Isostrip mouse monoclonal antibody isotyping kit

Immunization of BALB/c mice and generation of hybridomas producing anti-HBc mAbs were carried out as previously described [28 (link)]. Briefly, purified His-precore/core was injected into BALB/c mice using keyhole limpet hemocyanin as a carrier protein. Four weeks later, lymphocytes were isolated and fused to myeloma cell. Isotype determination was then performed using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit according to the manufacturer’s protocol (Roche Diagnostics, Basel, Switzerland). Purification of mAbs from hybridoma supernatant was performed using a Spin column based Antibody Purification Kit (Protein G) (Cosmo Bio Co., Ltd., Tokyo, Japan). The protein concentration was calculated using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific).

Hybridoma cells were grown in CD hybridoma medium AGT medium (Thermo Fisher Scientific, Rockford, IL, USA). Primary antibodies in the culture supernatant of each clone were separated by centrifugation at 8,000 rpm for 15 min and eluted with AcroSep Hyper DF columns (Pall, New York, NY, USA). Samples were then further concentrated 10–20-fold using Amicon Ultra centrifugal filters (Millipore, Bedford, MA, USA). Concentrations of purified IgG were determined by measuring the absorbance at OD₂₈₀. Immunoglobulin characterization was carried out using the IsoStrip mouse monoclonal antibody isotyping kit (Roche Diagnostics, Basel, Switzerland).

IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics) was used for the analysis. Isotype strips were immersed after instilling 1 mg/ml of the mAb 67-2 into a tube and stirring. After standing for 5 min, the isotype was determined based on the blue band on the strip.

C45 CTOSs were washed and suspended in HBSS (GIBCO). A 10-week-old female Balb/c mouse (CLEA Japan, Inc., Tokyo, Japan) was immunized by intraperitoneally injecting 1–2.5 × 10⁴ CTOSs. A boost injection was performed eight times at 1-week intervals. Three days after the final boost injection, spleen cells were harvested and fused to mouse myeloma cells (P3-X63Ag8.653) using polyethylene glycol (Sigma, St. Louis, MO). The fused cells were cultured with CM-B medium (EIDIA, Tokyo, Japan) and hybridoma cells selected by HAT selection. Clone 5G2 was cloned by limiting dilution (twice) and expanded with SF-B medium (EIDIA). Antibody was purified using the MAbTrap kit (GE Healthcare, Buckinghamshire, UK). The immunoglobulin class was determined using the IsoStrip mouse monoclonal antibody isotyping kit (Roche Diagnostics GmbH, Mannheim, Germany).

Isotyping of mAbs was carried out using the Isostrip Mouse Monoclonal Antibody Isotyping Kit (Roche). Hemagglutination inhibition titres were determined using a stock solution of 1 mg/ml of mAb, determined by spectroscopy and confirmed by SDS gel electrophoresis, as previously described (4HA units/25 uL, 1% chicken red blood cells in PBSa)52 . Microneutralization assays were performed in quadruplicate using a standard solution of 1 mg/ml of monoclonal antibody as described elsewhere52 . Linearity of epitopes was determined by running an excess of purified UDL1/08 virus against an excess of mAb on a standard reducing Western blot, presence of a band indicated the epitope recognised was linear. Positive control polyclonal anti-H9HA chicken antiserum was generated by immunizing chickens with rAd-H9HA in a similar manor to mice as detailed above.

Selected cloned hybridomas were cultured in RPMI 1640 medium supplemented with 10% IgG-depleted fetal bovine serum in a 1-L Erlenmeyer flask (Corning®) with stirring at 80 rpm for 4 days at 37°C and 5% CO₂ in an agitator. Clone supernatant cultures were collected and canine CD22-specific antibodies were purified over a HiTrap Protein G HP column (GE Healthcare, ref 29-0485-81). Briefly, supernatants from hybridoma cultures were diluted in phosphate buffer (1:1) to adjust the pH to 7. After passage through the column, antibodies were eluted using a glycine-HCl buffer pH 2.7 and dialyzed overnight against PBS pH 7.4 using 30,000 MWCO dialysis cassettes (Thermo Scientific). Purified antibodies were filtered through 0.2-μm filters and stored at 4°C and their production yields were determined.
Isotypes and light chains of purified antibodies were characterized using the IsoStrip™ Mouse Monoclonal Antibody Isotyping Kit (Roche, ref 11493027001) according to the kit instructions.

Eight-week-old female BALB/c mice were immunized every 2 weeks for a total of six times and then boosted twice in a week. Fifty micrograms of antigen was prepared with an equal volume of TiterMax Gold adjuvant (Sigma-Aldrich) according to the manufacturers’ instructions. Four days after boosting, the splenocytes of immunized mice were collected and fused with SP2/O myeloma using electro cell fusion generator ECFG21 (Nepa Gene) according to the manufacturers’ instructions. The fused cells were cultured in GIT/IL-6/HT supplements and aminopterin medium [GIT medium (FUJIFILM Wako) supplemented with recombinant human interleukin-6 (IL-6) (1 ng/ml; PeproTech), hypoxanthine thymidine (HT) supplement (Gibco), and 0.4 μM aminopterin (Sigma-Aldrich)] for 1 week to select hybridomas. We performed enzyme-linked immunosorbent assay, WB, and IP to screen hybridomas using culture supernatant. Serial dilution was performed to monoclonize selected hybridomas. Monoclonal hybridomas were cultured in GIT medium (FUJIFILM Wako) supplemented with IL-6 (1 ng/ml) for antibody production. The isotype of antibodies was determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche). The animal experiments were approved by the Animal Care and Use Committee of Keio University and were conducted in compliance with the Keio University Code of Research Ethics.