A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase
Published: March 29, 2024
Abstract
Source: Xia, P., et al. Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium. J. Vis. Exp. (2021).
This video demonstrates an assay to produce monoclonal antibodies against porcine aminopeptidase N (APN), a membrane-bound metallopeptidase, using a traditional hybridoma technology. Upon immunizing a mouse with APN, the spleen cells are harvested and fused with immortal myeloma cells to produce hybridomas. The hybridomas are then selected and proliferated using the HAT (hypoxanthine-aminopterin-thymidine) medium to produce monoclonal antibodies specific to different APN epitopes.
Protocol
All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Animal immunization 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 Freu…
Declarações
The authors have nothing to disclose.
Materials
| 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 |
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A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase. J. Vis. Exp. (Pending Publication), e22087, doi: (2024).