2.4 ELISA assays:
PLAP concentrations in GCF and plasma were quantified using the commercially available Placental Alkaline Phosphatase ELISA kit (catalog no. MBS701995; MyBiosource, San Diego, CA, USA). The sensitivity was 0.39 ng/ml, the intra-assay coefficient of variation (CV) was < 8%, and the inter-assay precision CV was < 10%. The GCF and plasma samples were incubated in a pre-coated microplate according to the manufacturer’s instructions. The samples were read at a wavelength of 450 nm in an automatic ELISA plate reader (CM Sunrise™ 350-700 nm, Tecan US, Inc., AG, Switzerland).