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.Дendritic cells (DC) are central players of the immune system. We have successfully used chemically crosslinked О±DEC-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. Successful conjugation of OVA to О±DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). The resulting О±DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4- 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). 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. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (О±DEC-205)В for in vivo DC targeting studies in mice. Chemical conjugation ofВ full-length protein to purified antibodies is one possible strategy. 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.Д®fficient 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. Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach inВ vaccine design.

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