High throughput generation of antibody secreting B-cell clones from peripheral blood of rabbits
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1
Roche Diagnostics GmbH, Germany
We have developed a robust high throughput B-cell cultivation method using peripheral rabbit B cells. This rapid procedure generates a diverse set of antibodies using blood of immunized rabbits. The entire workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (v) the recombinant expression and purification of IgG antibodies. Rabbits immunized with the human IL1RL1 antigen (also known as ST2, DER4, FIT-1, IL33R, ST2L, ST2V, T1) were used for the proof-of-concept study. The IL33-IL1RL1 ligand-receptor system plays an important role in autoinflammatory diseases such as asthma, ulcerative colitis or arthritis. The fully integrated and to a large degree automated platform yielded clonal and very diverse IL1RL1-specific antibodies. Due to the high IgG productivity of the cultivated B-cell clones the IL1RL1-inhibiting IgGs from individual animals could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. In summary, this powerful B-cell cultivation method has proven to be an efficient and robust tool for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.
Acknowledgements
We would like to express our sincere gratitude and thanks for expert technical assistance to Sabrina Kettenbach and Marco Friedrich (immunization of rabbits), Daniela Buntefuss (preparation of rbTSN), Basile Siewe and Ireneus Jonetzko (B-Cell Cloning), Manuela Paul and Sven Kroemmelbein (screening, biochemical assays), Dominique Ostler, Simone Hoege (B-Cell PCR), Peter Kern (in vitro NK-cell IL1RL1 inhibition assay), and Florian Lipsmeier (statistical analyses).
Keywords:
B-cell cultivation,
antibody production,
high throughput,
rabbit,
Peripheral Blood,
B lymphocyte,
costimulation,
B-cell PCR,
IL1RL1,
peripheral B cell
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Immune receptors and signaling
Citation:
Offner
S,
Seeber
S,
Ros
F,
Thorey
I,
Tiefenthaler
G,
Kaluza
K,
Lifke
V,
Fischer
J,
Klostermann
S,
Platzer
J and
Kaluza
B
(2013). High throughput generation of antibody secreting B-cell clones from peripheral blood of rabbits.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.01170
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Received:
30 Jul 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Dr. Sonja Offner, Roche Diagnostics GmbH, Penzberg, Germany, sonja.offner@roche.com