Hypoxanthine aminopterin thymidine culture medium

A citrullinated peptide (CCP: HQCHQESTXGRSRGRCGRSGS; X=citrulline) and a noncitrullinated peptide (CRP: HQCHQESTRGRSRGRCGRSGS) were synthesized. The CCP synthetic peptide was intraperitoneally injected into four 6-week-old female BALB/c mice in conjunction with complete Freund's adjuvant (Chondrex, Redmond, WA, USA) to generate antibody to the CCP peptide. As a booster, the mice were injected using CCP diluted in phosphate-buffered saline (PBS) 4 and 8 weeks after the first immunization. After 3 days, B cells were isolated from the mouse that demonstrated the highest binding affinity for CCP via serum ELISA (method described below) and fused with myeloma cells (Sp2/0-Ag14) using polyethylene glycol (10 783 641001, Roche, Branchburg, NJ, USA).
The fused cells were cultured in 1 × hypoxanthine-aminopterin-thymidine culture medium (H0262, Sigma, St Louis, MO, USA). Cells displaying a positive ELISA test signal (see below) were transferred to a 24-well plate. After separating the cells into 96-well plates, they were cultured for 7–10 days in hypoxanthine and thymidine culture medium (11067-030, Gibco, Carlsbad, CA, USA) under 5% CO₂ at 37 °C in an incubator. Hybridoma cells were screened using ELISA (see below) and the cloning process was repeated until the final antibody secreting clone was selected.

A CCP (HQCHQESTXGRSRGRCGRSGS; X = citrulline) and a non-citrullinated peptide (NCP: HQCHQESTRGRSRGRCGRSGS) were synthesized. The synthetic CCP was mixed with complete Freund’s adjuvant and used to immunize four 6-week-old female BALB/c mice via injection into the abdominal cavity. For boosting, the mice were injected with CCPs diluted in phosphate-buffered saline (PBS) 4 and 8 weeks after the first immunization. Three days later, B cells were isolated from the mouse with the highest binding reactivity against the CCPs in serum, as measured by ELISA (details are provided in the method described below) and fused with myeloma cells (Sp2/0-Ag14) using polyethylene glycol (Roche, Basel, Switzerland).
The fused cells were then cultured in hypoxanthine–aminopterin–thymidine culture medium (Sigma, St. Louis, MO, USA). Cells showing a positive signal in the ELISA were transferred to a 24-well plate. After individual cells were placed into separate wells in 96-well plates, the cells were cultured for 7–10 days in hypoxanthine and thymidine culture medium (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) in a 5% CO2 incubator at 37°C. Hybridoma cells were screened by ELISA, and the cloning process was repeated until the final antibody-secreting clone was selected.