Canine crp elisa kit

The serum CRP concentration was assessed using a canine CRP ELISA kit (Abcam, Cambridge, UK) following the manufacturer's instructions. Briefly, 100 μL of canine serum samples diluted in reagent diluent were added to 96‐well ELISA plates pre‐coated with anti‐canine CRP antibodies and incubated for 10 minutes at room temperature. After washing, 100 μL of the enzyme‐antibody conjugate was added to the plates, followed by incubation for 10 minutes at room temperature in the dark. The plates were then developed with 3,3′,5,5′‐tetramethylbenzidine substrate solution for 5 minutes at room temperature, and the reaction was stopped with 100 μL of stop solution. Absorbance was determined at 450 nm with a scanning multi‐well spectrophotometer (molecular devices).

CRP levels in both the serum and saliva samples were measured using a commercial canine CRP ELISA kit (Abcam, Cambridge, MA, USA). Dilutions of the serum and saliva samples used in analyses were 1:1000, 1:10, and undiluted. All thawed salivary samples were centrifuged at 1500× g for 15 min at 4 °C to remove cellular debris and minimize the turbidity of the saliva, which could negatively impact the accuracy of analysis [13 (link)]. Supernatants were transferred into fresh Eppendorf tubes and appropriately labeled. A total of 64 samples, consisting of 32 serum and 32 saliva samples, were assayed in duplicate. For ELISA analysis, all samples were thawed and mixed thoroughly; ELISA was then performed in accordance with the manufacturer’s instructions. Absorbance was detected at a wavelength of 450 nm on a microplate reader (Biotek Instruments Inc., Winooski, VT, USA).