Lecture 17+18: Introduction to Diagnostic Immunology Flashcards
antibody affinity
the strength of binding between an antibody recognition site and the antigenic epitope
antibody avidity
the sum of all binding affinities b/w an antibody and antigen
what antibody is most often used in diagnostic tests
IgG
(some tests target IgM for early detection)
what does antibody specificity depend on
conformational qualities and various molecular interactions that are refined through clonal selection
polyclonal antibodies
antibodies that recognize a single disease, w/ each antibody recognizing a different part of the disease
- sensitive b/c many of them recognize a range of epitopes
- relatively inexpensive, easy to develop
monoclonal antibodies
antibodies made by identical immune cells, all clones of a single parent cell
- specific b/c they recognize a single epitope
- expensive but easy to mass-produce
what is an antigen?
anything capable of eliciting an immune response or otherwise being recognized by antibodies
can be: non-host proteins, host proteins, antibodies, nucleic acid, hormones, etc.
how can we ‘see’ antibody-antigen complexing
conjugation = producing an antibody with an enzyme attached
ex: horseradish peroxidase (HRP) or alkaline phosphatase (AP)
what is serology
antibody-antigen based diagnostic tests
what is sensitivity
detection of increasingly small concentrations of ‘x’
ability to correctly identify animals WITH a specific disease
what is specificity
detection of ‘x’ in the midst of A-Z
ability to correctly identify animals WITHOUT a specific disease
how to calculate sensitivity?
True positives / (TPs + False Negatives)
how to calculate specificity?
True negatives / (TNs + False Positives)
direct fluorescent antibody (FA or DFA) test
looks for the presence of a specific ANTIGEN in a fresh sample
won’t work if no antigen present
indirect fluorescent antibody test (IFA)
- looks for the presence of ANTIBODIES to a specific antigen in fresh samples
- requires a secondary antibody that recognizes antibodies from your sample
Hemagglutination Assays
looks for the presence of antibodies or antigens that cause the agglutination of RBCs
- autoantibodies commonly cause agglutination but some viruses (parvo or influence) can as well
Hemagglutination Inhibition Assays
looks for the presence of antibodies that prevent the agglutination of RBCs
- influenza viruses may cause agglutination so you can test a patient for influenza antibodies by observing the inhibition of agglutination
Latex Agglutination
uses latex rather than RBCs to serve as a binding substrate for antibodies
What is Virus Neutralization
- looks for the presence of virus-neutralizing antibodies in a patient sample, commonly serum or CSF
- results are read as a titer - the minimum dilution required to prevent viral infection in cells
Enzyme-Linked Immunosorbed Assays (ELISA)
Looks for the presence of antigen or antibody in a sample usually blood or serum
- one platform is the BVDV antigen capture ELISA
- another is the SNAP test
Agar Gel Immunodiffusion (AGID)
- looks for the presence of patient antibodies in serum, CSF, etc
- patient antibodies and central antigen sample diffuse through an agar gel. Where they meet in the middle, Ab-Ag complexing occurs and precipitates
Immunohistochemistry (IHC)
requires FIXED tissues, often w/ special treatment to help exposure antigens
can be used for any antigen
western blotting (WB)
separates proteins based on size on a gel
uses specific antibodies to recognize a specific antigen
Flow Cytometry
cells stained w/ fluorescently labeled antibodies are recognized and sorted based on that label
- 4 different cell populations are sorted based on the presence or absence of 2 different markers
what are direct fluorescent antibody tests looking for
Viral antigen
what is antibody titer
diluting a patient sample to identify the lowest dilution required to prevent CPE
Anti-nuclear antibody (ANA) testing
used t6o test for autoimmune dieseases
NOT specific b/c only tests for antibodies against self DNA/ self cells
identifies the presence of antibodies targeting dsDNA / ssDNA and histones
Saline agglutination test
identifies AUTOantibodies directed against RBCs
Coombs test (indirect agglutination test)
identifies antibodies to exogenous RBCs
- used for blood typing for blood transfusions
ear notch test for BVDV uses what testing methods
Antigen capture ELISA (ACE)
- requires fresh tissue
Immunohistochemistry (IHC)
- requires FIXED tissue
test where light absorbance is measured using a spectrometer
ELISA
what test is used for blood typing
Coombs
what are tests that use natural processes to detect disease?
PCRs (like SNAP) and antigen-antibody tests
when can false negatives occur during disease timeline
after primary antigen exposure but before primary response
when can false negative occur during disease timeline
after the secondary antigen response
The fluorescent molecule and Western Blot tests are considered what 2 types of techniques (qualitative/quantitative/semi-quantitative)?
Qualitative and semi-qualitative
ELISA and titer testing are considered?
(qualitative/quantitative/semi-quantitative)
Semi-quantitative and qualitative
what test is used for bovine viral diarrhea disease
Antigen Capture ELISA (ACE)
rabies testing utilizes what kind of test
Direct fluorescent antibody testing
- requires FRESH brain
During early stages of infection, antigen is present however if specific immunity has not yet developed, a titer test can expect to be… (high or low)?
low
During very late stage of infection, viral antigens are gone, however the immune response is present and a titer test can expect to be …. (high or low)?
high
what could be the reason for a high titer when there is no active infection
previous vaccines
what tests could be used to test for tick-borne diseases
SNAP 4DX
Ehrlichia indirect fluorescent antibdoy (IFA) test
what test can be used to test for systemic lupus erythematosus (SLE)
Anti-nuclear antibody (ANA) test
what does a positive saline agglutination test indicate
anti-RBC antibodies are present in the blood = autoantibodies
you test a cow for BVDV and the results indicate a positive ELISA ACE test but there are no antibodies against BVDV…. you conclude this cow must be?
persistently infected
