Common immunological lab tests Flashcards
What does sensitivity mean
The ability of the test to identify all animals that are truly positive for the disease
A test with a 100% sensitivity will detect all of the positive cases
What does specificity mean
The ability of the test to identify all of the true negatives
A test with a 100% specificity will detect all of the negative cases
Why are SNAP tests one of the most common immunological tests run in clinics
They are simple to run
They provide rapid results
Relatively inexpensive
They come in a kit format with everything required to perform the test (except the sample)
In order to generate accurate results, it is essential to pay attention to quality control
How to collect and handel a sample for immunological testing
Usually require serum or plasma to run the test, occasionally body excretions, fluids or tissues are required
Use largest bore needle and appropriate volume for blood collection
EDTA or heparin are the most commonly used anticoagulants
Check requirements for storage temperature and sample temperature when running test
Many tests have very specific specimen or handling requirements
Always check with the reference lab or kit manufacturer
What are immunological tests based on
The tests are based on use of antibodies and antigens
Antibodies may be used to detect antigens
It may measure the concentration of antibodies specific to an antigen
Use antibodies to detect the presence of other antibodies
What can an immunological test tell you
Tests can be quantitative
How much antibody? How much antigen?
Tests can be qualitative
Which antigen is present
What are the two types of test for infectious agents
Direct-Detects antigen(s) associated with the presence of the organism
Positive test means the antigen (virus, bacteria, parasite… is present)
Indirect–Detects antibodies produced in response to the antigen
Means the immune system has seen the targeted antigen
Does not necessarily mean the bacteria, virus, etc is present right now
Serology is the science of detecting antibodies
monoclonal antibodies are used how in test
Basis of immunological tests
These are antibodies that are identical to one another
Commercially produced
Produced by a single clone of plasma cells
The antibody recognizes a specific antigen
Makes the tests highly sensitive and specific
Enzyme linked immunosorbent assay is used how in tests
The most common immunoassay performed in veterinary clinics
Basis of benchtop SNAP tests
Purpose: To test a sample for the presence of a specific antigen or antibody
How: The test contains a monoclonal antibody that will bind to the antigen if it is present OR the test contains an antigen that will bind to antibody if it is present in the sample
Tests for viruses, bacteria, parasites, hormones, enzymes, etc
Heartworm, FeLV/FIV, Parvovirus, Progesterone
How does a direct ELISA test work
Direct ELISA looks for presence of antigen in a sample
Monoclonal antibody that is specific to the antigen is bound to filter paper
A conjugate solution is then added to the patient sample
This solution contains more of the mAb which has been “tagged with an indicator (dye)
The indicator is visible to the eye
The patient sample and conjugate are added to the sample well
If antigen is present in the sample, it binds to the mAb on the test kit
Any remaining sample is washed away in a rinse step
No colour change = antigen –
Color change = antigen +
How does a indirect ELISA test work
Indirect ELISA looks for the presence of antibody in a sample
A specific antigen is bound to the filter paper
A conjugate solution is then added to the patient sample
Conjugate contains more of the antigen that has been “tagged” with an indicator (dye)
The indicator is visible to the eye
The patient sample and conjugate are added to the sample well
If antibody specific to the antigen is present in the serum, it will bind to the antigen test kit
The unbound sample is washed away in a rinse step
No color change = antibody –
Color change = antibody +
What are the limitations of an indirect ELISA
Test limitation: The presence of antibodies does not necessarily mean that there is an active infection
It just means that the animal was exposed to the antigen at some point
It does not differentiate between an active infection and previous exposure
What are the controls for an ELISA
Most ELISA tests have a control built right into the test procedure
The control ensures:
The “conjugate” binds to whatever is being tested for
The color reagent works
Can be a simple dye, but may require an enzymatic reaction to activate the dye
How does an ELISA snap test work and what are the limitations
ELISA tests that have been simplified into a single step
Designed for in-clinic use
Limitations of SNAP tests:
Not quantitative – Only give “yes” or “no” response OR
Semi-quantitative – The amount of antigen in the control is known, so the test can determine if there is more or less than the amount in the control
What are the different types of quantitative ELISA test
Can make serial dilutions of the sample
Can compare to quantitative controls
Competitive ELISA
Antigen from the patient competes with labelled antigen in the reagent to bind with antibody in the test kit
The degree of color change corresponds with antigen concentration in the sample
