A Sensitive and Specific EIA-based Chemiluminescence Assay for the Detection of the Severe Acute Respiratory Syndrome (SARS) Viral Infection

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

^* To whom correspondence should be addressed. Email: tokamoto{at}med.nagoya-cu.ac.jp.

	Abstract

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 (Co-V). The CLEIA was established with an optical combination of monoclonal antibodies (MoAb) against SARS Co-V N protein prepared from mice immunized with recombinant N protein without cultivating the virus. The capture and detecting MoAbs of the CLEIA reacted to the carboxyl-terminal and the 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 Co-V cell culture lysates at 0.087 of 50% tissue culture infective doses/ml (TCID₅₀/ml). The CLEIA showed no cross-reactivities to recombinant N proteins of common human Co-V (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 NPA by quantitative PCR (Q-PCR) and antibodies to SARS Co-V, revealed that all (18/18) were found positive by the CLEIA, thus the sensitivity of detection was 100%. When we tested NPA 20 SARS negative NPA 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 RT-PCR.