表位
多克隆抗体
适体
化学
表位定位
单克隆抗体
线性表位
表面等离子共振
分子生物学
抗体
生物化学
生物
材料科学
纳米颗粒
免疫学
纳米技术
作者
Loredana-Mirela Lupu,Pascal Wiegand,Daria Holdschick,Delia Mihoc,Stefan Maeser,Stephan Rawer,F. Völklein,Ebrahim Malek,Frederik Barka,Sascha Knauer,Christina Uth,Julia B. Hennermann,Wolfgang Kleinekofort,Andreas Hahn,Günes Barka,Michael Przybylski
标识
DOI:10.3390/ijms222312832
摘要
Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.
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