A novel sensitive radioimmunoassay for the measurement of soluble Flt-1 (soluble vascular endothelial growth factor receptor-1)
G McKeeman1, J Ardill2, C Caldwell2, N McFerran3, B Greer3 & N McClure1
Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in various disease states, particularly pre-eclampsia. VEGF exerts its biological activities through two different receptor tyrosine kinases: KDR and Flt-1. Alternative splicing of Flt-1 pre-mRNA produces two products, one encoding the full-length receptor and the other a soluble form, sFlt-1. As sFlt-1 inhibits VEGF activities the regulation of VEGF relies on the expression of Flt-1 and production of sFlt-1. Recent studies involving the measurement of sFlt-1 levels have used Enzyme-linked Immunosorbent Assays (ELISA's). This technique lacks sensitivity to detect sFlt-1 in normal serum and the measurement of large VEGF/sFlt-1 protein complexes is problematic. We have developed a sensitive radioimmunoassay (RIA) for the measurement of total sFlt-1 concentrations in biological fluids.
An 11 amino acid sequence from the mapped structure of human Flt-1 was chosen and synthesised. The peptide was constructed to a MAP sequence for antibody production. Antiserum is used in the assay at a final dilution of 1: 1.5 x 10(super)6(/super)and shows no cross-reaction with plasma proteins or VEGF. Pure peptide was radiolabelled with (super)125(/super)I using Iodogen and purified using reverse phase HPLC. Samples were prepared for assay by trypsinisation (0.2mls x 2mg/ml trypsin for 10mins at 37(super)o(/super)C / 0.8mls sample). Samples were extracted with ethanol. The assay was performed in an alcohol extract, protein free serum. Separation of antibody bound from free peptide is achieved using a secondary precipitating antibody.
The sensitivity of the assay is 0.3pmol/l (25ng/l sFlt-1), and the D50 is 7.0pmol/l (633ng/l sFlt-1). The assay measures total sFlt-1 (free sFlt-1 plus sFlt-1 bound to VEGF) and has been used to measure circulating sFlt-1 concentrations in pregnancy.
A sensitive and specific RIA for sFlt-1 has been developed which uses trypsin digest to liberate an assayable fragment from the sFlt-1 protein.