Clonacell hy medium d

A semisolid medium containing HAT (hypoxanthine, aminopterin, and thymidine) was used for hybridoma selection and cloning. Briefly, recovered cells were harvested (360×g at 25 °C for 7 min) and reconstituted (2×10⁷ cells/ml) in ClonaCell-HY medium C (STEMCELL, 03803). Cell solutions were then gently mixed with semisolid ClonaCell-HY medium D (STEMCELL, 03804) at a ratio of 1:9 (v/v) according to the ClonaCell-HY technical manual instructions. The cell mixture was plated (2.5 ml/well) into a 6-well cell culture plate (Nunc, 140,675) and incubated at 37°C in a 5% CO₂ humidified incubator for 7–10 days. Four to six hybridoma colonies were randomly picked by pipetting and transferred to each well (mini-pool) of a 96-well culture plate containing 200 µl of ClonaCell-HY medium E (STEMCELL, 03,805). Mini-pools were cultured for 2 days, and the culture supernatants were taken to determine the hPD-1-specific IgG levels by ELISA.

The animal studies were approved by the Institutional Animal Care and Use Committee at Chongqing Academy of Animal Sciences. CAMouse^HG mice were subjected to multiple subcutaneous points primed with the SARS-CoV-2 RBD protein (Sino Biological, Beijing, China) (100 μg/mouse) in complete Freund’ adjuvant (Sigma-Aldrich) plus 25 μg of CpG (Sangon Biotech, China) and 1% (v/v) Alhydrogel (vac-alu-50, In vivogen). The mice were then successively boosted with the SARS-CoV-2 RBD protein in incomplete Freund’s adjuvant (Sigma-Aldrich) plus CpG and Alhydrogel at 1 week interval. Four days after the last injection, spleen cells were isolated and mixed with SP2/0 cells at a ratio of 1:5 to obtain hybridoma cells by electrofusion. Electrofusion was performed using BTX ECM2001 (BTX, San Diego, CA) by application a single 40-μs pulses at 900 V. The fused cells were added into ClonaCell™-HY Medium D (STEMCELL Technologies) to a final concentration of 0.9 × 10⁷ cells/mL. and cultured at 37°C in a 5% CO2 incubator. After 8 days of culture, cell clones were picked and seeded into a 96-well plate. Cell supernatant was collected after 3-4 days culture for the further analysis.