Biotin conjugated mouse anti human igg1

Measurement of IgG subclass-specific pneumococcal antibodies was performed on sera collected from infants at 5 months of age using a previously described binding antibody multiplex assay [26 (link)]. Briefly, the 13 pneumococcal serotypes tested above were coupled to carboxylated fluorescent beads (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the DMTMM-coupling protocol [27 (link)]. The coupled beads were incubated with diluted plasma (at 1:100 dilution) for 30 minutes at 20°C. Antigen-specific subclass response was detected using biotin-conjugated mouse anti-human IgG1 (BD Pharmingen, San Diego, CA, USA; 4 μg/L), IgG2 (Southern Biotech, Birmingham, AL, USA; 5 μg/mL), IgG3 (Calbiochem, San Diego, CA, USA; 2 μg/mL), or IgG4 (BD Pharmingen; 2 μg/mL) and tertiary detection agent streptavidin-phycoerythrin (BD Biosciences, Franklin Lakes, New Jersey, USA) at 5 μg/mL. Beads were washed and read on a Bio-Plex 200 instrument (Bio-Rad Laboratories, Inc.). IgG levels were expressed as mean fluorescence intensity (MFI) and all MFI values were corrected by subtracting the intensity of a blank control. IgG levels below 100 MFI were considered to be below the lower limit of quantitation for the assay. Consistency between assays was measured by tracking the 50% effective concentration, MFI, and area under the curve of the positive control (007SP) by Levey-Jennings charts.