Identification of a common epitope in knottins and phospholipases D present in Loxosceles sp venom by a monoclonal antibody.

In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.

Acanthamoeba spp. monoclonal antibody against a CPA2 transporter: a promising molecular tool for acanthamoebiasis diagnosis and encystment study.

Free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa involved in opportunistic and non-opportunistic infection in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Both infections have challenging characteristics such as the formation of the resistant cysts in infected tissues, hampering the treatment and most usual diagnosis depending on time-consuming and/or low sensitivity techniques. The use of monoclonal antibodies presents itself as an opportunity for the development of more effective alternative diagnostic methods, as well as an important and useful tool in the search for new therapeutic targets. This study investigated the possibility of using a previously produced monoclonal antibody (mAb3), as a diagnostic tool for the detection of Acanthamoeba trophozoites by direct and indirect flow cytometry and immunofluorescence. Immunoprecipitation assay and mass spectrometry allowed the isolation of the antibody's target and suggested it is a transporter part of the CPA (cation: proton antiporter) superfamily. In vitro tests indicate an important role of this target in Acanthamoeba's encystment physiology. Our results support the importance of studying the role of CPA2 transporters in the context of acanthamoebiasis, as this may be a way to identify new therapeutic candidates.

Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential.

Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30-35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody's neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.

Biomolecular engineering of antidehydroepiandrosterone antibodies: a new perspective in cancer diagnosis and treatment using single-chain antibody variable fragment.

AIM: To develop a monoclonal antibody against dehydroepiandrosterone (DHEA) and miniaturize it, generating a single-chain antibody variable fragment (scFv) against DHEA as an adrenocortical carcinoma (ACC) marker. MATERIAL & METHODS: DHEA conjugated to keyhole limpet hemocyanin was used as an immunogen to obtain anti-DHEA hybridomas. Variable fragments were cloned from hybridoma 5B7 total RNA, and used to detect DHEA in normal adrenal tissue and ACC cells. RESULTS: IgM monoclonal antibody was highly specific, and the recombinant scFv preserved parental antibody characteristics, allowing tissue localization of DHEA. CONCLUSION: Undefined small lesions are challenges for clinicians and impact clinical adrenocortical tumor management. Generating an anti-DHEA scFv facilitates development of imaging tests for early diagnosis of pediatric ACC.

Sevelamer reduces endothelial inflammatory response to advanced glycation end products.

BACKGROUND: Advanced glycation end products (AGEs) have been related to the pathogenesis of cardiovascular diseases (CVD), chronic kidney disease (CKD) and diabetes mellitus. We sought to investigate the binding capacity of sevelamer to both AGEs and uremic serum in vitro and then test this pharmaceutical effect as a potential vascular anti-inflammatory strategy. METHODS: AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. Human endothelial cells were incubated in culture media containing AGEs and uremic serum with or without sevelamer. Receptor for advanced glycation end product (RAGE) expression was evaluated through immunocytochemistry and western blot to explore the interactions between AGEs and the endothelium. Inflammatory and endothelial dysfunction biomarkers, such as interleukin 6 (IL-6) and IL-8, monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and serum amyloid A (SAA) were also measured in cell supernatant. The chemotactic property of the supernatant was evaluated. RESULTS: AGEs significantly induced the expression of RAGE, inflammatory and endothelial activation biomarkers [IL-6, (P < 0.005); IL-8, MCP-1, PAI-1 and SAA (P < 0.001)] and monocyte chemotaxis as compared with controls. In addition, AGEs increased the levels of inflammatory biomarkers, which were observed after 6 h of endothelial cell incubation with uremic serum [IL-6 (P < 0.001) IL-8, MCP-1 and PAI-1 (P < 0.05)]. On the other hand, after 6 h of endothelial cell treatment with sevelamer, RAGE expression (P < 0.05) and levels of inflammatory biomarkers [IL-6 and IL-8 (P < 0.001), MCP-1 (P < 0.01), PAI-1 and SAA (P < 0.005)] significantly decreased compared with the AGEs/uremic serum treatment alone. CONCLUSIONS: Sevelamer decreased both endothelial expression of RAGE and endothelial dysfunction biomarkers, induced by AGEs, and uremic serum. Further studies are necessary for a better understanding of the potential protective role of sevelamer on uremic serum and AGEs-mediated endothelial dysfunction.

Synthetic peptides for in vitro evaluation of the neutralizing potency of Loxosceles antivenoms.

An important step in the development of therapeutic antivenoms is the pre-clinical testing using in vivo methods to assess their neutralizing potency. For spider antivenoms (Loxosceles species), horse serum potency against the necrotizing activities of Loxosceles intermedia crude venom is currently tested in rabbits. These procedures are time consuming and involve a large number of animals. The aim of this study was to develop an in vitro method to assess the neutralizing potency of anti-Loxosceles sera. We first demonstrated that it was not possible to establish a correlation between the ELISA antibody reactivity of horse anti-Loxosceles serum and their neutralizing potency. We then showed that the antivenoms recognized several peptide epitopes from different regions of SMase-D proteins, which are toxic antigens from Loxosceles venoms. The recognition of some peptides was observed only when high neutralizing potency sera was used. Based on these results, three peptides (peptide 1, DNRRPIWNLAHMVNA and peptide 3, DFSGPYLPSLPTLDA corresponding to residues 2-16 and 164-178, respectively, of SMase-1 protein from Loxosceles laeta, and peptide 2, EFVNLGANSIETDVS corresponding to residues 22-36 of A1H - LoxGa protein from Loxosceles gaucho and LiD1 protein from L. intermedia) were selected. The peptides were synthesized, coupled to bovine serum albumin (BSA), and used as antigens in indirect ELISA to test their reactivity with horse anti-Loxosceles serum of varying neutralizing potencies. We found certain assay conditions that discriminated between the high and low neutralizing potency sera. This study introduced an in vitro and peptide-based neutralization assay for anti-Loxosceles antivenoms.