الفهرس | Only 14 pages are availabe for public view |
Abstract 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. 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 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. Successful implementation of cation exchange chromatography had yielded an amount of 74.72 æg rhMBP after 5 column volume of 0.5 M NaCl with purity of 83.56%. The detection of rhMBP in different fractions of NaCl concentration indicated the presence of several post-translation modification isoforms. A 2-step purification protocol (Cation exchange chromatography followed by immobilized metal affinity chromatography) was sufficient to obtain rhMBP from transgenic milk with purity of 93.42% |