Generation Of Novel Monoclonal Antibodies Research Articles

A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins.

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL−1 and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for β-amanitin (β-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.

Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component.

The non-hemolytic enterotoxin complex (Nhe) is supposed to be the main virulence factor of B. cereus causing a diarrheal outcome of food poisoning. This tripartite toxin consists of the single components NheA, -B and -C all of them being necessary for maximum toxicity. In the past, research activities aiming to elucidate the mode-of-action of Nhe were mostly focused on the B- and C-component. In this study the generation of novel monoclonal antibodies (mAb) and their thorough characterization enabled the determination of key features for NheA. By the means of immunoaffinity chromatography it could be shown that NheA does not interact with -B and -C in solution. Additionally, the establishment of a highly sensitive sandwich-EIA now enables the detection of NheA in B. cereus supernatants down to 20 pg ml-1.Peptide-based epitope mapping in combination with partially deleted recombinant NheA fragments allowed the allocation of the binding regions for the three mAbs under study. Furthermore, by different EIA set-ups the conformational flexibility of NheA could be shown. For two of the antibodies under study different mechanisms of NheA neutralization were proven. Due to prevention of complete pore formation by one of the antibodies, NheA could be detected in an intermediate stage of the tripartite complex on the cell surface. Taken together, the results obtained in the present study allow a refinement of the mode-of-action for the Nhe toxin-complex.

Progress towards a broadly neutralizing vaccine against the asexual blood-stage of Plasmodium falciparum

Event Abstract Back to Event Progress towards a broadly neutralizing vaccine against the asexual blood-stage of Plasmodium falciparum Alexander D. Douglas1*, Andrew R. Williams1, Joseph J. Illingworth1, Kathryn A. Hjerrild1 and Simon J. Draper1 1 University of Oxford, Jenner Institute, Nuffield Department of Medicine, United Kingdom There is a need for a more effective vaccine against P. falciparum than the current leading candidate, RTS,S. Vaccines against the parasite’s asexual blood-stage may reduce mortality, morbidity and transmission of malaria, but face problems of antigenic polymorphism and the apparent requirement for exceptionally high antibody levels to achieve protection. We recently reported that vaccines based upon the blood-stage merozoite antigen PfRH5 induce antibodies capable of strain-transcending in vitro growth inhibition activity (GIA). Here we will now describe cross-strain protection achieved by these vaccines in the stringent Aotus nancymaae non-human primate – P. falciparum challenge model, which correlates strongly with vaccine-induced pre-challenge GIA and total IgG anti-PfRH5 ELISA titres. This is the first example of clinically meaningful cross-strain protection of Aotus by a human-compatible blood-stage vaccine regime, and supports the strategy of vaccine selection using in vitro GIA. We will also describe work to characterise the mechanism of parasite neutralisation by anti-PfRH5 antibodies. This includes generation of novel monoclonal antibodies with neutralisation potency among the highest described for any anti-merozoite antibody, and mapping of minimal linear epitopes for two of these. We will present data which suggest that fine epitope specificity and interaction kinetics are more important determinants of neutralisation potency than ability to block the interaction between PfRH5 and its receptor, basigin. These data shed important insight on the mechanism of blood-stage malaria parasite neutralisation, and support the continued clinical development of PfRH5-based vaccines. Phase Ia clinical trials of PfRH5 vaccine candidates are due to commence in Oxford in 2014. Keywords: Malaria, Falciparum, Malaria Vaccines, PfRh5, Antibodies, viral vector vaccines Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Douglas AD, Williams AR, Illingworth JJ, Hjerrild KA and Draper SJ (2013). Progress towards a broadly neutralizing vaccine against the asexual blood-stage of Plasmodium falciparum. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00574 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Alexander D Douglas, University of Oxford, Jenner Institute, Nuffield Department of Medicine, Oxford, OX3 7DQ, United Kingdom, sandy.douglas@ndm.ox.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alexander D Douglas Andrew R Williams Joseph J Illingworth Kathryn A Hjerrild Simon J Draper Google Alexander D Douglas Andrew R Williams Joseph J Illingworth Kathryn A Hjerrild Simon J Draper Google Scholar Alexander D Douglas Andrew R Williams Joseph J Illingworth Kathryn A Hjerrild Simon J Draper PubMed Alexander D Douglas Andrew R Williams Joseph J Illingworth Kathryn A Hjerrild Simon J Draper Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer

Arrest defective 1 (ARD1) is an acetyltransferase involved in cell cycle control in yeast. ARD1 interacts with human N-acetyltransferase (NATH) to form a functional N-terminal acetyltransferase complex. Recently it had been linked with proliferation and apoptosis in mammalian cells, but its function in cancer development remains unclear. To evaluate significance of ARD1 expression in human colorectal cancer, we generated a panel of monoclonal antibodies (mAbs) with high specificity and sensitivity against ARD1. All of the 10 different clones could be used in ELISA and Western blot, and clone 10C12, 13G2, and 4D10 can interact with ARD1 in eukaryotic cells by immunoprecipitation (IP). Clones of 14D4 and 10C12 were strongly reacted to ARD1 in immunocytochemistry (ICH) and immunohistochemistry (IHC). ARD1 expression was evaluated in human colorectal cancer and colitis tissues by immunohistochemical analysis with mAb 14D4. Forty-one were ARD1-positive in 50 colorectal cancer tissues and only 12 were weak positive in the 50 matched normal tissues ( P < 0.001). Moreover, ARD1 expression was not detectable in 20 cases of colitis tissue ( P < 0.001). Furthermore, all of the six human colorectal cancer cell lines we examined were also ARD1-positive at mRNA and protein levels. Taken together, the novel mAbs against ARD1 we generated could be good tools for both basic and clinical studies, and ARD1 could be a potential biomarker in colorectal cancer.

Antigen-Based Immunofluorescence Analysis of B-Cell Targeting: Advanced Technology for the Generation of Novel Monoclonal Antibodies with High Efficiency and Selectivity

On the basis of immunofluorescence analysis, an entire pathway of B-cell targeting was successfully identified, which can drive selective production of monoclonal antibodies with high efficiency and selectivity. The technique comprises three critical steps, antigen-based preselection of B lymphocytes, formation of antigenselected B lymphocyte and myeloma cell complexes, and selective fusion of B-cell-myeloma cell complexes with electrical pulses. Intriguingly, expression of surface immunoglobulin receptors on B lymphocytes was recognized even after immunization in vitro. The number of the antigen-selected B lymphocytes after in vitro immunization was in fact higher than that obtained after in vivo immunization, suggesting that such shortterm immunization is applicable for B-cell targeting. Immunofluorescence analysis revealed the targeting technique to demonstrate fivefold to tenfold higher efficiency for formation of hybridoma cells than a polyethylene glycol (PEG)-mediated method. This efficiency is in good agreement with production of hybridoma cells secreting desired monoclonal antibodies determined by an enzyme-linked immunosorbent assay (ELISA). The addition of a low concentration of PEG brought about enhanced fusion efficiency and reduced cell damage at even electric intensities as high as 4.0 kV/cm and 5.0 kV/cm. Here we demonstrate that immunofluorescence analysis can successfully clarify an entire pathway of B-cell targeting and provide advantages over the PEG-mediated method. This advanced technology may be applicable for rapid production of monoclonal antibodies based on in vitro immunization.