All measurements were performed in 25C using HBS-EP (10?mM Hepes, 150?mM NaCl, 3.4?mM EDTA, 0.005% Minocycline hydrochloride (v/v) Tween-20) as running buffer. growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27??10?7?M [humAb10.1 (H5F6:5E8)], 5.46??10?9?M [humAb10.2 (H5F6:5F6)] and 4.34??10?9?M [humAb10.3 (H5E8:5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1?mg/ml [95% confidence interval (CI) 2.6C6.6?mg/ml], 6.9?mg/ml (CI 5.5C8.6?mg/ml) and 9.5?mg/ml (CI 5.5C16.4?mg/ml), respectively. Conclusion This report describes a platform for the isolation of human antibodies Minocycline hydrochloride from semi-immune blood donors by EBV transformation and their subsequent characterization after transient expression in plants. To our knowledge, the presented antibodies are the first humAbs LIF directed against MSP10 to be described. They recognize the EGF-like folds of MSP10 and bind these with high affinity. Moreover, these antibodies inhibit 3D7A growth in vitro. Keywords: EBV transformation, Human monoclonal antibodies, MSP10, Plant-based expression Background Malaria still claims over half a million victims each year and poses a significant burden to global health care and to the economy of endemic countries [1]. In Minocycline hydrochloride humans, semi-immunity to clinical malaria develops slowly due to high allelic variations in many immuno-relevant plasmodial antigens, is incomplete and wanes quickly without frequent reinfections [2C4]. Several lines of defense contribute to the immune response against the erythrocytic stages of plasmodia, e.g. innate-like V9:V2 T cells [5] and anti-plasmodial antibodies. The repertoire and spectrum of antibodies which eventually can prevent clinical episodes of malaria gradually develops with cumulative exposure to infections [6C8]. Passive transfer of naturally acquired antibodies has been shown to reduce parasitaemia in infected individuals [9, 10]. Such anti-plasmodial antibodies may mediate protection by prevention of re-invasion of merozoites into new erythrocytes [11], by antibody-dependent cellular inhibition mediated by monocytes [3, 12] and by antibody-dependent respiratory burst mediated by neutrophil granulocytes [13]. Most of the antibodies used for studies in the malaria field originate from rodents or rabbits. Unquestionably, these antibodies are valuable tools. However, they do not reflect the human immunoglobulin repertoire of semi-immune individuals. Malaria vaccine development is focusing on targets from the three major life cycle stages of the parasite in humans, the liver stage, the blood stage and the sexual stage. Sterile immunity, induced by experimental malaria infection, is mainly conferred by immune Minocycline hydrochloride responses against the pre-erythrocytic stages [14]. However, immune responses in natural infection in hyper- and holoendemic regions are primarily focused on the blood stage and the protection is incomplete [15]. Targets of this immune response mediating partial protection are mainly surface proteins of the merozoite stage. Members of the merozoite surface protein (MSP) family, the reticulocyte homology (Rh) and the erythrocyte-binding like (EBL) proteins as well as the apical membrane antigen 1 (AMA1) play a central role. All of these proteins are in the focus of vaccine candidate studies [16]. One of the most recently identified members of the MSP family is MSP10. MSP10 was first described by Black et al., but until today there is little known about the function of this protein and whether it is essential [17]. Under the assumption that the genome of encodes more than one MSP that contains a double epidermal growth factor (EGF)-like domain, Black et al. sought to identify potential homologs of MSP1. It appears that MSP1 and MSP10 have several features in common; i.e. they share a double EGF-like domain and a glycosylphosphatidylinositol (GPI) anchor at their C-terminus and both are mainly expressed during the later blood stages. Furthermore, MSP10 is also subject to proteolytic processing similar to MSP1, i.e. in the case of MSP10 starting from 30?h post invasion a product of 36?kDa can be detected besides the intact protein of 80?kDa. Puentes et al. aimed at finding out if certain parts of MSP10 are capable of binding to erythrocytes and to inhibit the invasion of merozoites into new erythrocytes [18]. Three MSP10-derived 20-mer peptides showed these properties, thus arguing for a role of MSP10 in the attachment, re-orientation and/or invasion. Nevertheless, human monoclonal antibodies (humAbs) directed MSP10 have neither been generated nor characterized yet. The aim of this work was to isolate MSP10-specific humAbs from semi-immune donors living in the Ashanti region in Ghana, a holoendemic area. Three humAbs, humAb10.1, humAb10.2 and humAb10.3, which are specific for either the first or the second EGF-like domain of MSP10, were isolated and characterized in detail. Methods Recombinant plasmodial.