A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

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All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animal immunization

1. Subcutaneous (s.c) inject female BALB/c mice, 6-8 weeks of age, with 50 µg of APN protein or phosphate-buffered saline (PBS, negative control) mixed with adjuvants once every 2 weeks. Use complete Freund's adjuvant that contains the heat-killed Mycobacteria for initial immunization, and incomplete Freund's adjuvant for booster immunizations. Mix equal volumes of APN protein (or PBS) and Freund's adjuvant or incomplete Freund's adjuvant, respectively.
2. Detect antibody titers against APN in the sera of these mice by indirect enzyme-linked immunosorbent assay (ELISA) using a microtiter plate coated with 5 µg/mL APN protein diluted in 0.05 M PBS (pH 9.6). 

2. Hybridoma technology to produce monoclonal antibodies against APN

1. Intraperitoneally (i.p.) inject 100 µg of APN protein into the selected mice for a final antigen boost.
2. Three days later, euthanize the mice using pentobarbital sodium (50 mg/kg, v/v, intraperitoneal) and cervical dislocation.
3. Collect spleens, and wash with DMEM twice to remove blood and fat cells. Filter the spleen-cell suspension using a 200-mesh copper grid to remove tissue debris, and harvest spleen cells using centrifugation (1500 × g, 10 min) to remove the membrane of the spleen.
4. Seed mouse myeloma SP2/0 cells in a 25 cm² flask containing 5 mL of Dulbecco's modified Eagle medium (DMEM) supplemented with 6% fetal bovine serum (FBS) and culture at 37 °C, 6% CO₂ atmosphere to maintain cell viability. After 5-6 days of culture, the cells reach 80%-90% confluence post-resuscitation and are in growth log phase. Under the microscope, the cells are round, bright, and clear.
5. One day before hybridization, collect macrophages from peritoneal cavities of the mice according to a previously published method.
6. Seed peritoneal macrophages at a density of 0.1-0.2 × 10⁵/mL in 96-well plates, each well containing 100 µL of HAT medium (DMEM supplemented with 10% FBS and 1x HAT Supplement), and incubate at 37 °C, 6 % CO₂ humidified atmosphere overnight.
7. For hybridization, gently aspirate SP2/0 cells with a pipette from 8-10 bottles, and suspend in 10 mL of serum-free DMEM medium. Wash cells with fresh DMEM, centrifuge (1500 × g, 10 min) twice, and then re-suspend in 10 mL of DMEM.
8. Mix the quantified spleen cells with SP2/0 cells at a ratio of 10:1 and transfer into 50 mL tubes. Centrifuge (1500 × g, 10 min) and discard the supernatant. Collect the cell pellets at the bottom of the tubes and tap with palm to loosen the pellets prior to hybridization.
9. Add 1 mL of polyethylene glycol 1500 (PEG 1500), pre-warmed to 37 °C, dropwise using a dropper to the loosened cell pellet over the time period of 45 s while gently rotating the bottom of the tube.
10. Slowly add 1 mL of DMEM pre-warmed to 37 °C to the above mixture over the period of 90 s, followed by another 30 mL of fresh DMEM. Place the fusion tube into a 37 °C water bath for 30 min.
11. After incubation in the warm bath, harvest the cells and re-suspend in HAT medium. Then culture in a 96-well plate inoculated with peritoneal macrophages.
12. Five days later, add 100 µL of fresh HAT medium to each well, and incubate the plate for an additional 5 days, after which replace the medium with HT medium (DMEM supplemented with 10% FBS and 1x HT Supplement).
13. Use a microtiter plate coated with 5 µg/mL APN protein diluted in 0.05 M PBS (pH 9.6) to analyze monoclonal antibodies in the hybridoma supernatant using ELISA assay.
    1. When the medium in the wells of the 96-well plate turns yellow (due to cell growth and metabolite release, pH in the medium decreases to 6.8, and phenol red turns from fuchsia to yellow) or cell clusters are observed, acquire 100 µL supernatant from the selected wells and add to the wells of the coated ELISA plate. Use a microplate reader to measure the OD₄₅₀ values.
    2. Use the polyclonal antibodies against APN and non-infected mouse serum as positive and negative control, respectively, and use PBS as blank control. In this study, OD₄₅₀ ratio of sample to negative control (P/N) ≥ 2.1 was recognized as positive selection standard.

자료

Name	Company	Catalog Number	Comments
Complete Freund's adjuvant	Sigma-Aldrich	F5881	Animal immunization
DMEM	Gibco	11965092	Cell culture
DMEM-F12	Gibco	12634010	Cell culture
Enhanced Cell Counting Kit-8	Beyotime Biotechnology	C0042	Measurement of cell viability and vitality
Fetal bovine serum	Gibco	10091	Cell culture
Geneticin™ Selective Antibiotic	Gibco	11811098	Selective antibiotic
HAT Supplement (50X)	Gibco	21060017	Cell selection
HT Supplement (100X)	Gibco	11067030	Cell selection
Incomplete Freund's adjuvant	Sigma-Aldrich	F5506	Animal immunization
Microplate reader	BioTek	BOX 998	ELISA analysis
Polyethylene glycol 1500	Roche Diagnostics	10783641001	Cell fusion
96-well microplate	Corning	3599	Cell culture