Analytical Chemistry Department, Faculty of Pharmacy , Cairo University , Cairo 11562 , Egypt.
Bioanalysis Research Group, School of Pharmacy , Newgiza University , Giza 12256 , Egypt.
ACS Sens. 2019 Feb 22;4(2):413-420. doi: 10.1021/acssensors.8b01315. Epub 2019 Jan 16.
Recombinant human myelin basic protein (rhMBP) produced in the milk of transgenic cows was found exclusively associated with milk caseins. This hindered its direct determination without extensive sample pretreatment. Here, a label-free potentiometric immunosensor was developed and validated for the determination of rhMBP. An ion flux was generated under zero-current based on surface blocking of the polymeric membrane ion-selective electrode by anti-hMBP antibody and tetrabutylammonium bromide as a marker ion. The immunosensor was successfully employed in the quantitative determination of hMBP in the range of 0.10-20.00 μg/mL with a limit of detection of 50.00 ng/mL. The applicability of the passive ion flux immunosensor for determination of target analyte in complex matrices was investigated. Downstream purification of rhMBP from the milk of transgenic cows was achieved using cation exchange chromatography, immobilized metal affinity chromatography, and immunoaffinity chromatography. The specificity of the immunosensor along with matrix effect of milk proteins were demonstrated. Results obtained using the rhMBP immunosensor were further cross-validated using an orthogonal testing protocol assembled of RP-HPLC and SE-HPLC. It should be noted that the proposed ion flux immunosensor provided a feasible and specific tool for monitoring rhMBP concentration/purity, immunogenic activity, and stability. Such approach provides an attractive economic alternative to sophisticated biosensors required for in-process quality control of biopharmaceutical products.
转基因为人髓磷脂碱性蛋白（rhMBP）在奶牛的乳汁中被发现与乳清蛋白特异性结合，这极大地阻碍了对 rhMBP 的直接检测，除非进行广泛的样品预处理。本研究开发并验证了一种无需标记的 rhMBP 免疫测定的电流型电化学传感器。基于聚合膜离子选择电极上的抗 hMBP 抗体和四丁基溴化铵作为标记离子的表面阻断，在零电流下产生离子流。该免疫传感器可成功用于定量测定范围在 0.10-20.00 μg/mL 之间的 hMBP，检测限为 50.00 ng/mL。考察了该被动离子流免疫传感器在复杂基质中测定目标分析物的适用性。采用阳离子交换色谱、固定化金属亲和色谱和免疫亲和色谱从转基因奶牛的乳汁中对 rhMBP 进行下游纯化。验证了免疫传感器的特异性和乳蛋白的基质效应。利用正交试验方案组装的反相高效液相色谱（RP-HPLC）和凝胶排阻高效液相色谱（SE-HPLC）对 rhMBP 免疫传感器的检测结果进行了进一步的交叉验证。值得注意的是，所提出的离子流免疫传感器为监测 rhMBP 的浓度/纯度、免疫原性和稳定性提供了一种可行且特异的工具。该方法为生物制药产品的过程质量控制提供了一种有吸引力的经济替代方案，而无需复杂的生物传感器。
