Desenvolvimento de Enzyme Linked Immunosorbent Assay (ELISA) para determinação plasmática de tirosina quinase tipo 1 solúvel semelhante à FMS (sFlt-1) em gestantes com alto risco para desenvolver pré-eclâmpsia.

Abstract

Pre-eclampsia (PE) is a pregnancy disorder characterized by hypertension that occurs after 20 weeks of gestation, affecting 2 to 8% of pregnancies worldwide and leading to high maternal and perinatal morbidity and mortality. The pathogenesis of PE has not been fully elucidated, but it is known to involve maternal, genetic, and immunological factors, characterized by varying degrees of poor placental perfusion, leading to hypertension and multiple organ damage. The current gold standard for diagnosing the disease is based on the analysis of hypertension with or without recent-onset proteinuria during the second half of pregnancy, which has limited predictive capacity. In this context, as there is an imbalance between angiogenic factors, such as placental growth factor (PlGF), and antiangiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1), international guidelines suggest using the sFlt-1/PlGF ratio test to aid in the diagnosis of PE, wich demonstrates better predictive capacity. Currently, all ANVISA approved products to determine sFlt-1 and PlGF levels are imported, leading to external technology dependence and increases healthcare costs. Therefore, it was proposed the development of an ELISA to determine plasma sFlt-1 levels in pregnant women at high risk of PE onset. The following parameters were defined: capture antibody concentration of 2 μg/mL, biotin-conjugated antibody concentration of 500 ng/mL, 1:200 dilution for peroxidase-conjugated streptavidin, the use of undiluted samples obtained from serum or plasma collected in EDTA, heparin, or citrate tubes, an incubation temperature of 37°C for the sample, biotin-conjugated antibody, and peroxidase-conjugated streptavidin, a 45-minute incubation time for the sample and biotin-conjugated antibody, three cycles in the washing step, and solid phase and biotin-conjugated antibody stability of 1.09 years at 5°C. Formulations for sensitization, blocking, washing, stopping, and biotin-conjugated antibody and peroxidase-conjugated streptavidin diluent solutions were also defined. After establishing these parameters, the developed diagnostic kit was found to be suitable for determining the serum concentration of sFlt-1 in pregnant women who have developed PE.