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BMC Proceedings

Open Access

Novel monoclonal antibodies against pathogenic Leptospira

  • Bruno Moisés de Matos1,
  • Cláudia Pinho Hartleben1,
  • Leonardo Garcia Monte1,
  • Thais Farias Collares1,
  • Biancia Sica Siedler1 and
  • Francine Alves Sinnott1
BMC Proceedings20148(Suppl 4):P59

https://doi.org/10.1186/1753-6561-8-S4-P59

Published: 1 October 2014

Background

Leptospirosis is a zoonotic disease caused by pathogenic bacteria belonging to the genus Leptospira and more than 1 million cases occur worldwide annually. Several mammals may carry the agent, and rats are the most important source of human infection in urban settings. The Leptospira surface proteins have an important role during pathogen infection and some of these allow the differentiation between pathogenic and non-pathogenic species. Among these, the LigA and LigB adhesins are surface localized proteins that interact with the proteins of extracellular matrix and fibrinogen. Therefore, antibodies against these targets are useful tools in immunodiagnostic assays.

Methods

The goal of this study was to generated monoclonal antibodies (mAbs) against a truncated fragment of approximately 54 kDa, named rLigBrep, that comprise a identical portion of LigA and LigB (domains 2-7). For the mAbs production, two BALB/c mice were inoculated via intraperitoneal with 150 µg of rLigBrep on days 0, 14, 21 and 28. Freund's complete adjuvant was used in the first dose and incomplete in the subsequent ones. Four days before cellular fusion the mouse with the highest titer in indirect ELISA (1:64000) was boosted with 20 µg of protein intravenously. Splenic lymphocytes were fused to murine Sp2/O-Ag14 myeloma cells in the presence of PEG 1450. Fused cells were cultivated in Dulbecco's modified Eagle medium containing 20% fetal calf serum and supplemented with hypoxanthine, aminopterin and thymidine (HAT). Hybridomas growing in HAT medium were screened by indirect ELISA and those positive for rLigBrep were cloned twice by limiting dilution, expanded and stored in liquid nitrogen.

Results and conclusion

Two hibridomas (ID-Ra and ID-Rg) were obtained and used for ascites production. An indirect ELISA was performed to verify antibodies presence in ascites fluid. The mAbs presented high titres against rLigBrep (ID-Ra 51,200 and ID-Rg 128,000, respectively). In conclusion, the mAbs produced in this study can be useful tools in immunodiagnostic assays for leptospirosis.

Declarations

Acknowledgements

We are grateful to Universidade Federal de Pelotas, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) and Cnpq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).

Authors’ Affiliations

(1)
Laboratório de Imunodiagnóstico, Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas

References

  1. Adler B, de la Peña Moctezuma A: Leptospira and leptospirosis. Veterinary Microbiology. 2010, 140 (3-4): 287-296.View ArticlePubMedGoogle Scholar
  2. Harlow E, Lane D: Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press. 1988, NY: Cold Spring HarborGoogle Scholar
  3. Monte LG, Conceicao FR, Coutinho ML, Seixas FK, da Silva EF, Vasconcellos FA, deCastro LA, Hartleben CP, Dellagostin OA, Aleixo JA: Monoclonal antibodies against the leptospiral immunoglobulin-like proteins A and B conserved regions. Comparative Immunology. Microbiology & Infectious Diseases. 2011, 34 (5): 441-446.Google Scholar

Copyright

© de Matos et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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