Germline-reverted Stem Antibody Expression

Germline-reverted variants of stem antibodies or VRC-01 were synthesised (GeneArt) and used to express receptor IgM on the surface of Expi293 cells as previously described (17 (link)). 24 hours after transfection, cells were stained with AQUA Live/Dead amine reactive dye (Invitrogen) and binding to HA was assessed using PE-conjugated HAdSA probes derived from A/New Caledonia/20/1999 (13 (link)). Immunoglobulin surface expression was confirmed by staining transfected cells with anti-huIgM Cy5.5PerCP (BD Biosciences). The ability of IGHV1-69 alleles to differentially bind the anti-idiotypic monoclonal antibody G6 was confirmed by co-staining transfected cells with G6-Alexa Fluor 488 (Ax488).

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Wheatley A.K., Whittle J.R., Lingwood D., Kanekiyo M., Yassine H.M., Ma S.S., Narpala S.R., Prabhakaran M.S., Matus-Nicodemos R.A., Bailer R.T., Nabel G.J., Graham B.S., Ledgerwood J.E., Koup R.A, & McDermott A.B. (2015). H5N1 vaccine-elicited memory B cells are genetically constrained by the IGHV locus in the recognition of a neutralizing epitope in the HA stem. Journal of immunology (Baltimore, Md. : 1950), 195(2), 602-610.

Publication 2015

AQUA Live/Dead amine reactive dye

Alexa fluor 488 Alleles Amine Anti idiotypic antibody Antibodies Cells Germline variants Igm receptor Immunoglobulin Stem Transfection

Corresponding Organization :

Other organizations : National Institutes of Health, National Institute of Allergy and Infectious Diseases, Ragon Institute of MGH, MIT and Harvard, Sanofi (United States), AVEO Oncology (United States)

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Variable analysis

independent variables

Germline-reverted variants of stem antibodies or VRC-01

dependent variables

Binding to HA using PE-conjugated HAdSA probes derived from A/New Caledonia/20/1999
Immunoglobulin surface expression confirmed by staining transfected cells with anti-huIgM Cy5.5PerCP
Ability of IGHV1-69 alleles to differentially bind the anti-idiotypic monoclonal antibody G6 confirmed by co-staining transfected cells with G6-Alexa Fluor 488

control variables

Expression of receptor IgM on the surface of Expi293 cells as previously described (17 (link))

positive controls

None specified

negative controls

None specified

Annotations

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