目的: 建立定量检测A型肉毒抗毒素效价的酶联免疫吸附法(ELISA法),并进行验证。方法: 以A型肉毒类毒素为包被抗原包被酶标板,封闭后加入稀释的血清样品,以辣根过氧化物酶标记的兔抗马IgG(IgG-HRP)作为酶标抗体,加入四甲基联苯胺显色剂(TMB)显色10~15 min,2 mol·L-1硫酸终止反应,于酶标仪上读取A450 nm值,以标准曲线样品浓度的对数(X)为横坐标,A450 nm值(Y)为纵坐标绘制标准曲线,将待测血清样品A450 nm值代入曲线方程即可得到血清样品浓度,乘以稀释倍数即为血清样品效价。结果: 在优化的条件下,该方法具有较高的特异性,除了与A型肉毒抗毒素血清反应外,对B、C、D、E、F型肉毒抗毒素血清均无交叉反应;在线性范围50~400 mIU·mL-1时线性关系良好,r＞0.990 0;准确度介于80%~120%;精密度(重复性和中间精密度)RSD＜20%;采用该方法测定20批A型肉毒抗毒素血清的效价范围在525~2 450 IU·mL-1,中和试验法检测范围在675~2 400 IU·mL-1,二者的相关系数r=0.906 0(P＜0.001),呈显著性正相关。结论: 建立的ELISA定量检测方法在实验室条件下可以取得稳定的结果,为A型肉毒抗毒素效价的测定提供了一种简便的体外检测方法。
Objective: To establish and validate an ELISA method to measure the titer of botulinum antitoxin type A. Methods: Botulinum toxoid type A was used as coated antigen coated enzyme label plate, and diluted serum samples were added after plate blocking. The horseradish peroxidase labeled rabbit anti-horse IgG (IgG-HRP) was subsequently used as the enzyme-labeled antibody. Tetramethyl benzidine(TMB) was added for 10-15 min for color development, and the reaction was terminated by 2 mol·L-1 of sulfuric acid. Finally, the A450 nm value was read on the enzyme marker. The logarithm of concentration(X) of the sample concentration of standard curve was taken as the horizontal coordinate and the A450 nm value(Y) was taken as the vertical coordinate to draw the standard curve. The serum sample concentration could be obtained by substituting A450 nm value of serum samples to be tested into the curve equation, and the titer of the serum samples could be obtained by multiplying the dilution ratio. Results: Under the optimized conditions, the specificity of the method was high, there was no cross reaction with B, C, D, E and F type of antitoxin serum except botulinum antitoxin type A. In the linear range of 50-400 mIU·mL-1, the linear relationship was high with r＞0.990 0. The accuracy was between 80% and 120% and the RSD of precision (repeatability and intermediate precision) was less than 20%. The titers for 20 batches of botulinum antitoxin type A serum were determined by ELISA in the range of 525-2 450 IU·mL-1, and the neutralization test was in the range of 675-2 400 IU·mL-1. The coefficient of correlation r=0.906 0(P＜0.001), which was significantly positive correlated between the two methods. Conclusion: The established ELISA method could acquire stable results under laboratory conditions, which provides a simple detection method for botulinum antitoxin type A in vitro.
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