(T0930-01-06) Improved SPR Signal Enhancement in Bioanalytical Assays Using Micropolymer-Conjugated IHC Reagents

Purpose: Surface plasmon resonance (SPR) has long been demonstrated as a suitable and reliable platform for label-free, sensitive, and real-time monitoring of biomolecular interactions. SPR can be applied to the detection of clinical targets in bioanalytical contexts, even in complex matrices like blood serum, when innovative approaches are used to improve the SPR signal response [1]. Signal enhancement techniques aim to improve sensitivity—amplifying the signal by means of secondary, tertiary, or higher order binding events subsequent to the capture of analyte [2]. Here, we introduce a facile means for enhancing SPR signal with cross-linked polymers of the kind used in immunohistochemistry / immunocytochemistry (IHC) applications [3]. If properly developed, such molecules would join other critical reagents in the PK, PD, and IG assays associated with clinical and preclinical drug development.

Methods: SPR assays were performed on a Biacore 8K at 25 °C in HBS-EP+ running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20). Glycine, pH 2.0, was used as regeneration solution. Purified human beta-2-microglobulin (B2M) and mouse anti-human (MAH anti-B2M) monoclonal antibody were purchased from BioRad. Rabbit anti-human (RAH anti-B2M) polyclonal antibody was purchased from ThermoFisher. Polymerized horse anti-rabbit (HAR anti-IgG) reagent was purchased from Vector Laboratories. MAH anti-B2M was immobilized to ca. 250 RU using standard amine coupling methods, with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and a CM5 Sensor Chip. Following immobilization, B2M was captured onto the prepared surface to an initial level of 130 RU. RAH anti-B2M was next bound to the captured analyte in a second injection shortly following the B2M. Lastly, the polymeric HAR anti-IgG (IHC enhancement reagent) was introduced onto the surface. The overall signal increase induced by the enhancer combination of RAH anti-B2M + HAR anti-IgG polymer was calculated as a percentage of the initial response from B2M alone.

Results: Strategies for signal enhancement in SPR assays often turn to conjugation of antibodies (or fragments thereof), onto a nanoparticle scaffold. Nanoparticles are large, high-MW structures that, when sequestered near the biosensor surface, can have a strong effect on the SPR signal. However, complications in using a nanoparticle-based enhancement mechanism include low yields during antibody conjugation and purification, poor solubility of the conjugate, and nonspecific interactions with the biosensor surface [4,5]. When we encountered these obstacles, we turned to a novel approach, whereby cross-linked polymers of the kind used in IHC applications were employed in the SPR binding sequence. Conjugated IHC reporters are comprised of separate antibody and enzyme molecules cross-linked via a micro-polymer. The design of these reagents is intended to provide increased staining contrast by multiple enzyme copies localized to the region of antibody binding. We find that the features which lead to improved performance as an IHC staining reagent also benefit SPR signal enhancement, namely the large MW and high index of refraction of the polymer and enzyme groups. We demonstrate effectiveness with SPR detection of beta-2-microglobulin (B2M) analyte, which is a known biomarker for kidney disease, hematological malignancies, and multiple myeloma. When B2M is captured at low levels on an SPR biosensor surface, introduction of the IHC polymer reagent (after initial RAH anti-B2M binding), improves enhancement over two-fold, and gives a ca. 15x response increase overall (FIG 1). This result indicates the broader utility of the reagent class, and opens the door to further advancements in SPR signal enhancement.

Conclusion: A novel and effective means of using an IHC polymerized reporter enzyme reagent is demonstrated for improved signal enhancement in SPR assays. The detected signal of the biomarker analyte B2M was improved two-fold by use of the commercially available IHC reagent. Further enhancement is predicted, by optimization of the polymer and protein conjugate properties. Polymer-conjugated antibody-based, like the type introduced here, are therefore a promising class of critical reagents, analogous to the tool antibodies used in conventional plate-based ligand binding assays.

References: [1] Olaru A, Bala C, Jaffrezic-Renault N, Aboul-Enein HY. Surface plasmon resonance (SPR) biosensors in pharmaceutical analysis. Critical reviews in analytical chemistry. 2015 Apr 3;45(2):97-105.
[2] Shalabney A, Abdulhalim I. Sensitivity‐enhancement methods for surface plasmon sensors. Laser & Photonics Reviews. 2011 Jul 1;5(4):571-606.
[3] Buchwalow I, Boecker W, Wolf E, Samoilova V, Tiemann M. Signal amplification in immunohistochemistry: loose-jointed deformable heteropolymeric HRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histochemica. 2013 Jul 1;115(6):587-94.
[4] Liang RP, Yao GH, Fan LX, Qiu JD. Magnetic Fe3O4@ Au composite-enhanced surface plasmon resonance for ultrasensitive detection of magnetic nanoparticle-enriched α-fetoprotein. Analytica chimica acta. 2012 Aug 6;737:22-8.
[5] Wang Y, Partridge A, Wu Y. Improving nanoparticle-enhanced surface plasmon resonance detection of small molecules by reducing steric hindrance via molecular linkers. Talanta. 2019 Jun 1;198:350-7.

Acknowledgements: The authors would like to thank the Critical Reagents Characterization group within Novartis Biomedical Research, including Lijuan Su and Melissa Grippo for helpful discussions, and Maria Jadhav and Jeniffer Cunliffe of Novartis BA Laboratories, for review of the submission.

A graphical representation of the poster abstract.

FIG 1: Sensorgram of B2M binding to immobilized mouse anti-human (MAH anti-B2M) monoclonal antibody. Subsequent binding of rabbit anti-human (RAH anti-B2M) polyclonal antibody amplifies the response approximately seven-fold, while adding the IHC reagent has an overall 15x signal enhancement.