Coupled solid phase extraction and microparticle-based stability and purity-indicating immunosensor for the determination of recombinant human myelin basic protein in transgenic milk

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• 作者：Medhat A. Al-Ghobashy^a ; ^b ; ^{medhat.alghobashy@cu.edu.eg} ; Martin A.K. Williams^c ; Gö ; tz Laible^d ; David R.K. Harding^c
• 关键词：Biopharmaceuticals ; Optical immunosensors ; Bio-Plex ; Myelin basic protein ; Solid phase extraction
• 刊名：Talanta
• 出版年：2013
• 出版时间：15 May, 2013
• 年：2013
• 卷：109
• 期：Complete
• 页码：7-12
• 全文大小：494 K

文摘

An optical immunosensor was developed and validated on the surface of microparticles for the determination of a biopharmaceutical protein. The recombinant human myelin basic protein (rhMBP) was produced in milk of transgenic cows as a His-tagged fusion protein. Previous work indicated exclusive association of rhMBP with milk casein micelles that hindered direct determination of the protein in milk. In this work, a solid phase extraction using a cation exchange matrix was developed in order to liberate rhMBP from casein micelles. A sandwich-type immunoassay was then used for in-process monitoring of the full-length protein in the presence of major milk proteins. The assay was successfully employed for detection of ultra-traces of rhMBP (LOD=6.04 ng mL^?1¡Ö0.3 nmol L^?1) and for quantitative determination over a wide concentration range (10.00-10,000.00 ng mL^?1). The assay was able also to detect the rhMBP in the presence of its human counterpart that lacks the His-tag. The high sensitivity along with the ability of the assay to determine the full length protein enabled monitoring of the stability of rhMBP. The testing protocol is particularly useful for intrinsically unstructured proteins that are extremely sensitive to proteolysis and lack a traceable enzymatic activity. This immunosensor provides a specific, ultrasensitive high throughput tool for in-process monitoring in biopharmaceutical industry.