A型肉毒抗毒素ELISA方法的建立及验证

Establishment and validation of an ELISA method for detection of botulinum antitoxin type A

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A_(450 nm)值,以标准曲线样品浓度的对数(X)为横坐标,A_(450 nm)值(Y)为纵坐标绘制标准曲线,将待测血清样品A_(450 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 A_(450 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 A_(450 nm) value(Y)was taken as the vertical coordinate to draw the standard curve.The serum sample concentration could be obtained by substituting A_(450 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 m IU·mL^(-1),the linear relationship was high with r>0.9900.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 525-2450 IU·mL^(-1),and the neu-tralization test was in the range of 675-2400 IU·mL^(-1).The coefficient of correlation r=0.9060(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.