Method Article

A Method to Produce Recombinant Antibodies Against Metallopeptidase

July 8th, 2025

In This Article

Abstract

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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 a procedure for the production of recombinant antibodies against metallopeptidase. Recombinant plasmids carrying genes for the antibody, green fluorescent protein, and antibiotic resistance are transfected into epithelial cells with lipofectamine. The transfected cells are selected and cultivated to generate recombinant antibodies.

Protocol

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1. Preparation of porcine aminopeptidase N (APN) protein antigen

NOTE: The pET28a (+)-APN-BL21 (DE3) strain and the APN stably expressed cells pEGFP-C1-APN-IPEC-J2 were constructed in a previous study.

  1. Recover bacteria from a frozen glycerol stock and streak onto Luria-Bertani (LB) plates containing 50 µg/mL kanamycin (Km+) for single colony isolation.
  2. Select a single colony from the freshly streaked plate, culture in 4 mL of LB medium (10 g/L tryptone, 10 g/L sodium chloride (NaCl) and 5 g/L yeast extract, pH 7.2) supplemented with Km+ (50 µg/mL), ....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Complete Freund's adjuvantSigma-AldrichF5881Animal immunization
DAPIBeyotime  BiotechnologyC1002Nuclear counterstain
DMEMGibco11965092Cell culture
DMEM-F12Gibco12634010Cell culture
Dylight 549-conjugated goat anti-mouse IgG secondary antibodyAbbkineA23310Indirect immunofluorescence analysis
Enhanced Cell Counting Kit-8Beyotime  BiotechnologyC0042Measurement of cell viability and vitality
Fetal bovine serumGibco10091Cell culture
Geneticinâ„¢ Selective AntibioticGibco11811098Selective antibiotic
HAT Supplement (50X)Gibco21060017Cell selection
HT Supplement (100X)Gibco11067030Cell selection
Incomplete Freund's adjuvantSigma-AldrichF5506Animal immunization
Isopropyl β-d-1-thiogalactopyranosideSigma-AldrichI5502Protein expression
KanamycinBeyotime  BiotechnologyST102Bactericidal antibiotic
Leica TCS SP8 STED confocal microscopeLeica Microsystems SP8 STEDFluorescence imaging
Lipofectamine® 2000 ReagentThermofisher11668019Transfection
LSRFortessaâ„¢ fluorescence-activated cell sortingBDFACS LSRFortessaFlow cytometry
Microplate readerBioTekBOX 998ELISA analysis
Micro spectrophotometerThermo FisherNano Drop oneNucleic acid concentration detection
NaClSinopharm Chemical Reagent10019308Culture broth
(NH4)2SO4Sinopharm Chemical Reagent10002917Culture broth
Opti-MEMGibco31985088Cell culture
Polyethylene glycol 1500Roche Diagnostics10783641001Cell fusion
PrimeScriptâ„¢ 1st strand cDNA Synthesis KitTakara BioRR047qPCR
Protein A agaroseBeyotime  BiotechnologyP2006Antibody protein purification
Protino® Ni+-TED 2000 Packed ColumnsMACHEREY-NAGEL745120.5Protein purification
SBA Clonotyping System-HRPSouthern BiotechMay-00Isotyping of mouse monoclonal antibodies
Seamless Cloning KitBeyotime  BiotechnologyD7010SConstruction of plasmids
Shake flasksBeyotime  BiotechnologyE3285Cell culture
Sodium carbonate-sodium bicarbonate bufferBeyotime  BiotechnologyC0221ACell culture
Trans-Blot SD Semi-Dry Transfer CellBio-rad 170-3940Western blot
TryptoneOxoidLP0042Culture broth
Ultrasonic HomogenizerNingbo Xinzhi BiotechnologyJY92-IINSample homogenization
Yeast extractOxoidLP0021Culture broth
96-well microplateCorning3599Cell culture

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Tags

Recombinant AntibodiesMetallopeptidase TargetingPlasmid TransfectionLipofectamine 2000Fluorescence Activated Cell SortingAntibiotic SelectionGreen Fluorescent ProteinChinese Hamster Ovary CellsSerum Free MediumG418 Selection

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