The software used for designing the arrays was from DIGEN, Jerini Biotools GmbH, Berlin, Germany

The software used for designing the arrays was from DIGEN, Jerini Biotools GmbH, Berlin, Germany. reported to WHO by August 2003 including 916 deaths (http://www.who.int/csr/sars/en/). The viral genome has been sequenced Marra et al., 2003, Rota et al., 2003. It consists of 29?751 nucleotides which contain 15 identifiable open reading frames (“type”:”entrez-nucleotide”,”attrs”:”text”:”AY274119″,”term_id”:”30248028″,”term_text”:”AY274119″AY274119). Peptides incorporating all of the sequences predicted in the open reading frames of the SARS-CoV genome were prepared on derivatized cellulose membranes using a robotic peptide synthesizer (Autospot ASP 222, Intavis Bioanalytical Instruments, Lagenfeld, Germany). The peptides were 10 amino acids long and overlapped by eight residues. Each peptide on a membrane was therefore shifted from the one previous by two amino acids towards the C-terminal end. The arrangement of the 4942 peptides on sets of four membranes covering the 15 open reading frames (Orfs) is illustrated in Fig. 1 . Open in a separate window Fig. 1 (a) Outline of the overlapping peptide set covering membranes M1, M2, M3, and M4. Panels on the membranes are designated from left to right P1, P2, P3, and P4. Numbers in the boxes Targapremir-210 designate the location of each open reading frame. The note to the right of M4P1 shows the key to the Orfs with peptide totals in brackets. This membrane was spotted with dye instead of amino acids. In actual membranes, each spot is a 10-amino-acid peptide with adjacent spots being shifted by two amino acids. Characterization of the immune response against these single case epitopes promises to provide important insights into their role in the resolution of infection. However, epitopes recognized by multiple convalescent sera may be the most important targets of neutralizing antibodies. (bCd) Examples of membrane panels probed with various serum samples and developed with peroxidase-labeled goat antihuman IgA; (b) M1P2 probed with control serum, acute as well as convalescent serum from case 2, convalescent serum from case 1, and serum from the deceased case. Notice the triad of spots recognized only in the serum of the two convalescent cases. The peptide sequences from Orf 1a are SDDYIKLNGP, DYIKLNGPLT, and IKLNGPLTVG. (c) P3M2 probed with acute and chronic serum from case 2 and serum from the deceased case. Panel 3 has peptides from S-protein. Notice the triad of spots recognized only by the convalescent serum. The peptide sequences from S-protein are FQPFQQFGRD, PFQQFGRDVS, and QQFGRDVSDF. (d) P4M3 probed with acute and convalescent serum from case 2 and convalescent serum from case 1. Notice the triad of spots recognized in the two convalescent sera. The peptide sequences from N-protein are QLPQGTTLPK, PQGTTLPKGF, and GTTLPKGFYA. Membranes were probed with pairs of acute and convalescent sera from four cases who recovered from a SARS infection. As controls, serum from one case that failed to survive, and one from a healthy, nonexposed volunteer was utilized. Samples were diluted 100-fold in buffer and then applied to a membrane for 1 h at 37 C. The membranes were developed with horseradish peroxidase-conjugated goat antibodies against human IgG, IgM, or IgA. Positive spots were then identified following treatment with ECL chemiluminescence reagents. Sera were tested for the presence of antibodies against SARS-CoV in a standard virus neutralizing test. Serial 2-fold dilutions of each serum from 1/8 to 1/1024 were incubated with 100 plaque-forming units of SARS-CoV (Tor-2 isolate, Marra et al., 2003) for 2 h and then added to monolayers of Vero E6 cells. The cultures were examined after 72 h for the presence of characteristic cytopathic effects. The dilution before the one at which cytopathic effects were first noted was recorded as the antivirus titer. All sera, Targapremir-210 which showed evidence of antivirus activity, were retested following heat treatment at 56 C for 30 min. This process confirmed the Rabbit Polyclonal to GLRB presence of neutralizing Targapremir-210 antibodies and eliminated the possibility of nonspecific, heat-sensitive factors contributing to the neutralizing activity. Results and discussion The results of neutralization assays for the various sera are shown in Table 1 , along with the age and sex of the respective patients. Acute sera were collected on presentation, and convalescent sera collected 1 month later in case 1, and at least 2 weeks later in cases 2, 3, and 4. The acute serum of case 3 had a neutralizing antibody titer of 1/8, which indicates the.

---