An antibody microarray assay was developed for Salmonella serotyping based on the Kauffmann-White scheme. A model (8 by 15) array was constructed using 35 antibodies for identification of 20 common Salmonella serovars and evaluated using 117 target and 73 nontarget Salmonella strains. The assay allowed complete serovar identification of 86 target strains and partial identification of 30 target strains and allowed exclusion of the 73 nontarget strains from the target serovars.The genus Salmonella consists of over 2,500 serovars, as determined by its somatic (O) and flagellar (H) antigens. A slide agglutination test is commonly used in serotyping for Salmonella spp. based on the Kauffmann-White scheme (11,12), which involves over 250 antisera. The current serotyping method only allows detection of a single antibody-antigen reaction at a time, requires well-experienced technologists to perform, consumes relatively high volumes of reagents, and takes a minimum of 3 days to perform a minimum of three antibody-antigen reactions to determine a serotype. The number of reactions and the time required can be many times greater if a less-common serovar is tested.DNA-based alternative approaches, such as PCR, have been developed to identify a particular serovar (1, 7). However, the PCR methods only detect a limited number of serovars at a time, and many different genetic markers are still to be developed or verified for identification of various serovars (8). In this research, a new antibody microarray-based assay that allows parallel analysis of multiple antigens was investigated for Salmonella serotyping.Salmonella antisera were purchased from Statens Serum Institut (Copenhagen, Denmark) or provided by the Office International des Épizooties Reference Laboratory for Salmonellosis, Public Health Agency of Canada (Guelph, Ontario, Canada). The antisera were diluted to 1 to 5 mg protein per ml in Micro Printing buffer (TeleChem International, Sunnyvale, CA), and then spotted in quadruplets at a density of 400 spots/cm 2 onto SuperEpoxy microarray slides (TeleChem International) under a humidity of 58 to 60% with SMP8 spotting pins (TeleChem International) using the SpotBot Protein Edition arrayer (TeleChem International). The epoxy-functionalized glass slide allowed completion of the coupling reaction within 10 min after printing. Cy5-labeled dCTP (Amersham Biosciences, Baie d'Urfe, Quebec, Canada) was included in the spotting solution at a concentration of 20 fmol/l to monitor spotting quality. The slides were scanned after spotting under the Cy5 channel (670 nm) of the scanner so that the slides with compromised spotting quality were identified prior to their use.Salmonella strains (Table 1) were obtained from the OIE Reference Laboratory for Salmonellosis, Public Health Agency of Canada. Overnight cultures (0.5 ml) were inactivated at 63°C for 10 min and washed with 1.0 ml phosphatebuffered saline (PBS). The cells were fluorescently labeled by incubating the cells for 30 min in 100 l PBS containing 5 l Eosin Y solution [0...

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