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Clin. Vaccine Immunol. doi:10.1128/CVI.00347-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Utilization of Complement-Dependent Cytotoxicity to Measure Low Levels of Antibodies: Evaluation in a Model of Japanese Encephalitis Nonstructural Protein 1

Department of Health Sciences, Kobe University School of Medicine, Kobe, and Epizootic Research Center, Equine Research Institute, Japan Racing Association, Tochigi, Japan

^* To whom correspondence should be addressed. Email: ekon{at}kobe-u.ac.jp.

	Abstract

Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce "nonspecific" reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) mixing of test samples with a suspension of cells expressing the antigen of interest on their surface, (ii) addition of rabbit complement and (iii) measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate in the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody detecting immunostaining and ELISA methods. As well, the assay method detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV: specific, but not natural IgM antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.