Comparative Study of Random and Oriented Antibody Immobilization as Measured by Dual Polarization Interferometry and Surface Plasmon Resonance Spectroscopy

Abstract

Dual polarization interferometry (DPI) is used for a detailed study of antibody immobilization with and without orientation control, using prostate specific antigen (PSA) and its antibody as model. Thiol modified DPI chips were activated by a heterobifunctional cross-linker (sulfo-GMBS). PSA antibody was either directly immobilized via covalent binding or coupled via the Fc-fragment to protein G covalently attached to the activated chip. The direct covalent binding leads to a random antibody orientation and the coupling through protein G leads to an end-on orientation. Ethanolamine (ETH) was used to block remaining active sites following the direct antibody immobilization and protein G immobilization. A homobifunctional cross-linker (BS3) was used to stabilize the antibody layer coupled on protein G. DPI provides a real-time measurement of the stepwise molecular binding processes and gives detailed geometrical and structural values of each layer, i.e., thickness, mass, and density. These values evidence the end-on orientation of closely packed antibody on protein G layer and reveal structural effects of ETH blocking/deactivation and BS3 stabilization. With the end-on immobilized antibody, PSA at 10 pg/mL can be detected by DPI through a sandwich complex that satisfies the clinical requirement (assuming <30 pg/mL as clinically safe). However, the randomly immobilized antibody failed to detect PSA at 1 ng/mL. In a parallel study using surface plasmon resonance (SPR) spectroscopy, random and end-on antibody immobilization on streptavidin-modified gold surface was evaluated to further validate the importance of antibody orientation control. With the closely packed antibody layer on protein G surface, SPR can also detect PSA at 10 pg/mL.

Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

“…The need to detect low levels of target analyte further compounds the issue. 20,21 Such glass slides with functionalized surface groups (eg, amine, aldehyde, and N-hydroxysuccinimide [NHS] ester groups) bind proteins and antibodies either through the formation of covalent bonds or by their adsorption or electrostatic interactions. 18 The demand for a higher binding capacity for analytes and the need for decreased sample consumption while performing precise analyte quantification are leading to the application of these functionalized glass slides in single-cell microarrays.…”

Direct and label-free monitoring oligonucleotide immobilization, non-specific binding and DNA biorecognition

“…In order to avoid non-22 specific binding of target molecules, ethanolamine and inert proteins were assayed, and 23 successful results were obtained when using small size proteins such as gelatine. Hybridization 24 efficiency was around 20% for aminosilane-based immobilized probes, and more than 4-fold 25 this value when the other immobilization methods were employed. The ability for recognition 26 complementary DNA strands discriminating non-complementary ones was applied for species 27 identification in mixtures.…”

Orientation and characterization of immobilized antibodies for improved immunoassays (Review)

“…The combined beams create an interference pattern that provides information regarding mass, film thickness, refractive index, and density. 138 Song et al 139 used DPI to investigate anti-PSA antibody (anti-PSA) immobilized covalently via lysines to thiolated DPI chips, or captured via the Fc using immobilized Protein G. The thickness of the two systems was monitored as the antibody bound the PSA antigen and a detection limit of 10 pg/ml was achieved. Song went on to investigate different methods for anti-PSA immobilization with DPI, comparing boronate chelation, TCEP reduction with maleimide covalent linkage, Protein G, and random immobilization methods to PEG:Thiol or amine modified DPI chips.…”