Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Abstract Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody – a potential biomarker of Bothrops jararacussu venom – against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.

摘要 背景
洞蛇属（Bothrops，俗称颊窝蝰蛇（pit viper））是巴西绝大多数蛇伤事件的主要致病蛇类。该属下的贾拉库苏洞蛇（Bothrops jararacussu）与贾拉拉卡洞蛇（Bothrops jararaca）因咬伤症状严重且栖息于人口稠密区域而备受关注。针对贾拉库苏洞蛇咬伤的治疗，现有研究对专用洞蛇抗蛇毒血清中和其肌毒性的效果提出了质疑，但目前尚无针对人类的准确临床数据。因此，针对该物种的鉴别诊断试剂盒研发具有重要应用价值：其可为医护人员提供可精准判定咬伤系贾拉库苏洞蛇还是该属其他蛇类所致的检测工具，同时还可用于评估治疗方案的特异性，并为流行病学研究提供关键数据。

方法
首先，我们针对洞蛇毒素-I（bothropstoxin-I，简称BthTx-I）制备了物种特异性多克隆抗体，该抗体可作为贾拉库苏洞蛇蛇毒的潜在生物标志物；而在巴西南部地区的贾拉拉卡洞蛇（Bothrops jararaca）蛇毒中，该毒素的含量相对较低。

结果
针对洞蛇毒素-I的多克隆抗体可分离得到多种物种特异性免疫球蛋白。为构建安全且标准化的免疫检测体系，我们进一步制备了单克隆抗体，最终获得7株杂交瘤细胞。其中5株仅与贾拉库苏洞蛇蛇毒发生特异性结合，2株可识别巴西东南部种群的贾拉拉卡洞蛇蛇毒；利用该单克隆抗体还可区分巴西南部种群的贾拉拉卡洞蛇蛇毒。通过检测单克隆抗体与其他洞蛇属蛇毒的反应性，我们发现单克隆抗体Bt-3对贾拉库苏洞蛇蛇毒的特异性优于其余抗体。

结论
本研究结果证实，洞蛇毒素-I（BthTx-I）具备制备单克隆抗体的潜力，可用于区分贾拉库苏洞蛇与其他洞蛇属物种的蛇毒。