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).
Clonotyping system hrp kit
Manufactured by Southern Biotech
Sourced in United States
The Clonotyping system-HRP kit is a lab equipment product designed for the analysis of T-cell receptor (TCR) repertoire. The kit provides reagents and protocols for the identification and quantification of specific TCR clonotypes within a sample.
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Lab products found in correlation
Sba clonotyping system horseradish peroxidase hrp kit, by Southern Biotech (1 mentions)
Goat anti mouse igκ hrp, by Southern Biotech (1 mentions)
Immunospot analyzer, by Cellular Technology (1 mentions)
Goat anti mouse igg1 hrp, by Southern Biotech (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Protein g column, by GE Healthcare (1 mentions)
2 protocols using clonotyping system hrp kit
1
Quantification of Isotype-Specific NP-Reactive Antibodies
2
Characterization of Monoclonal Antibody Binding
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Native reactivity and peptide binding of mAbs was evaluated in a preliminary competitive ELISA. mAbs in hybridoma supernatant competed with samples of human, rat or mouse serum and plasma, for biotinylated peptide (5 ng/mL), bound to a streptavidin coated microtitre plate. Clones were also tested against the free synthetic peptide (GAPGFRGPAG), a non-sense synthetic peptide, and a synthetic peptide elongated with one amino acid at the free N-terminal end (RGAPGFRGPAG). Isotyping of the mAbs was performed using the Clonotyping System-HRP kit, cat.5300-05 (Southern Biotech, Birmingham, AL, USA). The selected clones were purified using Protein G columns according to manufacturer's instructions (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK).
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