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Journal of Clinical Microbiology, March 2004, p. 1082-1088, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1082-1088.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Molecular Cloning, Expression, and Serological Evaluation of an 8-Kilodalton Subunit of Antigen B from Echinococcus multilocularis

Wulamu Mamuti,^1,2 Hiroshi Yamasaki,¹ Yasuhito Sako,¹ Minoru Nakao,¹ Ning Xiao,^1,3 Kazuhiro Nakaya,⁴ Naoki Sato,⁵ Dominique A. Vuitton,⁶ Renaud Piarroux,⁶ Marshall W. Lightowlers,⁷ Philip S. Craig,⁸ and Akira Ito^1*

Department of Parasitology,¹ Animal Laboratory for Medical Research, Asahikawa Medical College, Asahikawa,⁴ Surgical Center, Hokkaido University Hospital, Sapporo, Japan,⁵ Department of Parasitology, Xinjiang Medical University, Urumqi,² Sichuan Institute of Parasitic Diseases, Chengdu, China,³ Université de Franche-Comte, World Health Organization Collaborating Center on Prevention and Treatment of Human Echinococcosis, Besançon, France,⁶ Veterinary Clinical Center, University of Melbourne, Melbourne, Victoria, Australia,⁷ Bioscience Research Institute, School of Environment and Life Science, University of Salford, Greater Manchester, United Kingdom⁸

Received 16 September 2003/ Returned for modification 5 November 2003/ Accepted 24 November 2003

Full-length cDNA and genomic DNA encoding an 8-kDa subunit of antigen B from Echinococcus multilocularis (designated EmAgB8/1) were isolated from an E. multilocularis metacestode cDNA library and a protoscolex genomic DNA library, respectively. The open reading frame of the cDNA clone encodes a polypeptide comprising 85 amino acids with a 20-amino-acid NH₂-terminal signal sequence, which was confirmed following N-terminal sequencing of the native antigen. Reverse transcription-PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E. multilocularis. The gene consists of two exons (encoding the signal sequence and mature protein) separated by a 91-bp intron. The mature form was expressed in Escherichia coli, and its antigenic reactivity was compared with that of a counterpart, an 8-kDa subunit of antigen B from Echinococcus granulosus (EgAgB8/1) by Western blotting and enzyme-linked immunosorbent assay (ELISA) with serum samples from patients confirmed to have cystic echinococcosis (CE) and alveolar echinococcosis (AE). Recombinant EmAgB8/1 showed positive reactions in Western blots with 81.3% (65 of 80) of serum samples from CE patients and 40.6% (26 of 64) of serum samples from AE patients, while recombinant EgAgB8/1 showed positive reactions with 86% (43 of 50) and 42% (19 of 45) of the serum samples from these CE and AE patients, respectively. By the ELISA, both EmAgB8/1 and EgAgB8/1 exhibited similar positive reactions with 88% (44 of 50) of serum samples from CE patients and 37.8% (17 of 45) serum samples from AE patients. Statistical analysis revealed that the sensitivity of EmAgB8/1 was comparable to that of EgAgB8/1 for the serodiagnosis of echinococcal diseases. There was no cross-reaction with sera from patients with cysticercosis, which often cross-react when native antigens are used for serodiagnosis.

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* Corresponding author. Mailing address: Department of Parasitology, Asahikawa Medical College, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan. Phone: 81-166-68-2420. Fax: 81-166-68-2429. E-mail: akiraito{at}asahikawa-med.ac.jp.

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Journal of Clinical Microbiology, March 2004, p. 1082-1088, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1082-1088.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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