Sample preparation and analysis

GCF/PICF in the Periopaper strip was eluted by adding 320 μl quantity of phosphate-buffered saline (PBS) into the sample tube and incubated at 4 °C, overnight. The eluted protein solution from each gingival fluid sample was used for the biochemical analysis.

Total protein in the gingival fluid sample was measured by the Bradford analysis. Briefly, a 10 μl volume of sample or protein standard (0–500 μg/ml) was added into each well of a 96-well microplate and then 200 μl of Bradford working reagent were added to each well. The microplate was shaken for 5 min. After that, the absorbance was measured at 620 nm. The concentrations of total protein in the samples were detected and calculated from a standard curve.

The level of OC was measured by using commercially available ELISA kits (Human Osteocalcin Quantikine ELISA Kit, R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. A 100 μl volume of Assay Diluent RD1-117 (R&D Systems) was added into each well of the microplate and then 50 μl of standard (0–64 ng/ml) or sample was added to each well. The microplate was incubated for 2 h at room temperature on a horizontal orbital microplate shaker (Labnet International, Inc., Edison, NJ, USA) set at 500 rpm. After that, the solutions were aspirated and each well was washed with 400 μl of Wash Buffer (R&D Systems). This step was repeated three times for a total of four washes. Then, 200 μl of Human Osteocalcin Conjugate were added into each well, and the solutions were incubated for 2 h at room temperature on the shaker. After that, the solutions were aspirated and washed again. A 200 μl volume of substrate solution (tetramethylbenzidine) was added into each well, and the solutions were incubated for 30 min at room temperature on the bench-top. Next, 50 μl of stop solution (2 N sulfuric acid) were added into each well.