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      A novel in vitro potency assay of antisera against Thai Naja kaouthia based on nicotinic acetylcholine receptor binding

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          Abstract

          Snake envenomation is an important medical problem. One of the hurdles in antivenom development is the in vivo assay of antivenom potency which is expensive, gives variable results and kills many animals. We report a novel in vitro assay involving the specific binding of the postsynaptic neurotoxins (PSNTs) of elapid snakes with purified Torpedo californica nicotinic acetylcholine receptor (nAChR). The potency of an antivenom is determined by its antibody ability to bind and neutralize the PSNT, thus preventing it from binding to nAChR. The PSNT of Naja kaouthia (NK3) was immobilized on microtiter wells and nAChR was added to bind with it. The in vitro IC 50 of N. kaouthia venom that inhibited 50% of nAChR binding to the immobilized NK3 was determined. Varying concentrations of antisera against N. kaouthia were separately pre-incubated with 5xIC 50 of N. kaouthia venom. The remaining free NK3 were incubated with nAChR before adding to the NK3 coated plates. The in vitro and in vivo median effective ratio, ER 50s of 12 batches of antisera showed correlation ( R 2) of 0.9809 ( p < 0.0001). This in vitro assay should be applicable to antisera against other elapid venoms and should reduce the use of live animals and accelerate development of life-saving antivenoms.

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          Assessment of the prozone effect in malaria rapid diagnostic tests

          Background The prozone effect (or high doses-hook phenomenon) consists of false-negative or false-low results in immunological tests, due to an excess of either antigens or antibodies. Although frequently cited as a cause of false-negative results in malaria rapid diagnostic tests (RDTs), especially at high parasite densities of Plasmodium falciparum, it has been poorly documented. In this study, a panel of malaria RDTs was challenged with clinical samples with P. falciparum hyperparasitaemia (> 5% infected red blood cells). Methods Twenty-two RDT brands were tested with seven samples, both undiluted and upon 10 ×, 50 × and 100 × dilutions in NaCl 0.9%. The P. falciparum targets included histidine-rich protein-2 (HRP-2, n = 17) and P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH, n = 5). Test lines intensities were recorded in the following categories: negative, faint, weak, medium or strong. The prozone effect was defined as an increase in test line intensity of at least one category after dilution, if observed upon duplicate testing and by two readers. Results Sixteen of the 17 HRP-2 based RDTs were affected by prozone: the prozone effect was observed in at least one RDT sample/brand combination for 16/17 HRP-2 based RDTs in 6/7 samples, but not for any of the Pf-pLDH tests. The HRP-2 line intensities of the undiluted sample/brand combinations with prozone effect (n = 51) included a single negative (1.9%) and 29 faint and weak readings (56.9%). The other target lens (P. vivax-pLDH, pan-specific pLDH and aldolase) did not show a prozone effect. Conclusion This study confirms the prozone effect as a cause of false-negative HRP-2 RDTs in samples with hyperparasitaemia.
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            Characterization of a new polyvalent antivenom (Antivipmyn Africa) against African vipers and elapids.

            As a response to the antivenom shortage in Sub-Saharan Africa, evident for well over a decade, we developed a new polyvalent anti-ophidian antivenom (Antivipmyn((R)) Africa) designed for use in the region. We report a detailed characterization of its biochemical composition (protein content and profiling by size-exclusion chromatography and electrophoresis) as well as the specific and para-specific neutralization potencies (as median effective dose in the mouse lethality test). Additionally, we studied the neutralization of hemorrhagic, anti-hemostatic and necrotic activities of Echis ocellatus venom, responsible for a majority of severe envenomations in the continent according to existing epidemiological data. The antivenom is currently under production and has already been employed in the field in a pragmatic Phase III clinical trial in the Republic of Benin. It is a purified lyophilized polyvalent equine F(ab')(2)-based product obtained by immunization with the venoms of eleven species of African snakes of the Genera Echis, Bitis, Naja and Dendroaspis. The criteria for its design are discussed, particularly in terms of the implementation of realistic public health policies targeting mostly rural populations in the continent.
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              In vitro neuromuscular activity of snake venoms.

