De Novo MS/MS Sequencing of Native Human Antibodies

详细信息    查看全文

• 作者：Adrian Guthals ; Yutian Gan ; Laura Murray ; Yongmei Chen ; Jeremy Stinson ; Gerald Nakamura ; Jennie R Lill ; Wendy Sandoval ; Nuno Bandeira
• 刊名：Journal of Proteome Research
• 出版年：2017
• 出版时间：January 6, 2017
• 年：2017
• 卷：16
• 期：1
• 页码：45-54
• 全文大小：501K
• ISSN：1535-3907

文摘

One direct route for the discovery of therapeutic human monoclonal antibodies (mAbs) involves the isolation of peripheral B cells from survivors/sero-positive individuals after exposure to an infectious reagent or disease etiology, followed by single-cell sequencing or hybridoma generation. Peripheral B cells, however, are not always easy to obtain and represent only a small percentage of the total B-cell population across all bodily tissues. Although it has been demonstrated that tandem mass spectrometry (MS/MS) techniques can interrogate the full polyclonal antibody (pAb) response to an antigen in vivo, all current approaches identify MS/MS spectra against databases derived from genetic sequencing of B cells from the same patient. In this proof-of-concept study, we demonstrate the feasibility of a novel MS/MS antibody discovery approach in which only serum antibodies are required without the need for sequencing of genetic material. Peripheral pAbs from a cytomegalovirus-exposed individual were purified by glycoprotein B antigen affinity and de novo sequenced from MS/MS data. Purely MS-derived mAbs were then manufactured in mammalian cells to validate potency via antigen-binding ELISA. Interestingly, we found that these mAbs accounted for 1 to 2% of total donor IgG but were not detected in parallel sequencing of memory B cells from the same patient.