Liu Sihong, Dong Weihua, Kong Tianhan
Department of Pathophysiology, Guangzhou Medical College, #195 Dong Feng West Road, Guangzhou, Guangdong, 510082, China.
Indian J Exp Biol. 2010 Aug;48(8):778-85.
Chinese Cobra (Naja naja atra) bite is one of the leading causes of snake-bite mortality in China. The traditional anti-cobra venom serum therapy was found to be expensive and with high frequency of side effects. Therefore attempts were made to generate a high titer immunoglobulin from egg yolk (IgY) of crude cobra-venom immunized Leghorn hens, and to standardize an effective method for producing avian antivenom in relatively pure form. The IgY was isolated first by water dilution method to remove the lipid, then extracted by ammonium sulfate precipitation, and purified through anion exchange chromatogram. The different purities of IgY from different isolating stages were submitted to enzyme-linked immunosorbent assay and SDS-PAGE to determine their titers. Immunoblotting showed that the purified IgY (ion exchange chromatography fraction, IECF) recognized several antigenic fractions of cobra venom, and presented with the character of polyclonal antibody. IECF on SDS-PAGE under reducing conditions migrated as a 65 kDa heavy chain and a 35 kDa light chain, respectively. The LD50 of the N. naja atra venom was 0.62 mg/kg body weight in mice. Four times the LD50 dose of venom was selected as challenge dose, and the ED50 of IgY was 3.04 mg IECF/mg venom. The results indicate that the activity of anti-snake venom IgY could be obviously elevated by ion exchange chromatography, thus possessing therapeutic significance for snakebite envenomation.
中华眼镜蛇（舟山眼镜蛇）咬伤是中国蛇咬伤致死的主要原因之一。传统的抗眼镜蛇毒血清疗法被发现成本高昂且副作用频发。因此，人们尝试从用粗眼镜蛇毒免疫的来航鸡的蛋黄中制备高滴度免疫球蛋白（IgY），并规范一种以相对纯的形式生产禽抗蛇毒血清的有效方法。首先通过水稀释法分离IgY以去除脂质，然后通过硫酸铵沉淀提取，并通过阴离子交换色谱法纯化。将不同分离阶段的不同纯度的IgY进行酶联免疫吸附测定和SDS-PAGE以确定其滴度。免疫印迹显示纯化的IgY（离子交换色谱级分，IECF）识别眼镜蛇毒的几种抗原级分，并呈现多克隆抗体的特征。在还原条件下，IECF在SDS-PAGE上分别迁移为65 kDa的重链和35 kDa的轻链。中华眼镜蛇毒对小鼠的半数致死量（LD50）为0.62 mg/kg体重。选择四倍LD50剂量的毒液作为攻击剂量，IgY的半数有效量（ED50）为3.04 mg IECF/mg毒液。结果表明，离子交换色谱法可明显提高抗蛇毒IgY的活性，因此对蛇咬伤中毒具有治疗意义。
