Measurement of chromogranin A in porcine saliva: validation of a time-resolved immunofluorometric assay and evaluation of its application as a marker of acute stress

Abstract

The objective of this study was to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour in an acute stress model. Polyclonal antibodies were produced in rabbits immunized with a synthetic porcine fragment of CgA3592379 and used to develop a sandwich TR-IFMA. This TR-IFMA was analytically validated and showed intra- and inter-assay coefficients of variation of 6.23% and 5.82%, respectively, an analytical limit of detection of 4.27 3 1023 mg/ml and a limit of quantification of 24.5 3 1023 mg/ml. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution ( r 5 0.975) and recovery tests. When a model of experimental acute stress, in which animals were immobilized for 3 min with a nose snare (stressor stimulus), was applied, a significant increase ( P , 0.05) in CgA levels in saliva was detected at 15 min post-stressor stimulus. These results indicate that the assay developed in this study could measure CgA in porcine saliva in a reliable way and that the concentrations of CgA in saliva samples of pigs increase after an acute stress situation.