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).

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