A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

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

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.

Materials

List of materials used in this article

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