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Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine

Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide... Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine Dian Widiyanti a , e , Nobuo Koizumi b , Takashi Fukui c , Lisa T. Muslich a , Takaya Segawa a , Sharon Y. A. M. Villanueva a , Mitsumasa Saito a , Toshiyuki Masuzawa c , Nina G. Gloriani d and Shin-ichi Yoshida a Department of Bacteriology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan a Department of Bacteriology, National Institute of Infectious Diseases, Tokyo, Japan b Laboratory of Microbiology and Immunology, School of Pharmaceutical Sciences, Chiba Institute of Sciences, Chiba, Japan c Department of Medical Microbiology, College of Public Health, University of the Philippines—Manila, Manila, Philippines d Department of Microbiology-Parasitology, Faculty of Medicine, YARSI University, Jakarta, Indonesia e ABSTRACT Leptospirosis is an infectious disease caused by the spirochete bacteria Leptospira spp. and is commonly found throughout the world. Diagnosis of leptospirosis performed by culture and microscopic agglutination tests is laborious and time-consuming. Therefore, we aimed to develop a novel immunochromatography (ICG)-based method for detecting Leptospira antigen in the urine of patients and animals. We used the 1H6 monoclonal antibody (MAb), which is specific to the lipopolysaccharide (LPS) that is common among Leptospira spp. The MAb was coupled to 40-nm-diameter colloidal gold, and the amounts of labeled antibody and immobilized antibody were 23 μg and 2 μg per test, respectively. Several strains of Leptospira and other bacterial species were used to evaluate the sensitivities and specificities of the assays we developed. The detection limit of the assays was 10 6 cells/ml when disrupted whole bacterial cells were used. The assays were Leptospira specific since they did not cross-react with non- Leptospira bacteria used in the study. Application of diagnostic assays was done on the urine samples of 46 Leptospira -infected hamsters, 44 patients with suspected leptospirosis, and 14 healthy individuals. Pretreatment of the urine samples by boiling and centrifugation (for ultrafiltration and concentration) eliminated nonspecific reactions that occurred in the assay. The sensitivity and specificity of the ICG-based lateral flow assay (LFA) were 89% and 87%, respectively, which were higher than those of the dipstick assay, which were 80% and 74%, respectively. In summary, this ICG-based LFA can be used as an alternative diagnostic assay for leptospirosis. Further development is still necessary to improve the assay. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical and Vaccine Immunology American Society For Microbiology

Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine

Clinical and Vaccine Immunology , Volume 20 (5): 683 – May 1, 2013

Abstract

Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine Dian Widiyanti a , e , Nobuo Koizumi b , Takashi Fukui c , Lisa T. Muslich a , Takaya Segawa a , Sharon Y. A. M. Villanueva a , Mitsumasa Saito a , Toshiyuki Masuzawa c , Nina G. Gloriani d and Shin-ichi Yoshida a Department of Bacteriology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan a Department of Bacteriology, National Institute of Infectious Diseases, Tokyo, Japan b Laboratory of Microbiology and Immunology, School of Pharmaceutical Sciences, Chiba Institute of Sciences, Chiba, Japan c Department of Medical Microbiology, College of Public Health, University of the Philippines—Manila, Manila, Philippines d Department of Microbiology-Parasitology, Faculty of Medicine, YARSI University, Jakarta, Indonesia e ABSTRACT Leptospirosis is an infectious disease caused by the spirochete bacteria Leptospira spp. and is commonly found throughout the world. Diagnosis of leptospirosis performed by culture and microscopic agglutination tests is laborious and time-consuming. Therefore, we aimed to develop a novel immunochromatography (ICG)-based method for detecting Leptospira antigen in the urine of patients and animals. We used the 1H6 monoclonal antibody (MAb), which is specific to the lipopolysaccharide (LPS) that is common among Leptospira spp. The MAb was coupled to 40-nm-diameter colloidal gold, and the amounts of labeled antibody and immobilized antibody were 23 μg and 2 μg per test, respectively. Several strains of Leptospira and other bacterial species were used to evaluate the sensitivities and specificities of the assays we developed. The detection limit of the assays was 10 6 cells/ml when disrupted whole bacterial cells were used. The assays were Leptospira specific since they did not cross-react with non- Leptospira bacteria used in the study. Application of diagnostic assays was done on the urine samples of 46 Leptospira -infected hamsters, 44 patients with suspected leptospirosis, and 14 healthy individuals. Pretreatment of the urine samples by boiling and centrifugation (for ultrafiltration and concentration) eliminated nonspecific reactions that occurred in the assay. The sensitivity and specificity of the ICG-based lateral flow assay (LFA) were 89% and 87%, respectively, which were higher than those of the dipstick assay, which were 80% and 74%, respectively. In summary, this ICG-based LFA can be used as an alternative diagnostic assay for leptospirosis. Further development is still necessary to improve the assay.

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References (43)

Publisher
American Society For Microbiology
Copyright
Copyright © 2013 by the American society for Microbiology.
ISSN
1556-6811
eISSN
1556-679X
DOI
10.1128/CVI.00756-12
pmid
23467776
Publisher site
See Article on Publisher Site

Abstract

Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine Dian Widiyanti a , e , Nobuo Koizumi b , Takashi Fukui c , Lisa T. Muslich a , Takaya Segawa a , Sharon Y. A. M. Villanueva a , Mitsumasa Saito a , Toshiyuki Masuzawa c , Nina G. Gloriani d and Shin-ichi Yoshida a Department of Bacteriology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan a Department of Bacteriology, National Institute of Infectious Diseases, Tokyo, Japan b Laboratory of Microbiology and Immunology, School of Pharmaceutical Sciences, Chiba Institute of Sciences, Chiba, Japan c Department of Medical Microbiology, College of Public Health, University of the Philippines—Manila, Manila, Philippines d Department of Microbiology-Parasitology, Faculty of Medicine, YARSI University, Jakarta, Indonesia e ABSTRACT Leptospirosis is an infectious disease caused by the spirochete bacteria Leptospira spp. and is commonly found throughout the world. Diagnosis of leptospirosis performed by culture and microscopic agglutination tests is laborious and time-consuming. Therefore, we aimed to develop a novel immunochromatography (ICG)-based method for detecting Leptospira antigen in the urine of patients and animals. We used the 1H6 monoclonal antibody (MAb), which is specific to the lipopolysaccharide (LPS) that is common among Leptospira spp. The MAb was coupled to 40-nm-diameter colloidal gold, and the amounts of labeled antibody and immobilized antibody were 23 μg and 2 μg per test, respectively. Several strains of Leptospira and other bacterial species were used to evaluate the sensitivities and specificities of the assays we developed. The detection limit of the assays was 10 6 cells/ml when disrupted whole bacterial cells were used. The assays were Leptospira specific since they did not cross-react with non- Leptospira bacteria used in the study. Application of diagnostic assays was done on the urine samples of 46 Leptospira -infected hamsters, 44 patients with suspected leptospirosis, and 14 healthy individuals. Pretreatment of the urine samples by boiling and centrifugation (for ultrafiltration and concentration) eliminated nonspecific reactions that occurred in the assay. The sensitivity and specificity of the ICG-based lateral flow assay (LFA) were 89% and 87%, respectively, which were higher than those of the dipstick assay, which were 80% and 74%, respectively. In summary, this ICG-based LFA can be used as an alternative diagnostic assay for leptospirosis. Further development is still necessary to improve the assay.

Journal

Clinical and Vaccine ImmunologyAmerican Society For Microbiology

Published: May 1, 2013

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