Serological detection of antibodies against Salmonella polysaccarides in ELISA employing a new method for coupling of polysaccharides

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Jauho, E.
Wiuff, C.
Boas, U.
Wredstrøm, K.
Pedersen, B.
Andresen, L.
Heegaard, P.
Jakobsen, M.
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International Conference on the Epidemiology and Control of Biological, Chemical and Physical Hazards in Pigs and Pork
Iowa State University Conferences and Symposia

The SafePork conference series began in 1996 to bring together international researchers, industry, and government agencies to discuss current Salmonella research and identify research needs pertaining to both pig and pork production. In subsequent years topics of research presented at these conferences expanded to include other chemical and biological hazards to pig and pork production.


The ELISA (enzyme-linked immunosorbent assay) known as the mix-ELISA is used extensively for the screening of pig serum and meat juice samples for the presence of antibodies against Salmonella Typhimurium and Salmonella Infantis which are the most important Salmonella types occurring in Danish pig herds. Lipopolysaccharide (LPS) from S. Typhimurium and S. Choleraesuis is used as coating antigens in this assay. We undertook an effort to develop a robust and standardized version of this assay based on covalent coupling of antigenic Salmonella lipopolysaccharide-derived polysaccharides to microliter plates by UVirradiation. The polysaccharides were derived from lipopolysaccharide (LPS) by acid hydrolysis and then conjugated to a photochemically active anthraquinone (AQ) derivative which is capable of forming active radicals when exposed to UV -irradiation. The polysaccharide-AQ (PS-AQ) conjugate could then be activated by UV -irradiation and coupled covalently to the polymer plastic surface of the microtiter plates. Polysaccharides derived from S. Typhimurium and S. Choleraesuis lipopolysaccharides, representing the 0-antigens I, 4, 5, 6, 7, and 12, were used. Covalent coupling of polysaccharides by this technique ensures a regiospecific binding that does not influence the antigenicity of the polysaccharide antigen, as the antigen is bound to the plastic surface through the AQ part of the AQpolysaccharide conjugate. In addition, conserved LPS domains such as lipid A which may give rise to crossreactivity is avoided. Plates coated by this technique allow the employment of harsh assay conditions, such as extensive washing procedures and buffers with high salt concentrations when used in ELISA. The PS-AQ ELISA repeatedly had OD-values closely corresponding to those of the conventional mix-ELISA. The obtained functional polysaccharide surface was shown to be reproducible, with very low inter- and intra-plate variation in ELISA and was stable at room temperature for more than 6 months.

Fri Jan 01 00:00:00 UTC 1999