| Summary: | Antibody-conjugated magnetic nanoparticles (Ab-MNPs) have received considerable attention in bioseparation and clinical diagnostics assays due to their unique ability to detect and isolate a variety of biomolecules and cells. Because antibodies can be expensive, a key challenge for bioconjugation is to determine the optimal amount of antibodies with reasonable antigen-capturing activity. We designed an approach to determine the minimum amounts of antibodies for efficient coating. Different quantities of Herceptin (anti-human epidermal growth factor receptor 2: HER2) antibody were applied and immobilized on the surface of MNPs. Antibody binding was then checked by using an anti-human antibody conjugated with fluorochrome and flow cytometry. When the ratio of MNPs to antibodies increased from 0.79 to 795.45, mean fluorescence intensity (MFI) of conjugated MNPs decreased markedly from 185.56 to 20.07, indicating lower surface antibody coverage. We then investigated the relation between antibody content and isolation efficiency. Three Ab-MNP samples with different MFI were used to isolate SK-BR-3, a HER2-positive breast cancer cell line, from mixtures of whole blood or mononuclear cells. After isolation in a magnetic field, separation efficiency was evaluated by fluorescence microscopy and flow cytometry-based techniques. Our results collectively showed that the amount of anti-HER2 antibodies for conjugation with MNPs could be decreased by as much as one-fifteenth without compromising isolation efficiency, which in turn can reduce the cost of immunoassay biosensors.
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