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An ELISA for pigMAP

A sandwich type ELISA for the quantification of Pig-MAP has been developed and validated. The assay is based in two monoclonal antibodies, specific for pig-MAP and showing no crossreactivity with other plasma proteins when tested by western-blot. To increase the probability of obtaining antibodies adequate for sandwich ELISA, intact protein and a fragment of 30K corresponding to the carboxy-terminal end where used during the selection of hybridomas secreting antibodies that reacted with Pig-MAP. This 30K fragment belongs to the region of Pig-MAP showing less homology with the ITI family, and probably containing the most inmunogenic part of the protein.

The performance of the ELISA was good, the assay kept linearity under dilution, and precision was adequate, with coefficients of variation lowest of 8% for both, inter and intra assay. The test was validated by comparing the pig-MAP concentration values obtained with this technique with values determined by radial immunodiffusion, the technique routinely used in our laboratory for the quantification of Pig-MAP. Although radial immunodiffusion is a very precise technique, it has the inconvenience of being time- and reaction consuming method, not adequate for automatisation.

The concentration of Pig-MAP was measured with both techniques in 80 serum samples with Pig-MAP values ranging from 0.33 to 4.5mg/mL. A high degree of correlation between the two immunochemical methods was found (R=0,978, b=1,02), confirming the accuracy of the assay.

The assay described here appears as an adequate alternative to radial immunodifussion for the quantification of Pig-MAP, and inceases the set of specific assays availables for pig APP (acute phase proteins). Further studies have been performed elsewhere in the project to confirm the reliability of Pig-MAP as a parameter of health status or pig welfare. The results of these studies are described under individual result headings.

More information on the APP IN PIG PRODUCTIO -project can be found at: http://www.gla.ac.uk/appinpigs/

Reported by

University of Zaragoza
Department of Biochemistry, Fac Sciences, University of Zaragoza
50009 Zaragoza
Spain
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