Freund s complete incomplete adjuvant

Bovine type II collagen was purchased from Chondrex (Redmond, WA, USA). Acetic acid was purchased from Nanjing Chemical Reagents Co., LTD (Nanjing, Jiangsu, China). Freund's complete/incomplete adjuvant, methoxyamine hydrochloride, pyridine, 1, 2-¹³C-myristic acid, and N, O-Bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Both n-hexane and acetone were purchased from ROE Scientific (St. Louis, MO, USA). Methanol of MS grade was supplied by Merck Millipore (Billerica, MA, USA). ZT was purchased from Shanhaiguan Pharmaceutical Co., Ltd (Qinhuangdao, Hebei, China, batch number: 20161202). The quality of ZT was also evaluated and the method is shown in the Supplementary File. Carboxymethylcellulose sodium (CMC-Na; 300–800 mPa.S) was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Distilled water was purified using a Milli-Q purification system (Millipore, Milford, MA, USA).

Six-week-old male BALB/c mice (n = 2) were purchased from Koatech (Pyeongraek, Korea) and mice were injected subcutaneously with 50 μg of antigen mixed with 50 μl of Freund’s complete/incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) at three week intervals. Three weeks after 3^rd immunization, mice were anesthetized and cardiac puncture was performed for serum collection. All animal procedures performed in this study were reviewed, approved, and supervised by Kyung Hee University Institutional Animal Care and Use Committee (permit number: KHSASP-19-035).

The plasmid pET-32a-crp was transformed to E. coli BL21 (DE3) cells (Tiangen Biotech) with ampicillin selection. Recombinants were harvested after 6 h of induction with 1 mM IPTG (isopropyl b-D-1-Thiogalactopyranoside). The Crp protein was purified using 6× His/Ni-NTA affinity chromatography. To prepare the polyclonal anti-Crp antibody, two female New Zealand white rabbits were subcutaneously immunized with Crp protein (1 mg) adjuvanted with Freund’s complete/incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) four times at 14-day intervals. The titers of the antisera were then analyzed using the immune agar diffusion test. When the titers of the two rabbit antisera reached at least 1:32, blood samples were collected within approximately 14 days after the last immunization to obtain the anti-Crp antibody.

BSA, OVA, Freund’s complete/incomplete adjuvant, HEPES, L-Glu, HT/HAT (50×), PEG1450, and DMSO (bio-sterile grade) were purchased from Sigma, (St. Louis, MO, USA). AFB1, AFB2, AFM1, AFM2, AFG1, and AFG2 were purchased from Tianjin Alta. Double antibodies (penicillin) (100×), non-essential (100×), were purchased from Thermofisher, (Waltham, MA, USA). RPMI-1640 basal medium was purchased from Cytiva, (Beijing, China). Growth factors were purchased from Beijing Biodragon Immunotechnologies Co., Ltd, (Beijing, China). FBS was purchased from Zhejiang Tianhang Biotechnology, (Zhejiang Province, China). Goat anti-mouse IgG-HRP (2 mg/mL) was purchased from Shandong Lvdu Bio-science & technology CO., Ltd (Binzhou, China). AFB1-BSA was purchased from sigma, BeijingBoaolong Immunotech, USA, and provided by the Research Institute of China Agricultural University, respectively. The anti-AFB1 monoclonal antibody 1c11 was provided by the Research Institute of China Agricultural University and purchased from Shandong Lvdu Bio-science & technology Co., Ltd. The mouse myeloma cell line is SP2/0 provided by Shandong Fenghua Biological Co., Ltd, (Zibo, China). All other reagents were analytically pure. The 96-well polystyrene enzyme plates (3590) and cell culture plates (3599) were purchased from Costar, (New York, NY, USA).

Complete/incomplete Freund’s adjuvant (Sigma Aldrich, Shanghai, China) equal to the amount of purified His-Cap protein (100 μg/mouse) was added, fully emulsified, and subcutaneously inoculated into mice. The mice that were primed received two additional booster doses spaced two weeks apart. Serum samples were collected from immunized mice and an indirect ELISA was performed to determine the titer of antibodies specific to Cap protein. After three immunizations, a mouse with the highest antibody potency was selected for another immunization. The mice were then humanely executed, their splenocytes were fused with SP2/0 cells, and the treated cells were cultured in RPMI 1640 medium with HAT or HT for screening. Culture supernatants from individual hybridoma clones were detected using an indirect ELISA which used the differently labeled GST-Cap protein as an encapsulated antigen. Positive hybridomas were subcloned three times using limiting dilution. The resulting stable hybridomas were then inoculated into 10-week-old female BALB/c mice to prepare the ascites fluid. Ascites fluid was purified by protein G agarose (BioRad, Beijing, China) following the steps of the manufacturer’s instructions. Identification of heavy and light chains of mAbs was achieved using the mAb isotyping ELISA kit (Biodragon, Suzhou, China).

High glucose Dulbecco’s Modified Eagle Medium (DMEM), trypsin and penicillin–streptomycin, were obtained from Macgene (Beijing, China). Fetal bovine serum (FBS) was from PAN-Biotech (Aidenbach, Germany). Complete/Incomplete Freund’s adjuvant and LPS were from Sigma-Aldrich (St Louis, MO, USA). Bovine type II collagen was from Macklin (Shanghai, China). Methotrexate was obtained from Bidepharm (Shanghai, China). 4% paraformaldehyde was from Bioroyee Biotechnology (Beijing, China). EDTA and DAPI were from Solarbio (Beijing, China). Goat anti-rabbit secondary antibody, endogenous peroxidases blocker and goat serum albumin were from ZSGB-Biotech ((Beijing, China). PMA are purchased from Med Chem Express (MCE). Hoechst 33342 was obtained from TargetMol (Shanghai, China). Sytox Green was from Beyotime (Shanghai, China). Primary antibody of IL-6 was from Bioss (Beijing, China). Primary antibody of TNF-α was from Proteintech (Chicago, IL, USA). Primary antibodies against PI3K/p-PI3K were from Bioworld (St. Louis Park, MN, USA). Primary antibodies against p-Akt and p-mTOR were bought from Cell Signaling Technology (Beverly, MA, USA). Primary antibody against Akt was from Biogot (Nanjing, China). Antibodies of mTOR, MPO, CitH3 were from Abcam (Cambridge, UK).

We used standard hybridoma techniques to screen for specific anti-p61 MAbs (Chaithirayanon et al., 2002 (link)). Briefly, purified His-P61 protein was emulsified with equal volumes of complete/incomplete Freund's adjuvant (Sigma–Aldrich) at a final concentration of 0.25 mg/ml. Female BALB/c mice (6–8 weeks old) were subcutaneously immunized with purified His-P61 protein in 2 week intervals over 6 weeks to generate hybridoma lines that secreted antibodies. The spleen cells of immunized BALB/c mice were fused with mouse myeloma cells (SP2/0) 3 days after the last injection. The fused hybridoma clones were then screened by indirect enzyme-linked immunosorbent assays (ELISA) for mAbs exhibiting strong reactivity to the P61 protein. Selected clones that produced mAbs against the P61 protein were subcloned three times by limiting dilution. Ascites was induced in pristine-primed BALB/c mice. Finally, the mAbs were identified by Western blot and indirect ELISA analyses. Additionally, mAb subtypes were identified using a SBA Clonotyping System/HRP Kit (Southern Biotech, USA).