              1. Snake venoms consist of a multitude of pharmacologically active components used for the capture of prey. Neurotoxins are particularly important in this regard, producing paralysis of skeletal muscles. These neurotoxins can be classified according to their site of action (i.e. pre- or post-synaptic). 2. Presynaptic neurotoxins, which display varying phospholipase A2 activities, have been identified in the venoms of the four major families of venomous snakes (i.e. Crotalidae, Elapidae, Hydrophiidae and Viperidae). The blockade of transmission produced by these toxins is usually characterized by a triphasic effect on acetylcholine release. Considerable work has been directed at identifying the binding site(s) on the presynaptic nerve terminal for these toxins, although their mechanism of action remains unclear. 3. Post-synaptic neurotoxins are antagonists of the nicotinic receptor on the skeletal muscle. Depending on their sequence, post-synaptic toxins are subdivided into short- and long-chain toxins. These toxins display different binding kinetics and different affinity for subtypes of nicotinic receptors. Post-synaptic neurotoxins have only been identified in venoms from the families Elapidae and Hydrophiidae. 4. Due to the high cost of developing new antivenoms and the reluctance of many companies to engage in this area of research, new methodologies are required to test the efficacy of existing antivenoms to ensure their optimal use. While chicken eggs have proven useful for the examination of haemorrhagic venoms, this procedure is not suited to venoms that primarily display neurotoxic activity. The chick biventer cervicis muscle has proven useful for this procedure, enabling the rapid screening of antivenoms against a range of venoms. 5. Historically, the lethality of snake venoms has been based on murine LD50 studies. Due to ethical reasons, these studies are being superseded by in vitro studies. Instead, the time taken to produce 90% inhibition of nerve-mediated twitches (i.e. t90) in skeletal muscle preparations can be determined. However, these two procedures result in different rank orders because they are measuring two different parameters. While murine LD50 determinations are based on "quantity", t90 values are based on how "quick" a venom acts. Therefore, knowledge of both parameters is still desirable. 6. In vitro neuromuscular preparations have proven to be invaluable tools in the examination of snake venoms and isolated neurotoxins. They will continue to play a role in further elucidating the mechanism of action of these highly potent toxins. Further study of these toxins may provide more highly specific research tools or lead compounds for pharmaceutical agents.
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                Author and article information

                Contributors
                kavi.rtn.@mahidol.ac.th
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                17 August 2017
                17 August 2017
                2017
                : 7
                : 8545
                Affiliations
                [1 ]ISNI 0000 0004 0617 2559, GRID grid.418595.4, , Laboratory of Immunology, Chulabhorn Research Institute, ; Bangkok, Thailand
                [2 ]ISNI 0000 0004 0482 1383, GRID grid.452298.0, , Chulabhorn Graduate Institute, ; Bangkok, 10210 Thailand
                [3 ]ISNI 0000 0004 1937 0490, GRID grid.10223.32, , Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, ; Bangkok, 10400 Thailand
                [4 ]ISNI 0000 0000 9039 7662, GRID grid.7132.7, , Department of Microbiology, Faculty of Medicine, Chiang Mai University, ; Chiang Mai, Thailand
                [5 ]ISNI 0000 0001 2308 5949, GRID grid.10347.31, , Department of Molecular Medicine, Faculty of Medicine, University of Malaya, ; Kuala Lumpur, 50603 Malaysia
                [6 ]ISNI 0000 0001 2308 5949, GRID grid.10347.31, , Department of Pharmacology, Faculty of Medicine, University of Malaya, ; Kuala Lumpur, 50603 Malaysia
                [7 ]ISNI 0000 0004 1937 0490, GRID grid.10223.32, , Faculty of Veterinary Science, Mahidol University, ; Salaya, NakornPrathom 73170 Thailand
                Article
                8962
                10.1038/s41598-017-08962-3
                5561211
                28819275
                4266ad2a-3458-4d28-b1da-1d82099ee73c
                © The Author(s) 2017

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 24 January 2017
                : 17 July 2017
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