Fabrication of oriented antibody-conjugated magnetic nanoprobes and their immunoaffinity application.
In an attempt to fabricate highly active immunoprobes for serum biomarker detection, we report a simple and effective method for site-specific and self-oriented immobilization of antibodies on magnetic nanoparticles (MNPs). Through boronate formation, the carbohydrate moiety within the constant domain, Fc, of the antibody can be specifically and covalently linked to a boronic acid-functionalized MNP (BA@MNP) without hindering the antigen binding domain, Fab. The performance was evaluated by immunoaffinity extraction of multiple serum antigens. Compared with the random immobilization of antibody on a MNP, the antibody self-oriented immunoprobe provides long-term stability (>2 months) and 5-fold extraction efficiency. It also provides 5-fold improved sensitivity at a low nM range (0.4 nM), presumably through enhanced antibody@MNP activity. In addition, false-positive detections arising from nonspecific binding can be completely minimized by effective surface protection using concentration-dependent dextran blocking. Compared with conventional antibody site-specific immobilization through protein G, this new BA-mediated covalent antibody immobilization provides interference-free extraction resulting from noncovalent immobilization of antibody by protein G. The new immunoassay was applied in comparative profiling of serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP) in human serum. Our triple immunoassay revealed a distinct pattern among normal patients, patients with cancer, and patients with cardiovascular disease. Using the previously reported quantization capability of the MALDI MS readout, we expect that this site-specific immunonanoprobe-based immunoassay can be highly active, rapid, and accurate in nanodiagnosis.
Lin
Po-Chiao
PC
Department of Chemistry, National Tsing-Hua University, Hsinchu, Taiwan.
Chen
Shu-Hua
SH
Wang
Kai-Yi
KY
Chen
Mu-Lin
ML
Adak
Avijit Kumar
AK
Hwu
Jih-Ru R
JR
Chen
Yu-Ju
YJ
Lin
Chun-Cheng
CC
D055910:Antibodies, Immobilized / Q000276:immunology / Q000378:metabolism*; D002097:C-Reactive Protein / Q000032:analysis; D002318:Cardiovascular Diseases / Q000175:diagnosis; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry*; D009369:Neoplasms / Q000175:diagnosis; D000685:Serum Amyloid A Protein / Q000032:analysis; D000683:Serum Amyloid P-Component / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D055910:Antibodies, Immobilized; D000685:Serum Amyloid A Protein; D000683:Serum Amyloid P-Component; D002097:C-Reactive Protein