The time required for seroconversion, as determined by the two IFA assays for IgG, IgM and IgA, and by the two IFA assays and ELISA for IgG, was nearly identical among these patients (Fig.1). of the IgG, IgM and IgA responses between patients with or without underlying medical disease, steroid or intravenous immunoglobulin therapy, or mechanical ventilation. Keywords:Coronavirus, IgA, IgG, IgM, neutralisation antibody, SARS == Introduction == Severe acute respiratory distress syndrome (SARS) is an emerging infection that has affected more than 8000 patients in many countries [1]. This highly Xantocillin contagious infection has a propensity to spread to healthcare workers and household members, and may also cause outbreaks in the community [2,3,4,5,6,7]. As of 5 July 2003, when Taiwan was declared free of SARS by the World Health Organization, 346 laboratoryconfirmed SARS cases had been reported, and 37 (11%) of these patients had died [1]. The first SARS patient in Taiwan was identified in the National Taiwan University Hospital (NTUH) on 25 February 2003, and 76 patients with SARS were eventually identified in this hospital during the outbreak [2,7,8,9]. Among these patients, 18 had microbiological evidence of infection with SARSassociated coronavirus (SARSCoV), including positive RTPCR and realtime RTPCR assays from respiratory or serum samples. In all patients, an indirect enzymelinked immunosorbent assay (ELISA) revealed IgG antibody against SARSCoV in serum samples collected 2835 days after the onset of fever. The aim of this study was to evaluate the chronological development of IgM, IgA, IgG and neutralisation (NT) antibodies following SARSCoV illness of 30 individuals who have been treated at NTUH during the epidemic. == Individuals and methods == == Individuals == Of the 76 SARS individuals for whom serial serum samples were preserved, 30 were included in this study. Sera from these 30 individuals (612 samples from each patient) were collected from < 7 days to 23 weeks after the onset of illness (defined as 1st appearance of fever with body temperature 38.3C). The individuals were aged 2580 years (mean 43 years). Four individuals had underlying disease, namely diabetes mellitus (n= 2), hypertension (n= 1) and chronic hepatitis B disease carriage (n= 1), while the additional individuals were previously healthy. Sputum or throat swab specimens from 12 of these individuals were positive for SARSCoV RNA. == Immunofluorescent antibody assays == Specific antibodies Xantocillin (IgG, IgM and IgA) to SARSCoV were CR2 identified with two different immunofluorescent antibody (IFA) assays: an inhouse assay using wholecell lysate of infected Vero E6 cells as an antigen, or a commercial kit (AntiSARSCoVIIFT; Euroimmun, Lbeck, Germany) [6,10]. For the inhouse IFA assay, spot slides were prepared by applying 10 L of Vero E6 cell suspension, either infected or noninfected with the SARSCoV TW1 strain (GenBank accession no.AY291451). Slides were dried and fixed in acetone. The conjugates used were goat antihuman IgG, IgM and IgA conjugated to fluorescein isothiocyanate (Organon TeknikaCappel, Turnhout, Belgium). The starting dilutions of serum specimens were 1:25 for the inhouse IFA and 1:10 for the Euroimmun kit. Before dedication of IgM and IgA antibodies with IFA, IgG antibodies were removed from patient sera by immunosorption with antihuman IgG, using either a Eurosorb kit (Euroimmun) with the commercial IFA assay, or a Gullsorb kit (Meridian Bioscience, Cincinnati, OH, USA) with the inhouse assay. The cutoff ideals for any positive result were 1:25 for the inhouse IFA and 1:10 for the commercial IFA kit [2,10]. == ELISA == IgG antibody against SARSCoV was also measured with an indirect ELISA, with recombinant nucleocapsid as the coated antigen (SARS96 (TMB); General Biologicals, HsinChu, Taiwan) [10,11]. The cutoff value for any positive IgG result by ELISA was 0.26 [10,11]. == Control sera == Settings comprised 200 combined sera from individuals with communityacquired pneumonia seen at NTUH from October 2001 to December 2002, 70 sera from hospitalised individuals with acute respiratory distress syndrome treated in 2002 at the hospital, and ten sera from ten pregnant women obtained during routine prelabour checkups in 2002. The control sera were tested for the presence of IgG, IgM and IgA from the three methods explained above. == NT antibody assay == Briefly, sera from five individuals were incubated at 56C for 30 min, and then Xantocillin diluted twofold in cell tradition medium (revised Eagle medium). Aliquots (50 L) of diluted sera (from fourfold to 516folder) were added to 50 L of cell tradition medium comprising 100 the cells culture infective dose (TCID50) of the SARSCoV TW1 strain on a 96well microtitre plate and incubated at 37C for 2 h in CO25% v/v. Finally, 100 L of Vero E6 cells (2.5 105/mL) was added to each well of the plate. The plates were incubated at 37C for 35 days in CO25% v/v and examined daily for Xantocillin any cytopathic effect..
The time required for seroconversion, as determined by the two IFA assays for IgG, IgM and IgA, and by the two IFA assays and ELISA for IgG, was nearly identical among these patients (Fig
