The study was supported by grants from the Research Council of Norway and the University of Oslo. Conflict of interest disclosure B.B. various applications. The data suggest that the targeted DNA vaccine format can be utilized to enhance the number of Ab-producing hybridomas and thereby be a tool to improve the B cell hybridoma technology. Introduction The need for monoclonal antibodies (mAbs) both for scientific purposes and for treatment of patients is expanding. Since 1975, when K?hler and Milstein [1, 2] developed B cell hybridoma technology, several approaches have been tested with the aim to improve the immunization procedure and optimize the immune response . The basic methodology is based on multiple injection of protein antigen with or without adjuvant. Different adjuvants have been tested, and the protocols have been shortened by introducing single-step immunization and by using delivery systems securing sustained delivery (liposomes, polymers and virus particles) [4, 5]. The latest development is multiplex immunization that includes injection of several protein antigens in one mixture together with adjuvant followed by boost immunization [3, 6, 7]. By use of this procedure, several mAbs with specificity to distinct antigens can be generated in one animal. In other protocols, DNA encoding the antigen has been delivered into the muscle, peritoneum, spleen or veins, but only intrasplenic injection, when administering only a single dose, induced Abs and supported the generation of viable mAb-producing Mirodenafil dihydrochloride hybridomas [8, 9]. Mouse monoclonal to TNK1 Furthermore, codon optimization, fusion of the antigen Mirodenafil dihydrochloride to immune stimulatory sequences, as well as co-delivery of plasmids encoding adjuvant sequences have been performed to enhance the responses following genetic (DNA) immunization . DNA injection has also been potentiated by using electroporation immediately after delivery [11, 12] and by prime-boost strategies (DNA injection followed by injection of protein antigen) . Although DNA immunization holds great promise, low immunogenicity is a challenge. To enhance immune responses, we have generated a novel type of immunoglobulin (Ig)-based molecules (denoted vaccibodies) that are homodimers. These molecules have been bivalently targeted to antigen-presenting cells (APCs) by N-terminal expression of single-chain variable fragment (scFv) specific for mouse major histocompatibility complex (MHC) class II , CD40 , Toll-like receptor 2  or natural ligands such as chemokines (CCL3/MIP-1 and CCL5/RANTES) . In Mirodenafil dihydrochloride the C-terminal end, the APC-targeted vaccine molecules carry large bivalent antigens with intact B cell determinants [14, 17] for induction of potent T and B cell responses. Mirodenafil dihydrochloride Similar to complete Abs , vaccibodies [14, 17] may be delivered by injection of plasmid DNA into the dermis or skeletal muscle followed by electroporation. Transfected cells synthesize and secrete vaccibodies that can be found in serum and that target APC . In this study, we introduced the fluorescent protein (FP) mCherry  in the antigenic unit of the vaccibody molecule and utilized the natural ligand MIP-1 or scFv specific for MHC class II to target APCs. First, we generated mAbs against the antigen mCherry by two DNA immunizations performed with electroporation followed by a boost immunization with mCherry protein. Next, we tried to simplify the protocol by immunizing just once by delivering DNA into the muscle before electroporation. We observed that mCherry in the vaccibody format elicited a higher number of Ab-producing hybridomas compared with delivery of mCherry alone. The mAbs generated in the study were tested in different applications, and we found that they are usable in ELISA, Western blotting, immunocytochemistry and flow cytometry. Based on our findings, we suggest that the protocols for generating mAb-producing hybridomas can be shortened and improved by use of the targeted DNA vaccine format. We further hypothesize that the DNA vaccine format can overcome the challenges posed by weak and difficult-to-purify antigens. Materials and methods Mice Female BALB/mice were purchased from.