Substrate solution containing hydrogen peroxide and tetramethylbenzidine will then be added to each well and incubated for 30 minutes under protection from light with stop solution was added to each well also. The optical density will then be determined by subtracting readings at 540 nm from the reading at 450 nm. Protein levels will be calculated using a standard curve derived from a known concentration of respective recombinant proteins. The minimum detectable doses in the assay for sFlt-1, endoglin, and PlGF are 3.5, 7, and 5 pg/mL, respectively. Intraassay and interassay coefficients of variation of 3.5% and 5.5%, respectively, for sFlt-1, 3.2% and 6.5%, respectively, for endoglin, and 6% and 11%, respectively for PlGF.