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Serum antibody levels are typically quantified and reported as “titers.” In practice, test serum is diluted in a series of tubes and a known amount of antigen is added to each dilution. The most diluted sample in which a reaction occurs provides an estimate of the amount of antibody within the serum sample. The reciprocal of this dilution is reported as the titer (e.g. a sample in which the 1:32 dilution of serum was the highest sample dilution to display a reaction, has a titer of 32) (Figure 4.1). There are several caveats to the use and interpretation of antibody titers that should be understood:

 A titer from one laboratory may not be the equivalent of a titer at another laboratory.

 A titer determined by one method may not be the equivalent of a titer determined by another method.Figure 4.1 Antibody titration.

 What constitutes a “protective” titer differs for various pathogens and may vary between laboratories.

 The method in which a titer was determined to be “protective” may vary between laboratories.

 Lack of a “protective” titer may not indicate susceptibility.

 In most cases, a single titer measurement cannot distinguish between previous exposure and current infection.

 Titer detection alone cannot establish the cause of existing disease.

 In puppies and kittens, titers cannot distinguish between maternal antibodies and those derived from vaccination or previous exposure.

In‐house ELISA test kits can be used to measure antibody titers against canine distemper, canine adenovirus, canine parvovirus, and feline panleukopenia (see the discussion in the section on primary diagnostic testing) (Gray et al. 2012; Mazar et al. 2009; Mende et al. 2014); however, laboratory testing methods are considered the gold standard. Practitioners should be aware of the particular method utilized when interpreting clinical results, making case management decisions, and comparing literature references.

Agglutination, or clumping, of red blood cells in a serum sample occurs when antibodies cross‐link large particles of antigen (i.e. one antibody binds to two antigens). In the presence of excess antibody, the antigenic particles become saturated and further agglutination is inhibited. This reaction is the basis for antibody titration through HI. In these tests, a serial dilution of the test serum is exposed to soluble antigen (e.g. viral particles) cross‐linked to red blood cells. Antibodies within the test serum bind to the soluble antigen causing agglutination of the cross‐linked red blood cells—hemagglutination—until the antibodies are exhausted and further hemagglutination is inhibited. The reciprocal of the highest serum dilution that completely inhibited hemagglutination represents the HI titer for that sample. In addition to its use for measuring antibodies, HI can also be used to identify specific viruses (Tizard 2013). HI is commonly employed to measure antibodies against adenoviruses, coronaviruses, herpesviruses, influenza, and parainfluenza. HI is considered the gold standard titer testing method for canine and feline parvoviruses (Ford 2013).

CF testing relies on the classical complement pathway resulting in the binding of antibodies to red blood cells and their subsequent rupture and hemolysis. After the disruption of existing antigen–antibody complexes, test serum is mixed with a known amount of a new complement source, allowed to form new antigen–antibody complexes, and an indicator in the form of antibody‐linked red blood cells is added. If antibody is present in the sample, the complement is consumed and the red blood cells remain intact. In the absence of antibody, the complement binds to the antibody‐linked red blood cells and is lysed. Conducting the reaction within serial dilutions of test serum allows quantification of antibodies. The reciprocal of the highest dilution of serum in which no more than 50% of the red blood cells are lysed represents the CF titer for that sample (Tizard 2013). CF is commonly employed to measure antibodies against adenoviruses, parvoviruses, mycobacterial infections, and coccidioidomycosis.