Immunology: Reference Lab Tests Flashcards
What are the most common reference lab tests?
Coomb’s testing
Fluorescent Antibody Testing
Antibody titers
Polymerase Chain Reaction (PCR)
What is a very specific test for IMHA?
Coombs’ test
What does the Coombs’ test test for?
The presence of autoantibodies
What are autoantibodies
Antibodies against the body
What does a Direct Coombs’ test test for?
Detects antibodies that attack RBCs
What is Coombs’ test based on?
The level of agglutination present
What diseases does Immunodiffusion detect?
Equine Infectious anemia (retrovirus)
Johne disease
What is Johne (Yoh-nee) disease?
Ruminant GI disease caused by mycobacterium
What is present in the immunodiffusion test kit?
The antigen
What is placed in the in separate wells on an agar plate in an immunodiffusion test?
The antigen and patient serum
What diffue through the agar plate and form a band of precipitation where they meet in the Immunodiffusion test?
The antigen and patient serum
What does it mean if no band is present in an immunodiffusion test?
No antibody present
What is mainly used in research and diagnostic laboratories?
Radioimmunoassay
What is the difference between radioimmunoassay and CELISA tests?
Radioimmunoassy uses a radioisotope instead of an enzyme
How is Radioimmunoassay similar to CELISA?
It gives the amount of antigen present
What is not common in veterinary practice but available in reference laboratories and used to verify a tentative diagnosis?
Indirect Fluorescent Antibody Testing
Why do we run an IFA
To identify a specific antibody in a sample
How does the direct antibody fluorescent antibody test work?
The patient sample is added to a fluorescent dye-conjugated antigen and if antigen/antibody complexes form (the disease is present) the sample will fluoresce
What does an indirect Coombs test test for?
Detects antibodies that are already attached to red blood cells
What does a positive Coombs test mean?
Immune-mediated hemolytic disease
What kind of sample does the Radioimmunoassay test need?
Serum
In Radioimmunoassay testing, how do patient antigen and labeled antigen affect each other?
With increasing amounts of patient antigen, more labeled antigen is displaced from the antibody
In Radioimmunoassay testing what is compared with a standard curve to determine the concentration of the antigen?
Patient’s serum
What kind of disease do we run the IFA to rule out?
Diseases that have no other tests to detect them
What is a reason we run antibody titers?
To figure out if an animal has a current infection or if it has prior exposure to a specific antigen
What do antibody titers determine the need of?
Revaccination
How do you run an antibody titer?
WIth serial dilutions of each sample and examination of each dilution for the presence of antibodies
What does a high titer mean?
Indicates an active infection
What does a low titer mean?
Usually indicates a previous exposure to the specific antigen
What are molecular diagnostic testing based on?
The testing of DNA or RNA
What are DNA tests used for
Identify cancers
Detect genetic defects
Pedigrees
Bacterial contaminants in foods
What are the advantages of molecular diagnostics?
Increased sensitivity and specificity
Small samples with faster results
Factors that influence other procedures are not as crucial
What are the disadvantages of molecular diagnostics?
Contamination gives false positives
High levels of expertise to run tests
Need more than one room to perform
High costs
Polymerace Chain Reaction (PCR)
Detect DNA segments
Widely used
Reverse transcriptase polymerase chain reaction
Similar to PCR but tests single-stranded RNA
What must be converted to a double-stranced DNA before the PCR process can continue?
The single-stranded RNA
What are the benefits of running a Real-time Polymerase Chain Reaction?
Decreases the risk of contamination
More easily automated
Faster and easier to run
Fluorescence probe attaches to DNA segments
What else is the PCR called?
The amplification assay
Why is the PCR called an Amplification Assay?
Because a small amount of DNA segment detected in the sample is amplified to run the test better and to determine the results
What are the three steps of the amplification assay?
Denaturation
Annealing
Extension
How often is this process repeated?
25 to 30 times to gain enough DNA
Denaturation
Heated to break apart double stranded DNA
Each strand serves as a template for new nucleotides
Annealing
Temperature is lowered
Causes primers to bind to separate strands
Extension
Temperature raised again adn the Taq DNA polymerase causes new DNA segments to be produced
What is Taq DNA polymerase
The enzyme can read DNA code and assemble the nucleotide base to form new complementary strands
