In vitro generation of anti-hepatitis B monoclonal antibodies from a single plasma cell using single-cell RT-PCR and cell-free protein synthesis

Journal of Bioscience and Bioengineering
Yunita SabrinaHideo Nakano

Abstract

Monoclonal antibodies (mAbs) are an effective tool in therapeutics and diagnostics. A novel approach called the single-cell RT-PCR-linked in vitro expression system (SICREX) enables the high-throughput generation and screening of mAbs from single B cells. In this paper, instead of using B cells, cDNAs were synthesized from single plasma cells of an immunized mouse spleen. The light chain (Lc) and the Fd portion of the heavy chain (Hc) genes of each cell were amplified separately and followed by overlapping PCR to add a T7 promoter, a ribosome-binding site, and a T7 terminator. The paired Lc and Hc genes were simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by ELISA to measure their affinity for the antigen. A Fab fragment with affinity against the antigen was obtained from plasma cells of an immunized mouse with hepatitis B surface antigen (HBsAg).

References

May 21, 1992·Nature·E Gherardi, C Milstein
Oct 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·S Rudikoff, J G Pumphrey
Sep 1, 1980·The Biochemical Journal·T M Florence
Aug 1, 1994·Bioscience, Biotechnology, and Biochemistry·T KajinoS Udaka
Jan 1, 1994·Annual Review of Immunology·G WinterH R Hoogenboom
Jul 23, 1996·Proceedings of the National Academy of Sciences of the United States of America·J S BabcookJ W Schrader
Dec 1, 1996·Methods : a Companion to Methods in Enzymology·J Phillips, J H Eberwine
May 13, 1997·Proceedings of the National Academy of Sciences of the United States of America·J Hanes, A Plückthun
Aug 15, 1997·Nucleic Acids Research·J BrownieS Little
Mar 7, 2000·Lancet·F C Breedveld
Jul 18, 2000·Molecular Pathology : MP·P N NelsonP G Murray
Sep 14, 2000·Proceedings of the National Academy of Sciences of the United States of America·E T BoderK D Wittrup
Oct 12, 2000·Nucleic Acids Research·J A CoronellaR P Junghans
Oct 18, 2000·Journal of Immunological Methods·X Wang, B D Stollar
Oct 17, 1959·British Medical Journal·M BURNET
Mar 31, 2004·Biotechnology and Bioengineering·Gang Yin, James R Swartz
Jul 21, 2004·Journal of Immunological Methods·Dasa Lipovsek, Andreas Plückthun
Aug 31, 2004·Journal of Clinical Pathology·K A SmithJ Greenman
Oct 8, 2004·The New England Journal of Medicine·Jack R Wands
Dec 22, 2004·Nature Immunology·Joseph W Brewer, Linda M Hendershot
May 17, 2006·Clinical and Experimental Pharmacology & Physiology·John M Luk, Kwong-Fai Wong

❮ Previous
Next ❯

Citations

Feb 24, 2016·Protein Engineering, Design & Selection : PEDS·Teruyo Ojima-KatoHideo Nakano
Sep 4, 2020·International Journal of Molecular Sciences·Annamaria SandomenicoMenotti Ruvo

❮ Previous
Next ❯

Related Concepts

Related Feeds

Bioinformatics in Biomedicine

Bioinformatics in biomedicine incorporates computer science, biology, chemistry, medicine, mathematics and statistics. Discover the latest research on bioinformatics in biomedicine here.

B cell Differentiation

Depending on the signal received through the B cell receptor and other receptors, B cells differentiate into follicular or marginal zone B cells. Here is the latest research pertaining to this differentiation process.