Development and validation of an ELISA for the quantification of pig major acute phase protein (Pig-MAP).

ABSTRACT

Measurement of acute phase proteins (APPs) levels in blood is increasingly being used for monitoring health and welfare in farm animals. In this work a sandwich-type ELISA for the quantification of pig Major Acute phase Protein (Pig-MAP), one of the main APP in pigs, has been developed and validated. Two Pig-MAP specific monoclonal antibodies were developed in mouse. One of the monoclonal antibodies was fixed to microtiter plates and the other was coupled to horseradish peroxidase and used as detection antibody. To calibrate the assay dilutions of a standard pig serum of known Pig-MAP concentration were added to the plate in each assay. The assay showed good accuracy, kept linearity under dilution and recovery was proportional. The detection limit was 0.1 microg/mL. Precision was adequate with coefficients of variation lower than 8% for both inter and intra-assays. A good linear correlation between Pig-MAP concentration values obtained by ELISA and by radial immunodiffusion, used as reference method, was found (r = 0.978; beta = 1.02). Pig-MAP concentration was analysed in serum samples obtained from two pig herds of different health status (10 animals per age and herd, of 10, 12, 14, 18 weeks of age). Mean values obtained in the farm of low health status were higher than the obtained in the farm of high health status (p<0.001). In the farm of high health status, mean Pig-MAP concentration remained constant at the different ages analysed (mean values of 0.83+/-0.18 mg/mL) whereas in the farm of low health status differences between age groups were found. In this farm (low health status) mean values for the total of pigs analysed were of 1.68+/-0.74 mg/mL.