The calibrated Ku-0059436 mouse standard serum 89-SF was absorbed with 10 μg/mL CWPS. At least two internal controls were added to each plate. For serotypes 14 and 19F, a high-titre and a low-titre serum sample from
pneumococcal vaccine responders were used as internal controls, respectively. For serotype 23F, two high-titre and two low-titre serum samples from pneumococcal vaccine responders were used as internal controls. The coefficients of variation (CVs) for the high-titre controls were 19.53 and 16.55% for serotypes 14 and 19F and the CVs were 18.34 and 17.91% for serotype 23F. The CVs for the low-titre controls were 17.12 and 14.91% for serotypes 14 and 19F and the CVs were 15.39 and 14.23% for serotype 23F. Sera from persons with AIDS who had no antibodies to S. pneumoniae serotype 14, 19F, 23F or 6B capsules and <1 μg of antibody to CWPS per mL were used as negative controls. Capsular polysaccharides from S. pneumoniae serotypes 14 (catalogue number 197-X; lot #2038310), 19F (catalogue number 205-X; lot #2033178), 23F (catalogue number 217-X; lot #2048448) and 6B (catalogue number 225-X; lot #2045655) were obtained from the American
Type Culture Collection (ATCC; Manassas, VA, USA). These capsular polysaccharides were suspended in phosphate-buffered saline (PBS; pH 7.4) containing 0.02% NaN3 at a concentration of 10 μg/mL and used directly to coat wells
Selleck SB203580 by incubation at 4 °C overnight. After washing of the antigen-coated plates, the pre-absorbed patient sera or controls were added to plates and incubated at room temperature for 2 h. After thorough washing to remove antibodies not bound to the wells, horseradish peroxidase (HRP)-conjugated goat antibody to human IgG (Zymed Laboratories Inc., San Francisco, CA, USA) at a dilution of 1:2000 was used to detect IgG, and the reaction was developed for 10 min in the dark by the addition of K-blue substrate (Neogen Rucaparib research buy Corporation, Lexington, KY, USA), followed by the addition of 1N sulphuric acid to stop the reaction. Between incubations, all washings were performed with Tris buffered saline (TBS) buffer containing 0.1% Brij solution. Optical density was read in an ELISA reader (SpectraMAX 340; Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm, with subtraction of the optical density of the appropriate blank. The antibody concentrations were calculated relative to the 89-SF control serum, using the US Centers for Disease Control and Prevention (CDC) program elisa for Windows (version 2.15) . The detection limits for our assay for 14, 19F, 23F and 6B, determined using the control serum 89-SF, were 0.02, 0.015, 0.02 and 0.50 μg/mL, respectively.