Different Strategies for Bio-Conjugation of Anti-CD4 Monoclonal Antibodies to Silica-Coated Magnetic Nanoparticles and Their Application for the Positive Selection of CD4+ Lymphocytes

Efficient cell separation techniques are required for the isolation of various cellular populations in fundamental research. In the present study, five types of magnetic nanoparticles (MNPs) have been designed to isolate CD4⁺ T cells from peripheral blood mononuclear cells. MNPs were synthesized by a modified solvothermal method and subsequently coated by a silica shell through the sol-gel process. Then, the anti-CD4 antibody was conjugated on the surface of different groups of MNPs, which were functionalized with various cross-linkers (including the carboxylic and aldehyde groups) and proteins (such as protein A and streptavidin). The performance of prepared MNPs for the separation of CD4⁺ lymphocytes was investigated by optical microscopy and flow-cytometry. This study revealed that the cell isolation rates by different functionalized groups, including aldehyde, carboxyl, CHO-protein A, COOH-protein A, and streptavidin conjugated with nanoparticles, were 32 ± 3, 32 ± 1, 80 ± 4, 70 ± 1, and 71 ± 2%, respectively. Based on such data, the CHO-protein A-functionalized MNPs have been considered a practical choice to separate CD4⁺ T cells with over 92 ± 3% purity. MNPs obtained in the study provide an adequate tool for the cell isolation method and further present the dramatic potential to be applied to several fields of biomedical purpose.