Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification

Bumgarner, Gary W.; Zampell, Jamie C.; Nagarajan, Shanmugam; Poloso, Neil J.; Dorn, Amanda S.; D’Souza, Martin J.; Selvaraj, Periasamy

doi:10.1016/j.jbbm.2005.05.007

Cited by 12 publications

(7 citation statements)

References 14 publications

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“…Goat‐anti‐mouse‐FITC (Jackson Immunoresearch) was used as the secondary antibody. To determine HER‐2 specific IgG subtypes, a cell ELISA (Bumgarner et al, ; Cobern and Selvaraj ) using 5 × 10 4 D2F2/E2 cells per well with 50 μL of 1:100 diluted serum obtained from mice 7 days after boost was performed. The secondary antibody used was rat‐anti‐mouse‐IgG1‐HRP, rat‐anti‐mouse‐IgG2a‐HRP, or goat‐anti‐mouse‐IgG2b‐HRP (Southern Biotech) and the absorbance was measured at 450 nm.…”

Section: Methodsmentioning

confidence: 99%

Influenza virus‐like particles engineered by protein transfer with tumor‐associated antigens induces protective antitumor immunity

Patel

Vartabedian

Kim

et al. 2015

Biotech & Bioengineering

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Delivery of antigen in particulate form using either synthetic or natural particles induces stronger immunity than soluble forms of the antigen. Among naturally occurring particles, virus-like particles (VLPs) have been genetically engineered to express tumor-associated antigens (TAAs) and have shown to induce strong TAA-specific immune responses due to their nano-particulate size and ability to bind and activate antigen-presenting cells. In this report, we demonstrate that influenza VLPs can be modified by a protein transfer technology to express TAAs for induction of effective antitumor immune responses. We converted the breast cancer HER-2 antigen to a glycosylphosphatidylinositol (GPI)-anchored form and incorporated GPI-HER-2 onto VLPs by a rapid protein transfer process. Expression levels on VLPs depended on the GPI-HER-2 concentration added during protein transfer. Vaccination of mice with protein transferred GPI-HER-2-VLPs induced a strong Th1 and Th2-type anti-HER-2 antibody response and protected mice against a HER-2-expressing tumor challenge. Soluble form of GPI-HER-2 induced only a weak Th2 response under similar conditions. These results suggest that influenza VLPs can be enriched with TAAs by protein transfer to develop effective VLP-based subunit vaccines against cancer without chemical or genetic modifications and thus preserve the immune stimulating properties of VLPs for easier production of antigen-specific therapeutic cancer vaccines.

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Section: Methodsmentioning

confidence: 99%

Influenza virus‐like particles engineered by protein transfer with tumor‐associated antigens induces protective antitumor immunity

Patel

Vartabedian

Kim

et al. 2015

Biotech & Bioengineering

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“…The endogenous TEX101 protein present in pooled seminal plasma was used to calibrate the assay. Because limit of detection (LOD) of the immunoassay without seminal plasma pretreatment was not high enough, we examined literature (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) to identify conditions that would improve the assay sensitivity in seminal plasma. Thus, seminal plasma was subjected to pretreatment with the following detergents, salts and solvents: 1% SDS, 2-5% Triton X-100, 1% CHAPS, 2% sodium deoxycholate, 1 M urea, 3 M guanidine hydrochloride, 10% dimethyl sulfoxide, 10% dimethylformamide, 10% glycerol, 10% methanol, 10% acetonitrile.…”

