Sba clonotyping system horseradish peroxidase hrp kit

Total levels of immunoglobulins of different isotypes in the sera or culture supernatants were quantified using SBA Clonotyping system-horseradish peroxidase (HRP) kit (Southern Biotech, cat # 5300-05) according to the manufacturer’s recommendation. NP-specific IgM, IgG1, IgG2a, IgG2b, and IgG3 in the serum was measured using plates (Maxisorp, ThermoFisher, cat # 439454) coated with NP₂₀-BSA (Bioresearch Technologies, cat # N-5050H) and Clonotyping system-HRP kit (Southern Biotech). Pooled sera from NP-KLH-immunized WT mice, 14 days post immunization were used as standard control. ELISpot assays were performed for the detection of NP-specific ASCs. Multiscreen membrane filtration 96-well plates (EMD Millipore, cat # MAIPSWU10) were activated by 35% ethanol and coated with 25 µg/ml NP-BSA. Cells from spleen and bone marrow were seeded into wells with and incubated overnight at 37 °C. Wells were washed and developed using goat anti-mouse IgG1-HRP or goat anti-mouse Igκ-HRP (Southern Biotech, cat # 5300-05, 1:1000) and 3,3′-diaminobenzidine (Vectorlabs, cat # SK-4100). The spots were counted manually and photographed using ImmunoSpot analyzer (Cellular Technology Limited).

Proteins were expressed in E. coli BL21 (DE3) using previously described methods [34 (link)]. The GST-M fusion protein was purified using Glutathione Sepharose 4B (GE Healthcare, Amersham, UK) according to the manufacturer’s protocol. The mAbs against the M protein were prepared as previously described [35 (link)]. The SBA Clonotyping System-Horseradish Peroxidase (HRP) kit (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) was used to determine the IgG subtype of the mAbs.