Development of specific scFv antibodies to detect neurocysticercosis antigens and potential applications in immunodiagnosis.
We have shown previously that detection of circulating antibodies against mimotopes selected by phage display were useful in neurocysticercosis diagnosis. However, circulating antigens may also be useful in patients' clinical follow-up. Therefore, we aimed to select novel combinatorial antibodies, single-chain variable fragment (scFv), which can be used for specific antigens with pre-defined affinity and specificity without prior immunization. A phage scFv antibody library was selected against Taenia solium mimotopes displayed on phages coupled in beads and total saline extract of T. solium metacestodes (S) immobilized on microtiter plate wells. After two rounds of selection, 96 phage clones were evolved and validated against each target by enzyme linked immunosorbent assay (ELISA), and dot-blot, and three specific antibodies (B6, G10 and A4) were further characterized by sequencing and indirect immunofluorescence (IFI) assays. IFI revealed tegument staining for the B6, while the others showed a non-uniform staining in the whole parasite. The selected scFvs were used to capture their antigen targets that were elucidated through mass spectrometry, and used for antibody detection in NC patients' sera by ELISA, which achieved sensitivities greater than 97% and specificities above 95%. We have successfully developed scFv antibodies against important mimotopes used in NC diagnosis, and can be further explored to detect circulating antigens for clinical follow-up of patients with NC. Our strategy also highlighted the possibility of using this combinatorial approach to select, capture and characterize specific antigens to better understand this intriguing parasite infection and disease evolution.
Laboratório de Diagnóstico de Parasitoses, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, 38400-902 Uberlândia, MG, Brazil.
Vanessa da Silva
Vda S
Ribeiro
Thaise Gonçalves
TG
Araújo
Henrique Tomaz
HT
Gonzaga
Rafael
R
Nascimento
Luiz Ricardo
LR
Goulart
Julia Maria
JM
Costa-Cruz
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D000947:Antigens, Helminth / Q000097:blood / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006790:Host-Parasite Interactions / Q000276:immunology; D006801:Humans; D013058:Mass Spectrometry; D020019:Neurocysticercosis / Q000175:diagnosis / Q000276:immunology* / Q000469:parasitology; D019151:Peptide Library; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*; D041201:Taenia solium / Q000276:immunology* / Q000502:physiology
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D019151:Peptide Library; D057127:Single-Chain Antibodies
ELISA; Immunofluorescence; Phage displayed peptides; Taenia solium; scFv