Section: Production Purification and Analysis Of Recombinant Human mentioning

confidence: 99%

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids*

Korbakis

Brinc

Schiza

et al. 2015

Molecular & Cellular Proteomics

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Monoclonal antibodies that bind the native conformation of proteins are indispensable reagents for the development of immunoassays, production of therapeutic antibodies and delineating protein interaction networks by affinity purification-mass spectrometry. Antibodies generated against short peptides, protein fragments, or even full length recombinant proteins may not bind the native protein form in biological fluids, thus limiting their utility. Here, we report the application of immunocapture coupled with selected reaction monitoring measurements (immunocapture-SRM), in the rapid screening of hybridoma culture supernatants for monoclonal antibodies that bind the native protein conformation. We produced mouse monoclonal antibodies, which detect in human serum or seminal plasma the native form of the human testis-expressed sequence 101 (TEX101) protein-a recently proposed biomarker of male infertility. Pairing of two monoclonal antibodies against unique TEX101 epitopes led to the development of an ELISA for the measurement of TEX101 in seminal plasma (limit of detection: 20 pg/ml) and serum (limit of detection: 40 pg/ml). Measurements of matched seminal plasma samples, obtained from men pre-and post-vasectomy, confirmed the absolute diagnostic specificity and sensitivity of TEX101 for noninvasive identification of physical obstructions in the male reproductive tract. Measurement of male and female serum samples revealed undetectable levels of TEX101 in the systemic circulation of healthy individuals. Immunocapture-SRM screening may facilitate development of monoclonal antibodies and immunoassays against na-

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“…EPCR-GPI was purified from HEK-293 cells expressing N-terminal Histagged EPCR-GPI. Cells were harvested with citric saline (15 mM sodium citrate, 135 mM KCl) and cell pellets were lysed with 0.3 % saponin, 50 mM Tris pH 8.0 supplemented with EDTA-free protease inhibitor cocktail (Thermo Scientific) for 30 min on ice (39). Cell lysates were cleared by centrifugation for 30 min at 14,000g at 4 °C, diluted 1:10 in TBS, and EPCR-GPI was purified using Ni-NTA Sepharose (Invitrogen).…”

Section: Purified Proteinsmentioning

confidence: 99%

Cell painting with an engineered EPCR to augment the protein C system

Bouwens¹,

Stavenuiter²,

Mosnier³

2015

Thromb Haemost

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The protein C (PC) system conveys beneficial anticoagulant and cytoprotective effects in numerous in vivo disease models. The endothelial protein C receptor (EPCR) plays a central role in these pathways as cofactor for PC activation and by enhancing activated protein C (APC)-mediated protease-activated receptor (PAR) activation. During inflammatory disease, expression of EPCR on cell membranes is often diminished thereby limiting PC activation and APC’s effects on cells. Here a caveolae-targeting glycosylphosphatidylinositol (GPI)-anchored EPCR (EPCR-GPI) was engineered to restore EPCR’s bioavailability via “cell painting.” The painting efficiency of EPCR-GPI on EPCR-depleted endothelial cells was time- and dose-dependent. The EPCR-GPI bioavailability after painting was long lasting since EPCR surface levels reached 400% of wild-type cells after 2 hours and remained >200% for 24 hours. EPCR-GPI painting conveyed APC binding to EPCR-depleted endothelial cells where EPCR was lost due to shedding or shRNA. EPCR painting normalized PC activation on EPCR-depleted cells indicating that EPCR-GPI is functional active on painted cells. Caveolin-1 lipid rafts were enriched in EPCR after painting due to the GPI-anchor targeting caveolae. Accordingly, EPCR painting supported PAR1 and PAR3 cleavage by APC and augmented PAR1-dependent Akt phosphorylation by APC. Thus, EPCR-GPI painting achieved physiological relevant surface levels on endothelial cells, restored APC binding to EPCR-depleted cells, supported PC activation, and enhanced APC-mediated PAR cleavage and cytoprotective signaling. Therefore, EPCR-GPI provides a novel tool to restore the bioavailability and functionality of EPCR on EPCR-depleted and deficient cells.

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Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification

Cited by 12 publications

References 14 publications

Influenza virus‐like particles engineered by protein transfer with tumor‐associated antigens induces protective antitumor immunity

Influenza virus‐like particles engineered by protein transfer with tumor‐associated antigens induces protective antitumor immunity

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids*

Cell painting with an engineered EPCR to augment the protein C system

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