Mouse immunoglobulin isotyping kit

Hybridomas producing monoclonal antibodies (MAbs) against TSPyV VP1 VLPs were generated essentially as described by Kohler and Milstein (1975) [37 (link)]. BALB/c mice were immunized with non-modified recombinant TSPyV VP1 VLPs with an adjuvant as described above. On day 28 after the 2nd immunization, the mouse with the highest TSPyV VP1-specific antibody titer was boosted with 50 μg of TSPyV VP1 VLPs in PBS. Four days later, spleen cells of the boosted mouse were fused with mouse myeloma Sp2/0 cells using PEG 1500 as a fusion agent (PEG/DMSO solution, HybriMax, Sigma-Aldrich). Hybrid cells were selected in growth medium supplemented with hypoxantine, aminopterin and thymidine (50× HAT media supplement, Sigma-Aldrich). Viable clones were screened by an indirect ELISA using TSPyV VP1 protein immobilized on the microtiter plates. Hybridoma cells were cultivated in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal bovine serum (FBS, Merck-Millipore, Darmstadt, Germany) and antibiotics. MAb isotypes were determined by ELISA using Mouse Immunoglobulin Isotyping Kit (BD Pharmingen, Franklin Lakes, NJ, USA). MAb specificity was tested by an indirect ELISA and Western blotting, as described below.

Isotyping for the constant gene of the antibodies was done with the Mouse Immunoglobulin Isotyping Kit (BD, 550026) following the manufacture’s protocol.

To generate hybridomas producing antibodies specific to TGF-β1-treated A549 cells, one million TGF-β1-treated A549 cells were injected into the left hind footpads of female BALB/c mice (DBL, Chungbuk, Korea) 3 days after the decoy cells (A549 cells) were injected into the right hind footpad of the same mice. The following procedures were carried out as described previously [28 (link)]. Briefly, lymphocyte fractions isolated from the left popliteal lymph nodes were fused to FO myeloma cells (ATCC) by polyethylene glycol 1500 (Roche, Seoul, Korea). The fused cells were maintained in DMEM (Corning, Seoul, Korea) supplemented with 20% FBS (Welgene), HAT supplement (Sigma-Aldrich), and antibiotic-antimycotic solution (Welgene). Hybridoma clones specific to TGF-β1-treated A549 cells were selected by flow cytometry. Isotype of antibodies secreted from hybridomas was analyzed with the Mouse Immunoglobulin Isotyping Kit (BD Biosciences, Seoul, Korea) according to the manufacturer’s instructions. Antibody purification was performed by using Protein G agarose column chromatography (Merck Millipore). All animal experiments were approved by the Institutional Animal Care and Use Committee at Sejong University (SJ-21051104).

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.

The immunoglobulin subclasses in mouse serum were measured using Mouse Immunoglobulin Isotyping Kit (BD Cytometric Bead Array, 550026).