A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning

Sara Carbonetti, Brian G. Oliver, Vladimir Vigdorovich, Nicholas Dambrauskas, Brandon Wilder, Emilee Bergl, Stefan H.I. Kappe, D. Noah Sather

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40 years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12 days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4 days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens.

Original languageEnglish (US)
Pages (from-to)66-73
Number of pages8
JournalJournal of Immunological Methods
Volume448
DOIs
StatePublished - Sep 1 2017
Externally publishedYes

Fingerprint

Organism Cloning
B-Lymphocytes
Monoclonal Antibodies
Antigens
Infectious Disease Medicine
Interferometry
Polymerase Chain Reaction
Protein Array Analysis
CD40 Ligand
Modern 1601-history
Falciparum Malaria
HEK293 Cells
Conditioned Culture Medium
Coculture Techniques
Allergy and Immunology
Reverse Transcription
Antibody Formation
Ligation
Parasites
Complementary DNA

Keywords

  • Antibody
  • B cell
  • Cloning
  • FACS
  • Monoclonal
  • RACE
  • Recombinant

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning. / Carbonetti, Sara; Oliver, Brian G.; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Wilder, Brandon; Bergl, Emilee; Kappe, Stefan H.I.; Sather, D. Noah.

In: Journal of Immunological Methods, Vol. 448, 01.09.2017, p. 66-73.

Research output: Contribution to journalArticle

Carbonetti, Sara ; Oliver, Brian G. ; Vigdorovich, Vladimir ; Dambrauskas, Nicholas ; Wilder, Brandon ; Bergl, Emilee ; Kappe, Stefan H.I. ; Sather, D. Noah. / A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning. In: Journal of Immunological Methods. 2017 ; Vol. 448. pp. 66-73.
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