Id screen ruminant ifn g kit

Approximately 10 ml of whole blood was collected at the abovementioned time points from the jugular vein using heparinized tubes. Blood samples were maintained at room temperature for <1 h when they were stimulated in 96-well cell culture plates (Eppendorf, Hamburg, Germany) with PPD-B, P22 (except at week 7), and E/C at a final concentration of 20 μg/ml each. Phosphate-buffered saline was used as an unstimulated control. The samples were incubated at 37°C and 5% CO₂ for 18 ± 2 h. Plasma supernatants were collected after centrifugation at 18 g for 10 min, and the released IFN-γ was measured by ELISA (ID Screen^® Ruminant IFN-g kit, ID.vet, Grabels, France). Optical densities of the ELISA plates were read at 450 nm (OD_450nm) using a spectrophotometer (Biotek Power Wave XS^®, Agilent, Santa Clara, USA). Antigen-specific IFN-γ responses were calculated as OD_450nm of the antigen-stimulated well minus OD_450nm of the unstimulated well (ΔOD_450nm).

Whole blood samples were collected at time points mentioned above from the jugular vein using heparinized tubes and subsequently stimulated in 96-well cell culture plates (Eppendorf, Hamburg, Germany) with PPD-B, P22 (except at week 7) and E/C at a final concentration of 20 μg/ml. Phosphate-buffered saline was used as an unstimulated control. Samples were incubated at 37 ºC and 5% CO₂ for 18 ± 2 h. Plasma supernatants were collected after centrifugation at 18 g for 10 min and the released IFN-γ was measured by ELISA according to the ID.screen® Ruminant IFN-g kit (ID.vet, Grabels, France) instructions. ELISA plates were read as optical density obtained at 450 nm (OD_450nm) using a spectrophotometer (Biotek Power Wave XS®, Agilent, dana Clara, USA). Antigen-specific IFN-γ responses were calculated as ΔOD_450nm of antigen-stimulated well minus OD_450nm of non-stimulated well (ΔOD_450nm).

Assessment of IFN-γ concentration per sample was completed after the last stimulation day using a commercial sandwich ELISA (ID Screen Ruminant IFN-γ kit, IDVET, Montpellier, France). Procedures followed the manufacturer’s instructions, using 25 μl each of the kit’s negative and positive control (each diluted with 25 μl of the kit’s Dilution Buffer 1) and 10 μl of samples (each diluted with 90 μl of the kit’s Dilution Buffer 1). According to the procedure protocol, the test is valid if the mean optical density (OD) of the positive control is >0.5 and the ratio of the mean values of the positive and negative controls is >3. The results for each sample were interpreted as a sample to positive (S/P) ratio, or a ratio of IFN-γ concentration to the positive control using the following formula, as per the kit’s instructions: S/P (%) = [(OD activated sample–OD control sample)/(OD mean positive control–OD mean negative control)]*100. For both the preserved and unpreserved samples, the OD for the control sample was determined from the samples with only PBS added. According to this procedure protocol from the kit’s instructions, samples with S/P ratio >15% were considered positive for IFN-γ production.