Bioconjugation of Antibodies and Enzyme Labels onto Magnetic Beads

• Published in: Methods in enzymology Vol. 571; p. 135
• Main Authors: Otieno, B A, Krause, C E, Rusling, J F
• Format: Journal Article
• Language: English
• Published: United States 2016
• Subjects: Affinity Labels - chemistry; Antibodies, Immobilized - chemistry; Biosensing Techniques; Enzymes, Immobilized - chemistry; Horseradish Peroxidase - chemistry; Immunoassay; Magnets - chemistry; Microfluidic Analytical Techniques; Nanoparticles - chemistry; Streptavidin - chemistry; Enzymes; Immunoassay; Antibodies; Magnetic beads; Bicinchoninic acid assay; Bioconjugation; Enzyme kinetics
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2015.10.005

Immunoassays employ antibodies and labels to capture and detect target macromolecular analytes, often from complex sample matrices such as serum, plasma, or saliva. The high affinity and specificity of antibody-antigen interactions makes immunoassays critically important analytical techniques for clinical diagnostics as well as other research applications in the areas of pharmaceutical and environmental analysis. Integration of magnetic beads (MBs) into immunoassays and other bioanalytical methodologies is a valuable approach to allow efficient target capture, enrichment, and convenient separation. In addition, large signal amplification can be achieved by preconcentration of the target and by attaching many thousands of enzyme labels to the MBs. These features have enabled MB-based biosensors to achieve ultra-low detection limits needed for advanced clinical diagnostics that are challenging or impossible using traditional immunoassays. MBs are employed either as mobile substrates for target analyte capture, as detection labels (or label carriers), or simultaneously as substrates and labels. For optimal assay performance, it is crucial to apply an easy, efficient, and robust bead-probe conjugation protocol, and to thoroughly characterize the bioconjugated products. Herein, we describe methods used in our laboratory to functionalize MBs with antibodies and enzyme labels for ultrasensitive detection of protein analytes. We also present detailed strategies for characterizing the MB bioconjugates.