神经性贝类毒素免疫亲和柱的制备与应用

通过纯化PbTX-2单克隆抗体,以琼脂糖凝胶(sepharose 4B)为载体制备神经性贝类毒素(NSP)免疫亲和柱,并建立NSP的免疫亲和柱净化-液相色谱-串联质谱分析方法。样品以含80%甲醇水溶液提取,PBS缓冲液稀释,经过NSP免疫亲和柱净化和富集后进行LCMS/MS测试分析。同时对上样液、淋洗液、洗脱液等亲和柱参数进行优化。结果表明:NSP免疫亲和柱对贝类样品有很好的净化作用,降低了贝类基质中脂肪、蛋白质、色素、分泌黏液等杂质干扰,提高了样品回收率。在40~800μg/kg范围内样品加标平均回收率为82.95%~112.95%,相对标准偏差为1.27%~2.94%。该NSP免疫亲和柱净化效果强、灵敏度高且受基质干扰小,适用于贝类中神经性贝类毒素的分析测定。

The preparation and application of immunoaffinity column for neurotoxic shellfish poisoning

Objective:Prepare immunoaffinity column(IAC)for pre-processing Neurotoxic Shellfish Poisoning(NSP)samples,and to establish a method of IAC purification LC-MS/MS spectrometry for the NSP.Method:The IAC for the NSP was prepared by purification of PbTX-2 monoclonal antibody and agarose gel(Sepharose 4 B)as carrier.The samples were extracted with 80% methanol aqueous solution,diluted with PBS buffer,purified and enriched by the IAC,and analyzed by LC-MS/MS.Meanwhile,the parameters of IAC such as sample solution and eluent were optimized.The results showed:The IAC for the NSP had a good purification effect on shellfish samples,reduced the interference of fat,protein,pigment,mucus and other impurities in shellfish matrix,and improved the recovery.In the range of40~800 ng,the average recovery of standard addition was82.95% ~112.95%,and the relative standard deviation was1.27%~2.94%.Conclusion:The IAC for the NSP can meet the needs of daily monitoring,with small matrix interference,strong purification effect and high sensitivity.It’s suitable for the analysis and determination of the NSP.