Development of a cell-based in vitro assay as a possible alternative for determining bothropic antivenom potency
Development of a cell-based in vitro assay as a possible alternative for determining bothropic antivenom potency
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DOI：
10.1016/j.toxicon.2019.09.010
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发表时间：
2019-12-01
期刊：
影响因子：
2.8
通讯作者：
Chavez-Olortegui, Carlos
中科院分区：
文献类型：
--
作者：
Lopes-de-Souza, Leticia;Costal-Oliveira, Fernanda;Chavez-Olortegui, Carlos
在热带国家，与毒蛇发生的事故是一项主要的健康危害。巴西近80%的蛇咬伤都是由蛇属造成的。免疫疗法是唯一被批准的针对蛇毒素的特定治疗方法，而治疗性抗蛇毒血清的生产需要质量控制测试来确定其中和效力。目前，这些控制是通过体内致死性中和来进行的，然而，也需要抑制由人畜毒产生的特定事件，如凝血障碍、出血、水肿或细胞毒效应。这项工作的目的是建立一种用于抗蛇毒血清临床前评估的体外替代试验。在这个意义上，我们设计了一种细胞活力实验，用不同剂量(0.20-10mU/孔)的L低效抗血清和高效抗血清(按传统的体内试验分类)来评估抗Jaraca蛇毒细胞毒的抗毒能力(5×EC(50)=58.95mU/ml)。我们发现，与低效价的血清相比，高效价的血清在中和雅加拉第蛇毒细胞毒性方面更有效，这与它们预先确定的体内效力一致。考虑到血清的体外抑制浓度(IC50)可以防止50%的细胞死亡(IC50)和已知的体内效力，确定了区分低效价和高效价血清的分界点。我们的数据为体外方法的发展提供了启示，该方法可以在生产过程中测定抗人畜抗蛇毒血清的效力。
Accidents with venomous snakes are a major health hazard in tropical countries. Bothrops genus is responsible for almost 80% of snakebites in Brazil. Immunotherapy is the only approved specific treatment against snake toxins and the production of therapeutic antivenoms requires quality control tests to determine their neutralizing potency. Currently, these controls are performed by in vivo lethality neutralization, however, the inhibition of particular events produced by bothropic venoms such as coagulopathy, hemorrhage, edema or cytotoxic effects are also required. The aim of this work is to develop an in vitro alternative assay for antivenom pre-clinical evaluation. In this sense, we designed a cell viability assay using different amounts (0.2-10 mu L/well) of low and high potency anti-bothropic sera, previously classified by the traditional in vivo test, for assessing the antivenom capacity to protect the cells against B. jararaca venom cytotoxicity (5xEC(50) = 58.95 mu g/mL). We found that high potency sera are more effective in neutralizing B. jararaca venom cytotoxicity when compared to low potency sera, which is in accordance to their pre-determined in vivo potency. Considering sera in vitro inhibitory concentration able to prevent 50% cell death (IC50) and their known in vivo potency, a cut-off point was determined to discriminate low and high potency sera. Our data provide insights for the development of an in vitro method which can determine the anti-bothropic antivenom potency during its production.