Mouse typerisotyping panel kit

Nucleic acid gel imaging system, nucleic acid electrophoresis apparatus and protein gel electrophoresis apparatus were purchased from Shanghai Tanon company. Microplate reader was purchased from Thermo Fisher Technology Ltd (Shanghai, China). Polyethylene glycol(PEG) was purchased from Merck Co, Mouse typerisotyping panel kit from Bio-RAD Co, and RPMI MEDIEM 1640 medium, penicillin-Streptomycin double antibody solution, newborn calf serum and HEPES from Life Technologies Gibco Co. Hypoxanthine-Aminopterin-Thymidine (HAT) supplemented medium, Hypoxanthine- Thymidine (HT) supplemented medium, Freund's complete or incomplete adjutants were purchased from Sigma. Natural human cystatin C (N-HCC) was purchased from Enzo Life Sciences Ltd., Horseradish peroxidase-conjugated goat anti-mouse IgG from Santa Cruz Biotechnology Inc., or Tween-20 and Bovine serum albumin (BSA) from Amresco. BALB/c mice were from Shanghai Institutes for Biological Nutrition, according to the ethical permission approved by the committee of Animal Ethical Evaluation, Chinese Academy of Science. The natural camel single-domain heavy chain antibody library was kindly provided by Dr. Ario de Marco for Italian IFOM-IEO center.

Ninety-six-well microplates (PolySorp, Nunc International, Denmark) were coated with deleted recombinant atypical CPB2 toxin, diluted in 50 mM carbonate–bicarbonate buffer pH 9.6, to a final concentration of 10 μg/ml per well and were incubated ON at 4 °C. After blocking with 1% yeast extract in PBS containing 0.05% Tween 20 (PBST), hybridoma supernatants were added into the wells, and the plates were incubated for 1 h at 37 °C. After washing with PBST, HRP-conjugated goat anti-mouse IgG (GE Healthcare) was added, and the plates were incubated for 30 min at RT. After further washings, 100 μl/well of the chromogen substrate 3,3′, 5,5′-tetramethylbenzidine (TMB) (SIGMA-ALDRICH) was added to each well for 20 min. The reaction was stopped with H₂SO₄ 0.5 M, and the OD₄₅₀ was measured.
The MAb isotype was determined using the Mouse-Typer^® Isotyping Panel kit (BIO-RAD) according to the manufacturer’s instructions.