Reference immunoglobulin mixtures

The immunoglobulin isotype of each mAb was determined using the Mouse Immunoglobulin Isotyping Kit (BD Pharmingen), according to the supplier's protocol. Rat anti-mouse IgGs, IgM, IgA, Igκ, and Igλ were used for coating a multiwell plate, and a hybridoma supernatant was applied into each well. The reference immunoglobulin mixtures (BD Biosciences) were used as positive controls. For antibody gene sequencing, total RNA was extracted from hybridoma cells using the easy-spin™ Total RNA Extraction kit (Intron), and cDNA was synthesized using the Maxime RT-PCR PreMix kit (Intron). To amplify the variable regions of heavy and light chains, PCR primers were used as described previously [32 (link)]. For heavy chain sequencing, two variable heavy chain forward primers were combined with an isotype-specific constant region reverse primer. For light chain sequencing, three κ variable light chain forward primers were combined with the corresponding constant region reverse primer.

The immunoglobulin isotype of each mAbs was determined by the Mouse Immunoglobulin Isotyping Kit (BD Pharmingen), according to the supplier's protocol. Rat anti-mouse IgGs, IgM, IgA, Igκ, and Igλ were used for coating multiwell plate, and hybridoma supernatant was applied into each well. The reference immunoglobulin mixtures (BD Biosciences) were used as positive controls. HRP-labeled rat anti-mouse immunoglobulin was added into each well, and the isotype signals were determined by optical density of 450 nm. For antibody gene sequencing, total RNA was extracted from hybridoma cells by Easy-spin™ Total RNA Extraction kit (Intron), and cDNA was synthesized by Maxime RT-PCR PreMix Kit (Intron). To sequence variable regions of antibody heavy and light chains, PCR primers were synthesized and used (Table 1) as described previously [28 (link)]. For heavy chain sequencing, two variable heavy chain forward primers were combined with an isotype-specific constant region reverse primer. For light chain sequencing, three κ variable light chain forward primers were combined with the corresponding constant region reverse primer. The PCR products were cloned into pBluescript KS(+) vector, and sequencing was proceeded.