Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana

Abstract

Human neurocysticercosis (NC) is an important but neglected cause of epilepsy in developing countries where the parasite occurs. Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library was selected against peptides displayed on phages coupled to beads and total saline extract of Taenia solium metacestodes immobilized on microtiter plate wells. After two rounds of selection 96 phage clones of each panning were selected, tested for scFv expression and specificity to each target. Specific clones were further analyzed by ELISA (Enzyme-linked immunosorbent assay), Dot-blot, sequencing and immunofluorescence. After selection, three clones were used for antigen capture to characterize its targets for future immunodiagnostic assays development. Total saline extract was fractionated on ion exchange resin diethylaminoethyl (DEAE), and fractions were tested by ELISA to detect sera IgG from: NC, other parasites and health controls (40 each). The fractions with best diagnostic parameters (sensitivity, specificity, area under curve and likelihood ratio, calculated by TG-ROC) were selected and subjected to antigen capture using each purified scFv clone. Each captured fraction was tested by ELISA to detect IgG in 30 serum samples from each group. In immunofluorescence tests, no fluorescence was observed in negative controls, and all clones showed a non-uniform staining profile, and their targets were elucidated through mass spectrometry. After ion exchange fractionation and ELISA tests, DEAE S2 fraction showed to be the best one and was used to capture new antigens. DEAE S2 showed 93.3% specificity. Among all clones, A4 and B6 captured antigens from saline extract and DEAE S2 fraction, respectively, with the best diagnostic parameters. In conclusion, antibody phage display technology is a potential approach for the study of antigen-antibody interactions, which can be used to further elucidate the biology of interaction on neurocysticercosis and to capture new antigens with potential applications in NC diagnosis and therapeutics.