. ewingii
A. phagocytophilum/A.platys
C. Immitis (hearworm)
B. burgdorferi (Lyme)
E. Canis/E. ewingii (ehrliciosis): Antibody
A. phagocytophilum/A.platys (Anaplasmosis ): Antibody
C. Immitis (hearworm): Antigen
B. burgdorferi (Lyme): Antibody
How do you know if the dog has chronic ehrliciosis?
PCR negative due to circulating levels of bacteria being low, dog has inadequate treatment of acute infection, diagnose with quantitive titer
