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Journal of Clinical Microbiology, January 2008, p. 302-310, Vol. 46, No. 1
0095-1137/08/$08.00+0     doi:10.1128/JCM.01006-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Sensitive and Specific Enzyme-Linked Immunosorbent Assay Using Chemiluminescence for Detection of Severe Acute Respiratory Syndrome Viral Infection

Research and Development Division, Sysmex Co. Ltd., Kobe, Japan,¹ Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan,² Department of Microbiology, The University of Hong Kong and Queen Mary Hospital, Hong Kong³

Received 15 May 2007/ Returned for modification 16 July 2007/ Accepted 25 October 2007

Here we report the development of a more-sensitive immunoassay for severe acute respiratory syndrome (SARS) based on an enzyme-linked immunosorbent assay using chemiluminescence (CLEIA) to detect the viral nucleocapsid (N) antigen in nasopharyngeal aspirate (NPA) from patients infected with SARS coronavirus (CoV). The CLEIA was established with an optical combination of monoclonal antibodies (MAbs) against SARS CoV N protein prepared from mice immunized with recombinant N protein without cultivating the virus. The capture and detecting MAbs of the CLEIA reacted to the carboxyl-terminal and amino-terminal peptides of the N protein, respectively. The CLEIA was capable of detecting recombinant N protein at 1.56 pg/ml and viral N protein in SARS CoV cell culture lysates at 0.087 of 50% tissue culture infective doses/ml. The CLEIA showed no cross-reactivities to recombinant N proteins of common human CoV (229E, OC43, and NL63) or lysates of cells infected with 229E and OC43. In addition, an evaluation with 18 SARS-positive NPA samples, all confirmed SARS positive by quantitative PCR and antibodies to SARS CoV, revealed that all (18/18) were found positive by the CLEIA; thus, the sensitivity of detection was 100%. When we tested 20 SARS-negative NPA samples, the CLEIA was shown to have high specificity (100%). The sensitivity of our novel SARS CLEIA was significantly higher than the previous EIA and comparable to the other methods using reverse transcription-PCR.

Published ahead of print on 21 November 2007.