Determination of Prolactin in Canine Saliva: a validation and an experimental study using an ELISA kit

Prolactin is known to have high circulating levels in lactating females as well as to be a remarkable clinical index of stress response, both acute and chronic, in several species. The aims of this research were to investigate: study 1) the possibility of quantifying canine prolactin in saliva using a prolactin canine ELISA kit validated for plasmatic prolactin; study 2) the presence of a correlation between prolactin levels in saliva and serum samples. Study 1: saliva samples were collected from lactating and non-lactating bitches (N=29). An analytical validation method was developed. Repeatability was evaluated with an inter-assay variability between two kits. Matrix effect was evaluated by three identical canine prolactin controls in each kit (with high and low prolactin levels, respectively), one reconstituted with distilled water, one with artificial saliva and one with saliva from non-lactating dogs. Limit of quantification was evaluated with three dilutions of lactating dogs saliva (expected to contain higher values of prolactin). Study 2: saliva and serum samples were collected from shelter dogs (N=10) and prolactin concentrations were measured by using the same kit. Prolactin concentration in saliva samples without any additions resulted in very low values in both Study 1 and 2, often below the detection limit (0.4 ng/ml). Values of control samples reconstituted with non-lactating dogs saliva were slightly higher than values of the same control samples reconstituted with artificial saliva (18.71 versus 16.25 ng/ml; 12.07 versus 9.94 ng/ml). A reliable interassay repeatability was recorded for values whose concentrations were higher than 5 ng/ml, with coefficients of variation lower than 12%, whereas for those lower concentrations high coefficients of variation were recorded. In Study 2, prolactin values for blood samples tended to be low (<3-5 ng/ml), except for a phobic female (17.79 ng/ml). However, saliva prolactin levels were lower and in 50% of cases were not readable. No correlation between prolactin values in blood and saliva was found (ρ=0.482; p=0.274). Summarizing, for saliva we found: a matrix effect; very low prolactin concentrations, often under the limit of quantification; and a lack of correlation with prolactin plasmatic levels. These findings suggest that it is not reliable to measure prolactin in dog saliva through an ELISA Kit created for measuring prolactin in dog serum.