Hypoxanthine aminopterin thymidine

Two 5-week-old (Balb/C) mice were immunized using the recombinant antigen. The experimental protocol was approved by the Institutional Animal Care and Use Review Committee of Bore Da BioTECH (BDIACUC20180221). After preparing the antigen at 1 mg/mL, an emulsion was prepared by mixing with complete Freund’s adjuvant (CFA; Sigma-Aldrich, Darmstadt, Germany) at a ratio of 1:1. The first immunization was performed on the sole of each mouse (100 µL). The second immunization was performed two weeks after the first immunization and then weekly, starting with the third immunization. From the second immunization to the fourth and final immunization, 75 µL was used. One week later, the mice were sacrificed by cervical dislocation, and the lymph nodes were removed. The extracted lymph nodes were fused with Sp2/O, a mouse myeloma cell line, using polyethylene glycol 1500 (Roche, Switzerland). To selectively culture the fused cells, hypoxanthine aminopterin thymidine (HAT, Sigma-Aldrich) medium was used. After preparing a 96-well plate and dispensing 200 µL/well, each plate was incubated at 37 °C in a CO₂ incubator.

Penicillin G (PNG), ampicillin (AMP), amoxicillin (AMX), azlocillin (AZL), oxacillin (OXA), piperacillin (PIPC), cefalothin (KF), cefazolin (CFZ), ceftiofur (CFT), cefepime (CFP), and cefixime (CFM) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Cloxacillin (CLX), dicloxacillin (DCX), cefoperazone (CPZ) and cefotaxime (CTX) were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Cefalonium (CFN), CTRX, cefapirin (CFAP), horseradish peroxidase (HRP), poly (ethylene glycol) (PEG) 1500, hypoxanthine aminopterin thymidine (HAT), incomplete Freund’s adjuvant (IFA), and complete Freund’s adjuvant (CFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Gibco BRL (Carlsbad, CA, USA). The 3,3′,5,5′–tetramethylbenzidine (TMB) and goat anti-mouse IgG were acquired from Jackson ImmunoResearch (West Grove, PA, USA). The HRP conjugation labeling kit was from Abcam (Toronto, Canada). Reagent grade solvents and salts were supplied by Beijing Chemical Reagent Co. (Beijing, China). The buffer solutions are listed in the Supplementary Materials.

Alachlor and 3-mercaptopropionic acid were purchased from Macklin (Shanghai, China). Pretilachlor, propisochlor, acetochlor, and metalaxyl were provided from TMRM (Beijing, China) and butachlor was purchased from Aladdin (Shanghai, China). PEG1450, Freund’s complete adjuvant (FCA), hypoxanthine-aminopterin-thymidine (HAT), Freund’s incomplete adjuvant (FIA), Ovalbumin (OVA), Bovine serum albumin (BSA), Peroxidase labeled goat anti-mouse immunoglobulin (HRP-IgG), and 1-Ethyl-3(3-dimethylaminopropyl) Carbodiimide (EDC) were obtained from Sigma (St. Louis, MO, USA), fetal bovine serum were provided from ExCell Bio (Beijing, China). Cell culture plates were obtained from NEST Biotechnology Co., Ltd. (Wuxi, China), N-hydroxysuccinimide (NHS), and other organic chemical reagents were provided from Sinopharm (Shanghai, China).
UV spectrophotometer (UV2600) and high-performance liquid chromatography (LC-20AD) from Shimadzu, Kyoto, Japan. The multifunctional enzyme labeling instrument was from PerkinElmer (Waltham, MA, USA).
All experimental animals used in this study were approved by the Animal Ethics Committee. Female Balb/C mice (6~8 weeks old) were provided by the Laboratory Animal Center of Huazhong Agricultural University.

Six to eight-week-old female BALB/c mice were obtained from the Animal Research Center of Soochow University. All animal experiments were approved by the Ethics Committee of Soochow University (approval number: SUDA20210918A02). B7-H5 mAb generation was performed as previously described [19 (link), 20 (link)]. Briefly, 1 × 10⁷ B7-H5 overexpressing L929 cells (Bright Scistar Biotechnology Co., Ltd., Suzhou, China) pretreated with mitomycin were intraperitoneally administered to BALB/c mice four times every 21 days. Then, the splenocytes of immunized mice were harvested and used to fuse with SP2/0 cells in the presence of 50% polyethylene glycol (PEG). The fusion cells were cultured with Dulbecco's modified Eagle's medium (DMEM, Biological Industries, BeitHaemek, Israel) containing hypoxanthine-aminopterin-thymidine (HAT; Sigma, St Louis, MO, USA) and 15% fetal bovine serum (FBS, Gibco, Grand Island, New York, USA). To establish hybridoma cell lines, supernatants from mixed cell cultures were screened by flow cytometry. Finally, the protein G sepharose affinity column (GE Healthcare Biosciences AB, Uppsala, Sweden) was used to purify the ascite antibodies.

Sterigmatocystin, aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁), G₂ (AFG₂), and M₁ (AFM₁), glycolic acid (H₂O ∼1%), dioxane (H₂O ≤0.01%), goat anti-mouse immunoglobulin horseradish peroxidase (IgG–HRP), mouse monoclonal antibody ISO2-1 kits, BSA (≥98%, agarose gel electrophoresis grade, art. no. A3675), complete Freund's adjuvants (CFA), incomplete Freund's adjuvants (IFA), urea-hydrogen peroxide (97%), 3, 3′, 5, 5′-tetramethylbenzidine (TMB), hypoxanthine/aminopterin/thymidine (HAT), hypoxanthine/thymidine (HT), and polyethylene glycol 1450 (PEG 1450, 50%) were purchased from Sigma-Aldrich (St. Louis, MO). N-hydroxysuccinimide (NHS) and N, N-dicyclohexylcarbodiimide (DCC) were purchased from Fluka. RPMI-1640 medium with l-glutamine and HEPES (free acid, 283.3 g/L) were obtained from HyClone. Fetal bovine serum, penicillin (+10,000 U/mL), and streptomycin (+10,000 µg/mL) were from Gibco. Unless otherwise stated, all other inorganic chemicals and organic solvents were of analytical-reagent grade or better. Water was obtained from a MilliQ purification system (Millipore).