Robustness and reproducibility of SPR chips functionalized with anti-Rabbit antibodies on K-One surface chemistry

Introduction

Anti-rabbit antibodies, produced in host species such as goat or sheep, are highly valuable tools in biomedical research and diagnostics, as they specifically recognize and bind to rabbit immunoglobulins. Many therapeutic monoclonal (mAbs) and polyclonal antibodies (pAbs) are initially generated in rabbit or mouse models, and while mice remain the standard model, rabbits are often chosen when higher affinity or broader epitope coverage is needed, especially for targets that elicit weak responses in mice.

In diagnostics and immunoassays, anti-rabbit antibodies are commonly used as secondary antibodies, binding to rabbit primary antibodies to enable detection and signal amplification. In drug discovery, however, they can also be employed in Surface Plasmon Resonance (SPR) to serve as capture ligands. This enables the immobilization of rabbit-derived antibodies on the chip surface and allows for the screening and kinetic characterization of their interactions with target analytes in real time.

This application note presents an innovative use of SPR, as to our knowledge, no model currently exists using immobilized anti-rabbit antibodies as capture ligands for rabbit antibodies on sensor chips.

Objective of the Study: This study, conducted in collaboration with a major pharmaceutical company, aimed to evaluate the robustness and reproducibility of SPR chips functionalized with goat anti-rabbit antibodies on our K-One surface chemistry over multiple capture/regeneration cycles.

I- Material & Methods

• Instrument: Biacore T200;
• Ligand: goat anti-rabbit antibody;
• Captured ligand for reproducibility study: purified and non-purified rabbit antibodies;
• Control materials for binding efficiency study: control rabbit antibodies and control analyte;
• Surface chemistry: K-One
  
  • K-One is Kimialys' proprietary surface chemistry. By coating the surface with K-One, we gain precise control over ligand orientation, density, and distribution, while the surface remains fully protected from undesired interactions.

Description of the figure 1

Table summarizing the reproducibility study setup. Rabbit antibodies at 5 µg/mL concentration were injected during 50 cycles in multiple cycle kinetics mode. Each 10 cycles an injection of the control rabbit antibody followed by injection of the control analyte were performed to ensure the stability and efficiency of the performance of captured rabbit antibodies.

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II- Experimental results

Description of the figure 2

Table summarizing the reproducibility of rabbit antibodies capture efficiency as a function of surface density of immobilized anti-rabbit antibody.

Results

• The variability of rabbit Ab injections was very low across all tested chips, regardless of the antibody type (purified or non-purified), demonstrating the robustness of the K-One surface chemistry, even with complex matrices.
• The slightly higher CV observed on Chip #1 (with CV up to 6.3%) is attributed to fewer initial startup cycles, which were adjusted in subsequent chips resulting in CV ≤ 2.6%.

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Conclusion of the study

1. The objective of the study was to evaluate the robustness and reproducibility of K-One SPR sensor chips functionalized with goat anti-rabbit antibodies.

2. The system demonstrated consistent antibody capture over multiple capture/regeneration cycles, as reflected by a low coefficient of variation (CV ≤ 2.6% once the start-up cycles number was optimized).

3. The system also performed reliably with non-purified antibodies, demonstrating its suitability for complex sample matrices.

4. The Kimialys K-One SPR sensor chip rabbit model offers a reliable and robust solution for teams working with rabbit antibodies, thereby addressing the current lack of commercially available anti-rabbit capture chips.

Note: K-One surface chemistry enables direct ligand immobilization without intermediates and can integrate seamlessly with capture systems like streptavidin, protein A/G, anti-FC and anti-Fab.

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Kimialys in a Nutshell

Kimialys is an innovative industrial company specialized in biosensing, enabling laboratories around the world to develop and produce tomorrow’s diagnostics and therapeutics.

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Kimialys is leveraging a proprietary and patented surface chemistry, K-One, applied to nanoparticles and chips, to advance biomolecule detection and characterization to the next level. Our approach enhances key assay parameters, such as sensitivity, specificity, selectivity, reproducibility as well as cost-effectiveness, without compromising one for another.

Combining this technology platform with our expertise in ligand immobilization and biosensors, we enable cutting-edge applications in the field of diagnostic development, drug discovery and bioanalysis.

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