Desenvolvimento de Enzyme-linked Immunosorbet Assay (ELISA) para determinação plasmática de fator de crescimento placentário (PlGF) em gestantes com alto risco de desenvolver pré-eclâmpsia.

Abstract

Pre-eclampsia (PE) is characterized by the onset of hypertension after the 20th week of gestation associated with maternal target organ dysfunction and/or uteroplacental compromise. This condition can lead to serious complications that affect both maternal and fetal health, being the hypertensive disorder with the highest perinatal mortality. The etiology of PE is still poorly understood. It is believed that several pathogenic processes are involved and overlap, with syncytiotrophoblast stress being the main one, leading to the activation of metabolic pathways resulting in angiogenic imbalance, endothelial injury, oxidative stress, and an inflammatory state, resulting in the signs and symptoms of PE. It is worth noting that the imbalance between placental growth factor (PlGF), which has pro-angiogenic action and reduced plasma concentration in the context of PE, and soluble fms-like tyrosine kinase-1 (sFlt-1), which has anti-angiogenic action and increased plasma concentration in pregnant women with the disease, contributes considerably to endothelial dysfunction. Several studies have shown that the clinical use of the sFlt-1/PlGF ratio has predictive potential and in the early diagnosis of PE, allowing clinical interventions to prevent major maternal and fetal complications. Brazil lacks the technology necessary to produce and market the products used in obtaining the sFlt-1/PlGF ratio and depends on the importation of high-value kits, which makes them inaccessible for clinical use. Therefore, the objective was to develop an ELISA diagnostic kit for plasma determination of PlGF in pregnant women at high risk of developing PE, a novel proposal in Brazil aimed at reducing costs and increasing the clinical application of the sFlt-1/PlGF ratio. Several assays were performed to define the parameters to be applied in the diagnostic system: capture antibody concentration (4µg/mL), biotin-conjugated antibody concentration (60ng/mL), streptavidinperoxidase dilution (1:200), sample dilution (undiluted), incubation conditions (45 minutes at 37ºC), washing cycles (3 cycles), solution formulations, and reagent stability (most stable for storage for 1.09 years at 5ºC). An immunoassay system, capable of determining PlGF concentration in the range of 34 to 2186pg/mL, was obtained at the end of this project.