January 15, 2009 (Vol. 29, No. 2)

New Approach Advocates Interspot Referencing Using Multiplexed SPR Arrays

Surface plasmon resonance (SPR) has revolutionized the study of the intricate protein interactions that are required for the execution and maintenance of complex biological processes, enabling the elucidation of the roles that intracellular concentration, ionic environment, cofactors, and protein conformation play in maintaining those processes. SPR is also a powerful tool for the rapid identification of highly specific monoclonal antibodies with high affinity for the analyte of interest, detailed evaluation of drug lead compounds, and optimization of affinity protein purification methods.

Surface plasmon resonance optical biosensing requires neither radiochemical nor fluorescent labels to provide real-time data on the affinity, specificity, and kinetics of protein interactions, and it can detect picomolar levels of proteins and other molecules as small as a few hundred daltons. SPR occurs when light interacts with a metal film placed at the interface between two media with different refractive indices, such as glass and water.

SPR biosensors respond in real time to changes in the refractive index at the interface resulting from the binding and subsequent separation of two molecules, one of which is bound to the sensor surface. As the molecule in solution (analyte) flowing over the surface binds to the immobilized molecule (ligand), the refractive index near the sensor surface increases, leading to a shift in the SPR angle. When the complex on the sensor surface dissociates, the SPR angle shifts back.

These changes in refractive index, expressed in response units (RU), are proportional to mass changes at the surface of the sensor chip. Optimized association, dissociation, and equilibrium constants can be calculated by fitting response data for a range of analyte concentrations under identical reaction conditions to a computational model.

The Traditional Approach

SPR experiments for the determination of kinetic rate constants have often been run sequentially, using two flow cells, one as a reference and one for the binding interaction (Figure 1). The reference cell did not contain any ligand and served to measure nonspecific binding of the analyte to the surface of the chip, bulk effects, and signal drift. 

A single concentration of analyte was flowed over the ligand of interest bound to the surface in the sample flow cell, and the corresponding response data were collected. The reference cell signal was subtracted from the signal in the sample flow cell. The surface was then regenerated (analyte removed) to prepare the ligand for the next concentration of analyte. 

This sequence was repeated until a full analyte concentration series was measured.  Such a sequential approach necessitates that one flow cell always be used as a reference, rather than to produce additional binding interaction data. The end result is reduced sample throughput, as well as the risk of loss of ligand activity due to repeated regeneration cycles.

Figure 1. Flow Cell geometry of a traditional SPR system

Multiplexed SPR

An alternate approach utilizes Bio-Rad Laboratories’ ProteOn  XPR36 multiplexed SPR device, which integrates state-of-the-art microfluidics with a novel optical design. A specially designed Multi-Channel Module (MCM) is used to create a unique six channel by six channel interaction array on a sensor chip for the analysis of up to six ligands with panels of up to six analytes, producing 36 data points in a single experiment (Figure 2). Because multiple conditions can be tested in parallel, robust kinetic analysis of an analyte concentration series can be handled in one experiment. This One-shot Kinetics™ parallel approach can generate a complete kinetic profile of a biomolecular interaction, without the need for regeneration, in one experiment, using one injection of analyte and a single sensor chip.

Figure 2. The ProteOn interaction array

Interspot Referencing

The ProteOn XPR 36 protein interaction array system eliminates the need for a separate channel reference and allows all channels to be used to collect binding interaction data. The 6×6 interaction array pattern not only increases the number of interactions that can be monitored on a single chip, but it also creates unique interspot areas directly adjacent to the reaction spots (Figure 3). There are 36 interaction spots and 84 interspots, 42 vertical and 42 horizontal.

In a typical ProteOn One-Shot Kinetics experiment the six vertical channels are activated and the ligand of interest is bound to them. The analyte of interest is then injected into the horizontal channels, and the binding response is measured for each interaction spot.

This process gives rise to horizontal interspots that have not been exposed to the ligand or any of the reagents used to bind the ligand to the sensor chip. These interspots can be used to measure the nonspecific interaction of the analyte with the chip surface, bulk effects, and signal drift. The data are averaged from the two interspots adjacent to the interaction spot.

The horizontal interspot signal is then subtracted from the reaction spot signal, providing the kind of reference-corrected binding data previously obtained with sequential flow cell designs. This eliminates the need to dedicate a flow cell or channel for use as a reference. In addition, the interspots provide accurate and reliable reference data due to their close proximity to the interaction spots.

The vertical interspots can be used to provide a good measure of the uniformity of the immobilization of the ligand to the sensor chip, and to monitor the potential loss of a ligand that is not covalently bound to the sensor chip, such as an antibody captured by an immunoglobulin-binding protein (e.g., protein A).

Figure 3. The ProteOn sensor chip surface

Interspot vs. Channel Referencing

In order for interspot referencing to be utilized with multiplex SPR, it must provide kinetic data equivalent to that obtained using a separate channel to determine nonspecific binding.

To demonstrate the equivalence of these two referencing methods, a model system was constructed using interleukin-2 (IL-2) as the analyte and IL-2 antibody as the ligand. Five vertical channels were created on a sensor chip using the MCM, with each channel containing the ligand at a different density. An injection of buffer only into the last vertical channel created a channel reference devoid of ligand.

The IL-2 analyte was then injected into the horizontal channels at six different concentrations to obtain complete binding kinetic data in one experiment (One-Shot Kinetics). The data was referenced using either the vertical reference channel or the horizontal interspots (where no ligand was immobilized) and is presented in the Table.

The global association and dissociation kinetic rate constants (ka and kd), as well as the equilibrium dissociation constants (KD) are virtually identical, whether interspot or channel referencing is used. In addition, when comparing these three values across the five different ligand surfaces, all the CVs are between 2–7%.  

This data also illustrates the lack of dependence of kinetic data constant values on the amount of ligand bound to the sensor chip (ligand density), since the kinetic constants were virtually identical at all five densities, using either interspot or channel referencing. Thus, multiplexed SPR can produce reliable binding analysis data across a wide range of ligand densities.


Interspot referencing using multiplexed SPR arrays to study binding interactions can produce kinetic data that are as accurate as that generated using channel referencing, allowing all interaction spots on the array to be used. The resulting increase in throughput can be critical to such large-scale applications as screening of hybridomas supernatants, evaluating large numbers of drug lead candidates, or optimization of affinity protein purification methods. 

Laura Moriarty, Ph.D. ([email protected]),
is a product manager at Bio-Rad Laboratories. Web: www.bio-rad.com.

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