HIV p24 Antigen Expression and Purification

DNA for HIV gag-p24 (aa 133–363 derived from HIV isolate BH10) was cloned in frame into the COOH terminus of the heavy chains of α–mouse-DEC205 as described previously (19 (link)). The fusion α–CD205-p24 mAb was produced by transient transfection (calcium phosphate) in 293T cells in serum-free DMEM supplemented with Nutridoma SP (Roche Applied Science). The mAbs were purified on protein G columns (GE Healthcare Bio-Sciences Corp.) and characterized by SDS/PAGE and Western blotting using α-mouse IgG1-HRP (Southern Biotech) or HRP-α-gag-p24 (ImmunoDiagnostics). mAb binding was verified on CHO cells stably transfected with the respective receptor by FACS using phycoerythrin-conjugated goat α-mouse IgG (Jackson ImmunoResearch). Unconjugated α–DEC-205 mAb expressed by stably transfected CHO cells was similarly purified. Recombinant NYVAC-encoding HIV BX08gp120-IIIBGag/Pol/Nef is a replication incompetent vector, kindly provided by Dr. Giuseppe Pantaleo (Centre Hospitalier Universitaire Vaudois (CHUV) Lausanne, Switzerland) and prepared as previously described (75 (link)).

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Ruane D., Do Y., Brane L., Garg A., Bozzacco L., Kraus T., Caskey M., Salazar A., Trumpheller C, & Mehandru S. (2016). A Dendritic cell targeted vaccine induces long term HIV-specific immunity within the gastrointestinal tract. Mucosal immunology, 9(5), 1340-1352.

Publication 2016

Nutridoma SP protein G columns α-mouse IgG1-HRP phycoerythrin-conjugated goat α-mouse IgG

293t cells Calcium phosphate Cd205 Cho cells Dec 205 Frame Goat Igg1 Immunodiagnostics Mouse P24 hiv Phycoerythrin Protein g Replication Sds page Serum Transfection Transient Vector

Corresponding Organization : Rockefeller University

Other organizations : Ulsan National Institute of Science and Technology, Oncovir (United States)

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Variable analysis

independent variables

The fusion α–CD205-p24 mAb

dependent variables

MAb binding on CHO cells stably transfected with the respective receptor, as verified by FACS using phycoerythrin-conjugated goat α-mouse IgG

control variables

Unconjugated α–DEC-205 mAb expressed by stably transfected CHO cells

controls

Positive control: Unconjugated α–DEC-205 mAb expressed by stably transfected CHO cells
Negative control: Not explicitly mentioned

Annotations

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