磁性载体蛋白抗体阻断剂制备及性能测定

Preparation of Magnetic Carrier Protein Antibody Blocker and Determination of its Properties

为了有效去除血清中的白喉毒素跨膜结构域抗体,提高检测的精准度,建立四氧化三铁纳米磁珠共价偶联载体蛋白对血清中载体蛋白抗体的去除方法,并评估去除效果;采用高温多元醇合成法制备表面包裹葡聚糖的四氧化三铁纳米颗粒,利用溴化氰将葡聚糖分子上的羟基活化为亚氨基碳酸酯,并与白喉毒素跨膜结构域通过氨基共价偶联,制得纳米磁珠-白喉毒素跨膜结构域抗体阻断剂;纳米磁珠-白喉毒素跨膜结构域抗体阻断剂与免疫过白喉毒素跨膜结构域-血管内皮生长因子或白喉毒素跨膜结构域-肿瘤坏死因子融合蛋白疫苗的小鼠抗血清进行孵育,以吸附去除白喉毒素跨膜结构域特异性抗体;对孵育前、后的抗血清进行酶联免疫吸附测定。结果表明:白喉毒素跨膜结构域抗体的去除率高达90%以上,检测精准度显著提高;在37℃条件下,仅需孵育1 h即可充分去除血清中的白喉毒素跨膜结构域抗体。

To effectively remove the diphtheria toxin transmembrane domain antibody in serum and improve the accuracy of detection,a method for removing carrier protein antibody from serum by using covalently coupled carrier protein to ferroferric oxide magnetic nanoparticles was established,and the removal effect was evaluated.Ferroferric oxide magnetic nanoparticles coated dextran were prepared by using the high-temperature polyol method,and hydroxyl groups on dextran were activated by using cyanogen bromide.Hydroxyl groups coupled with amino groups of the transmembrane domain protein molecule of diphtheria toxoid were activated.The magnetic nanoparticles-transmembrane domain protein molecule of diphtheria toxoid antibody blocker was thus obtained.Magnetic nanoparticles-transmembrane domain protein molecule of diphtheria toxoid antibody blocker was incubated with mouse antiserum immunized with transmembrane domain protein molecule of diphtheria toxoid-vascular endothelial growth factor or transmembrane domain protein molecule of diphtheria toxoid-tumor necrosis factor fusion protein vaccine to adsorb and remove transmembrane domain protein molecule of diphtheria toxoid-specific antibodies.The antiserum before and after incubation was detected by using enzyme-linked immunosorbent assay.The results show that the removal rate of transmembrane domain protein molecule of diphtheria toxoid antibody is over 90%,and the detection accuracy is significantly improved.The antibody of diphtheria toxin transmembrane domain can be sufficiently removed through incubation at 37℃for only 1 h.