Immunodiagnostic assays REVISED

Principle of immunodiagnostic tests

Mix an antibody with the antigen in order to detect binding

To detect an antibody,

patient’s body fluid + a known antigen to detect binding

To detect antigen:

Patient’s Specimen + Known antibody to detect binding

Serology

detect/measure the presence of antibody to pathogens, good quality serum is best for test results 

How are immunodiagnostic tests reported?

Qualitative, semi-quantitative (titer) or quantitative tests

What is a titer?

The reciprocal of the most dilution of the sample that still gives a positive test result

Agar Gel Immunodiffusion

Precipitation of soluble solutions

What are the only tests that can detect antigens?

ELISAs, Immunohistochemistry, lateral flow immunoassay

What are the only immunoassay that can’t test for antibodies?

Immunohistochemistry

Agglutination

Clumping of particles

Hemagglutination inhibition

Inhibition of a virus’s ability to agglutinate RBCs

Enzyme-linked immunosorbent assay (ELISA)

Antigen or antibody is labeled with an enzyme, and the enzymatic reaction causes formation of a colored product

Complement fixation

Activation of complement by Ag+Ab complex

Serum or virus neutralization

Neutralization of virus infectivity or toxin activity

Fluorescent antibody

Antibody is labeled with a fluorescent molecule, presence of fluorescence indicated binding reaction (indirect tests for antibodies, direct tests for antigens)

Immunoblot

Antigen are separated by electrophoresis prior to reacting with the antibody; detection is similar to the ELISA

Immunohistochemistry

Similar to ELISA, except antigens are on a glass slide

Lateral flow immunoassay

Antigen or antibody is labeled with gold particles that migrate across a filter, Capture of binding complex leads to visible spot on the filter

Flow cytometry

A rapid way to measure cells tagged with fluorescent molecules in suspension; used to measure different T cell populations in blood

What is the direct Coombs test for?

Diagnoses of immune-mediated hemolytic anemia (IMHA)

What is the indirect Coombs test used for?

For cross-matching blood types

Sensitivity

Proportion of infected animals that test classifies as positive

Specificity

The proportion of uninfected animals the test classifies as negative

What are some possible reasons a test may have a false positive or a false negative?

Testing errors, diagnostic test characteristics or biological differences

Why might there be a presence of an antibody to a pathogen in the serum of an animal?

Current active infection, prior subclinical or clinical infection, prior vaccination, or infection by a cross-reacting organism

What must be done in order to get diagnostically useful results?

Test two serum samples taken 2 weeks apart and have an at least 4-fold increase in titer that indicated ongoing infection

PPV

The probability a positive test result is a true positive

NPV

The probability a negative test result is a true negative

What is the equation for sensitivity?

True positive / (all animals who tested positive)

What is the equation for specificity?

True negative / (all animals who tested negative)