Chelation-Controlled Oriented and Irreversible Immobilization of Native Antibodies on Photoreactive Magnetic
Yi-Ren Huo1, Avijit K Adak1, Sachin K Kawade1
1Department of Chemistry, National Tsing Hua University, Hsinchu 300044, Taiwan.
Abstract:
We present a strategy for the irreversible and oriented immobilization of native antibodies (Abs) onto magnetic nanoparticles (MNPs) by integrating Ni2+-NTA chelation with diazirine (Dia)-mediated photo-crosslinking. MNPs were co-functionalized with nitrilotriacetic acid (NTA) and photoreactive Dia-2 to create a mixed monolayer NTA/Dia-2@MNPs that selectively binds the His-rich Fc domain of unmodified Abs. Short UV exposure activates Dia-2, generating reactive carbenes that covalently anchor proximal residues and permanently lock the Ab in an oriented configuration. This dual-mode immobilization preserves Fab accessibility, enhances binding performance, and prevents Ab dissociation during stringent washing. We validated the platform using two cancer therapy Abs (trastuzumab and cetuximab) and one cancer biomarker (anti-serum amyloid A, anti-SAA) in cancer cells and human serum. Anti-SAA MNPs fabricated by the NTA-Ni2+ method showed a 1.5-fold increase in antigen binding in the serum sample compared to the boronate affinity-based method and a significant (22-fold) improvement over random immobilization. Cetuximab-functionalized oriented MNPs by the current immobilization strategy achieved a 4.7-6-fold enhancement in EGFR pulldown efficiency from human embryonic kidney (HEK293T) and non-small cell lung cancer (NSCLC) models, compared to randomly immobilized controls. Notably, the oriented MNPs enabled co-purification of markedly high interactome coverage of >1000 proteins and differential abundance of downstream proteins. Importantly, this platform requires no prior Ab modification and is compatible with full-length native Abs and stable in complex biological samples (cell or serum). By combining chelation-guided orientation with photoinduced covalent fixation, this strategy addresses key challenges in Ab surface engineering and offers a robust, versatile solution for applications in immunoprecipitation, proteomics, and biomarker discovery.
Related Concept Videos
01:14Reversible and Irreversible Processes
01:19Complexation Equilibria: The Chelate Effect
01:13Location and Orientation of the Heart
01:10Antibody Structure
Antibodies, also known as immunoglobulins (Ig), are essential players of the adaptive immune system. These antigen-binding proteins are produced by B cells and make up 20 percent of the total blood plasma by weight. In mammals, antibodies fall into five different classes, which each elicits a different biological response upon antigen binding.
The Y-Shaped Structure of Antibodies Consists of Four Polypeptide Chains
Antibodies consist of four polypeptide chains: two identical heavy...
01:18Methods of Documentation I: Source-Oriented Records
In an SOR, each discipline involved in patient care maintains a separate medical record section. This record-keeping method enables easy tracking of patient progress and ensures healthcare staff have access to up-to-date information.
Key Attributes include the following:
03:02Colors and Magnetism
When atoms or molecules absorb light at the proper frequency, their electrons are excited to higher-energy orbitals. For many main group atoms and molecules, the absorbed photons are in the ultraviolet range of the electromagnetic spectrum, which cannot be detected by the human eye. For coordination compounds, the energy difference between the d orbitals often allows photons in the visible range to be absorbed and emitted, which is seen as colors by the human...