Preparation of nitrocellulose (NC) immuno-affinity membrane for purification of rAPC antibody.
In this study, recombinant allophycocyanin (rAPC) with a purity of 98% was transferred from a gel to a nitrocellulose (NC) membrane to develop a simple and efficient immuno-affinity membrane. Atomic force microscopy (AFM) was used to investigate the surface topography of the affinity membrane and its characterization indicated that rAPC easily forms trimers or hexamers on the membrane surface on use of the given transfer method. The hydrodynamic radius (R(h)) of the rAPC aggregation was equal to 103 nm or 365 nm according to dynamic light scattering (DLS), which was in agreement with the result obtained by AFM. Based on the specific immunological reaction of antigen and antibody, anti-APC antibodies were purified from rabbit polyclonal serum in a single step. The amount of absorbed antibody was 5.79 mg/g membrane according to analysis by ELISA methods. The purity of antibodies was up to 98% according to SDS-PAGE. The adsorption-desorption cycle of rAPC was repeated six times using the same immuno-affinity membrane, and there was no significant loss in adsorption capacity. The method provides a novel and efficient immunological affinity membrane for the purification of antibodies.
Sun
Haixiang
H
College of Chemistry and Chemical Engineering, China University of Petroleum, Dongying, Shandong, PR China. shxgbs@yahoo.com.cn
Ge
Baosheng
B
Liu
Shengnan
S
Chen
Huanlin
H
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification*; D003101:Collodion; D004797:Enzyme-Linked Immunosorbent Assay; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D010798:Phycocyanin / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology
D000906:Antibodies; D011994:Recombinant Proteins; C002497:allophycocyanin; D010798:Phycocyanin; D003101:Collodion