Preparation of novel immunomagnetic cellulose microspheres via cellulose binding domain-protein A linkage and its use for the isolation of interferon alpha-2b.
We have developed a novel method for immobilizing antibodies onto magnetic cellulose microspheres (MCMS) using a cellulose binding domain-protein A (CBD-ProA) linkage. Biospecific connection between antibodies and MCMS exhibited significant advantages compared to chemical coupling, including convenient and simple preparation, elimination of toxic compounds, and highly efficient antibody utilization. To evaluate the application of this method, interferon alpha-2b (IFN alpha-2b) was chosen as a model target for detailed analysis of method parameters, such as protein adsorption, antibody efficiency, and reproducibility of the matrix. After optimization and characterization, IFN alpha-2b was successfully purified from crude cell lysate in a single step by cross-linked anti-IFN alpha-2b IgG protein A-CBD-MCMS, purifying 106.1 microg IFN alpha-2b/mL matrix, corresponding to a 13-fold increase over the chemical coupling method. Size-exclusion HPLC identified that the IFN alpha-2b isolated by this method had an overall purity of 95.5%, while immunological and biological assays showed an activity recovery of 91.9% and specific antiviral activity of 2.67 x 10(8)IU/mg. Overall, this study effectively illustrates the favorable qualities of this immobilization method with precisely defined properties that provide an attractive strategy for developing large-scale purification suitable for targeting compounds in highly complex samples.
Cao
Yu
Y
Department of Microbiology, College of Life Sciences, Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071, China.
Zhang
Qi
Q
Wang
Chao
C
Zhu
Yuanyuan
Y
Bai
Gang
G
D001483:Base Sequence; D002482:Cellulose / Q000737:chemistry* / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D018189:Immunomagnetic Separation / Q000295:instrumentation*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000302:isolation & purification*; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A / Q000378:metabolism*
D017931:DNA Primers; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D002482:Cellulose