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Label-Free Kinetic Measurements Using SoPRano™ Gold Nano Rods (GNRs) and a Spectrometer Microplate Reader

Meike Roskamp, Jessika Meuvis, PharmaDiagnostics, Zellik, Belgium; David Myszka, Biosensor Tools, Utah, USA; E.J. Dell, BMG LABTECH, GmbH, Ortenberg, Germany, 09/2012

  • Label-free kinetics without expensive, dedicated instrumentation
  • Easily measure the SoPRano™ label-free LSPR signal with a BMG LABTECH spectrometer-based microplate reader
  • Determine binding constants (KD), plus association (ka) and dissociation (kd) rates with MARS Data Analysis Software

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Introduction

Using Localized Surface Plasmon Resonance (LSPR), the SoPRano™ Gold Nano Rods (GNR) allow users to design and run label-free, microplate-based interaction analysis. The interaction of a ligand protein with an amine coupled GNR immobilized protein causes a concentration dependent and highly reproducible red-shift of the LSPR peak (Fig 1). The specificity of the LSPR signal was previously demonstrated by separately conjugating Human Serum Albumin (hSA) and Bovine Serum Albumin (bSA) to the SoPRano™ GNRs and checking the selectivity of the signal using two specific monoclonal antibodies raised against hSA and bSA, respectively(1).

Here a glycerol concentration series was used to demonstrate a linear change in response with respect to the change in refractive index (Fig 2). Furthermore, simulation data show the technology's potential to characterize binding constants for protein-protein interactions using the MARS Data Analysis software (Fig 3).

With MARS application specific templates, the SoPRano™ ratiometric calculations are easily extracted from the spectral measurements taken with a spectrometer based microplate reader. In figure 4, the SPECTROstar Omega was used show how binding rates can be calculated from an antibody-antigen interaction. The template enables the calculation of KD values, EC/IC50s, as well as the association (ka) and dissociation (kd) rates. The SoPRano™ Gold Nano Rod technology will open up new applications for label-free SPR analysis.

Assay Principle

The SoPRano™ platform enables the monitoring of binding events (i.e. between two proteins) based on the detection of refractive index changes. This LSPR (localized surface plasmon resonance) based technology is an adaptation of the widely-used SPR technology (2).

How it works

  • Conjugate your protein to the gold nanorods (GNRs).
  • Capture the full absorbance spectrum of the GNR-protein conjugate to determine the λmax, since each GNR-protein conjugate will be different.
  • Add the protein ligand to the GNR-protein conjugate and measure a full spectrum. A dose-dependent red shift in λmax will occur upon specific interaction.
  • For kinetic measurements, full spectrum were taken every ten seconds for 3000 seconds. For slow kinetics, measurements may need to be taken for a longer time period; and for fast kinetics, measurements may need to be taken more frequently.

Fig.1: Gold Nano Rods have an LSPR signal that can be detected with
full spectral absorbance. The LSPR peak red-shifts upon ligand binding
to the GNR surface, which is instantly captured by the SPECTROstar Nano.

Materials and Methods

  • Human serum albumin (hSA) (Sigma-Aldrich) and an hSA antibody (ab18081, Abcam) were used for kinetics studies
  • SoPRano™ Kit was from Pharma Diagnostics (Belgium)
  • SPECTROstar Omega and SPECTROstar Nano microplate reader and MARS data analysis from BMG LABTECH (Germany)
  • Non-binding, 384-well, black microplates with clear bottoms from Greiner (Germany)

Fig. 2: Absorbance peak increases and shifts to higher wavelengths with
increasing refractive index. Ratio of ODλmax+80nm/λmax shows a
linear response with the change in refractive index.
Based on the observed signal to noise of the SoPRano™ gold nanorods,
the lower limit of detection of the analyte would be ~9kDa for a 50kDa ligand.

 

hSA was conjugated to Gold Nano Rods (GNRs) via the proteins' free amine using EDC and sulfo-NHS. Using the SPECTROstar Nano, the λmax of both systems were determined at a 1 nm resolution (see figure 1 as an example). Using the MARS data analysis software and calculation templates, ratiometric analysis was easily done (ΔRu = λmax+80nm/λmax) and binding constants were determined.

 

Fig. 3: Determining kinetics with MARS Data Analysis. Simulated data
sets with 10% random noise using the same association rate constant
(ka = 1e5 M-1s-1) but different dissociation rates (kds from 1e-2 to 1e-6 s-1).
Using MARS, the correct ka was returned for each data set (1e5 M-1s-1).
The standard error in the output kd increases only below 1e-5 s-1.
This demonstrates that systems with slower kds will require longer data collection time.

Results and Discussion

In figure 3, simulated data was used with 10% noise to show that the MARS data Analysis software can readily calculate kinetic binding rates. In figure 4, kinetic curves of hSA-GNRs binding to different concentrations of an hSA antibody were created from full spectral measurements.

 

Fig. 4: Human serum albumin was coupled to the SoPRano™ gold nanorods.
An antihuman albumin antibody was tested for binding. MARS Data Analysis
software was used to produce binding kinetics from the spectral kinetic data.

 

Using a one-site hyperbola binding fit and the last kinetic cycle when equilibrium is reached, a dose response curve can be created which gives the binding constant KD (not shown). The kinetic measurements were used to produce association (ka) and dissociation (kd) rates via a binding kinetic equation in the MARS Data Analysis software.

Conclusion

The label-free SoPRano™ assay from Pharma Diagnostics was performed on BMG LABTECH's SPECTROstar Omega and Nano microplate readers. This platform enables high-throughput, plate-based, homogeneous LSPR assays to be performed for the determination of protein-protein interactions. Using the SPECTROstar Omega and Nano, full spectra are quickly captured in less than one second per well. Full spectra are needed for the SoPRano™ assay to determine the shift in the λmax upon binding. MARS Data Analysis software easily allows for the ratiometric analysis that is needed for this assay. In addition, binding kinetics algorithms in the MARS Software allow for the determination of binding constants (KD) and binding rates (ka and kd).

BMG LABTECH spectrometer-based microplate readers include: SPECTROstar Nano, SPECTROstar Omega, FLUOstar Omega, POLARstar Omega, and PHERAstar FS. The SPECTROstar Nano also has a cuvette port which will aid in SoPRano™ label-free assay development.

References

  1. E.J. Dell, Meike Roskamp, Frederik Van de Velde. Label-Free SoPRano™ Gold Nano-Rod (GNR) Assays on a Spectrometer-Based Microplate Reader. Application Note 230. July 2012.
  2. Advances in surface plasmon resonance biosensor analysis. Rich RL,Myszka DG. Curr Opin Biotechnol. 2000 Feb;11(1):54-61.Review.
  3. For more information on SoPRano™ label-free assays please refer to www.pharmadiagnostics.com.