Single-chain antibodies from alpaca for the detection of Fasciola hepatica antigens

Authors

  • Teresa Barreto Unidad de Biotecnología Molecular, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia. Lima, Perú. Bióloga, magíster en Bioquímica
  • Yenisey Alfonso Unidad de Biotecnología Molecular, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia. Lima, Perú. bioquímica, doctor en Ciencias.
  • Pierre Lafaye Plateforme d’Ingénierie des Anticorps, Institut Pasteur. París, Francia. farmacéutico, PhD en Bioquímica
  • Maria del Pilar García Lazaro Unidad de Biotecnología Molecular, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia. Lima, Perú. bióloga
  • L. Agueda Perez Unidad de Biotecnología Molecular, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia. Lima, Perú. bióloga
  • Patricia Herrera-Velit Unidad de Biotecnología Molecular, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia. Lima, Perú. bióloga, PhD en Inmunología
  • Jose R. Espinoza Unidad de Biotecnología Molecular, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia. Lima, Perú. biólogo, PhD en Genética molecular

DOI:

https://doi.org/10.17843/rpmesp.2018.354.3101

Keywords:

Single-Domain Antibodies, Fascioliasis, Antibodies, Camelids, New World, Peru

Abstract

Objectives. To produce recombinant single-chain antibodies from alpaca that will bind to the excreted-secreted (ES) Fasciola hepatica antigen with high affinity and specificity, so as to develop new diagnostic technologies of human and animal fascioliasis. Materials and Methods. A gene bank of DNA of the variable dominions of heavy single-chain antibodies (VHH) has been created, based on mononuclear cells of peripheral blood of an alpaca immunized with the ES antigen of F. hepatica. The gene bank was screened with the ES antigen by differential phage display, selecting ten VHH that bind specifically to ES. The anti-ES VHH was cloned in an expression vector, the recombinant protein (VHH-ES1) of 15.3 kDa was produced by fermentation in E. coli and purified to homogeneity by affinity chromatography. The binding of VHH-ES1 to the ES antigen was evaluated by ELISA using VHH-ES1 as capture antibody, policlonal anti-ES serum of rabbit and conjugated rabbit anti IgG with radish peroxidase. Results. A VHH that binds to the ES antigen (VHH-ES1) has been identified through differential phage display and produced by fermentation in E. coli; this corresponds to an antibody of the long-hinge IgG2 subclass. The binding of the VHH-ES1 antibody to the antigen shows linearity with respect to the concentration of ES in the 50-5,000 ng/mL range and the limit of detection value of the antigen is in the 30-170 ng/mL range of ES (R2=0.99). Conclusions. The VHH-ES1 binds with affinity and specificity to the ES antigen of F. hepatica and is a promissory antibody to be assessed for the development of new fascioliasis diagnostic technologies.

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Published

2018-12-21

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Section

Original Article

How to Cite

1.
Barreto T, Alfonso Y, Lafaye P, García Lazaro M del P, Agueda Perez L, Herrera-Velit P, et al. Single-chain antibodies from alpaca for the detection of Fasciola hepatica antigens. Rev Peru Med Exp Salud Publica [Internet]. 2018 Dec. 21 [cited 2024 Nov. 2];35(4):573-80. Available from: https://rpmesp.ins.gob.pe/index.php/rpmesp/article/view/3101

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