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dc.contributor.authorVolckmar, Julia
dc.contributor.authorKnop, Laura
dc.contributor.authorHirsch, Tatjana
dc.contributor.authorFrentzel, Sarah
dc.contributor.authorErck, Christian
dc.contributor.authorvan Ham, Marco
dc.contributor.authorStegemann-Koniszewski, Sabine
dc.contributor.authorBruder, Dunja
dc.date.accessioned2021-03-26T12:21:46Z
dc.date.available2021-03-26T12:21:46Z
dc.date.issued2021-02-05
dc.identifier.citationJ Vis Exp. 2021 Feb 5;(168). doi: 10.3791/62018.en_US
dc.identifier.pmid33616089
dc.identifier.doi10.3791/62018
dc.identifier.urihttp://hdl.handle.net/10033/622799
dc.description.abstractTargeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.en_US
dc.language.isoenen_US
dc.publisherMyJove Corporationen_US
dc.rightsAttribution-NonCommercial 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc/4.0/*
dc.titleChemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.en_US
dc.typeArticleen_US
dc.identifier.eissn1940-087X
dc.contributor.departmentHZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.en_US
dc.identifier.journalJournal of visualized experiments : JoVEen_US
dc.source.issue168
dc.source.journaltitleJournal of visualized experiments : JoVE
dc.source.countryUnited States


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