Label-Free SoPRano™ Gold Nano-Rod (GNR) Assays on a Spectrometer-Based Microplate Reader

E.J. Dell, BMG LABTECH, GmbH, Ortenberg, Germany
Meike Roskamp and Frederik Van de Velde, PharmaDiagnostics, Zellik, Belgium

The SoPRano™ label-free assay requires full spectral analysis to determine the maximum absorbance wavelength (λmax)

In <1 sec/well, BMG LABTECH spectrometer-based instruments can capture a full spectrum (220-1000 nm)

With full spectral analysis the change in the spectrum that is seen upon binding to GNRs is instantly captured

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Introduction

Label-Free SoPRano™ Gold Nano-Rod (GNR) kits enable users to design and run label-free, microplate-based homogenous assays for high quality protein-protein interaction analysis based on Localized Surface Plasmon Resonance (LSPR). The interaction of a ligand with surface immobilized protein causes a concentration dependent and highly reproducible redshift of the LSPR peak of the GNR. Using the SPECTROstar Omega spectrometer-based microplate reader from BMG LABTECH, full spectra were captured and the specificity of the signal is demonstrated here. Human Serum Albumin (hSA) and Bovine Serum Albumin (bSA) were separately conjugated to the SoPRano™ GNRs and their respective monoclonal antibodies were bound at various concentrations. The Kd values derived from BMG LABTECH's MARS analysis software correspond well with Kd values obtained with other analysis software.

Using BMG LABTECH's proprietary ultra-fast CCD-based spectrometer, a full spectrum is captured in less than 1 second per well. A full spectrum measurement is necessary to define the λmax of each SoPRano™assay as this changes depending on the entity conjugated to the GNRs. BMG LABTECH spectrometer-based instruments are currently the only microplate readers that can capture full-spectrum measurements this quickly. Therefore, the SPECTROstar Omega and other BMG LABTECH spectrometer-based instruments are ideal microplate readers to perform the SoPRano™ label-free assay.

Assay Principle
The SoPRano™ platform enables the monitoring of binding events (for example 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 (1).

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 andmeasure a full spectrum. A dose-dependent red shift in λmax will occur upon specific interaction of the protein ligand with the conjugated protein.
This change in the spectrum upon interaction is due to a change in local refractive index at the GNR surface and is very reproducible.

Fig. 1: Proteins are conjugated to Gold Nano Rods via the free amine of the protein using EDC and sulfo-NHS. The graphs show how the λmax red-shifts upon conjugation.

Materials and Methods

bSA, hSA and their corresponding mono-clonal antibodies were purchased from Abcam (United Kingdom)
SoPRano™ Kit was from Pharma Diagnostics (Belgium)
SPECTROstar Omega microplate reader and MARS data analysis from BMG LABTECH (Germany)
Non-binding, 384-well, black microplates with clear bottoms from Greiner (Germany)

Fig. 2: SPECTROstar Omega microplate reader from BMG LABTECH is a spectrometer-based microplate reader that can capture a full spectrum in less than one second per well. App-style buttons and calculation templates can be created to easily perform the SoPRano™ assay on a BMG LABTECH microplate reader.

bSA and hSA were separately conjugated to Gold Nano Rods (GNRs) via the proteins' free amine using EDC and sulfo-NHS (see Figure 1). Using the SPECTROstar Omega, the λmax of both systems were determined at a 1 nm resolution (see figure 3 as an example). Increasing amounts of the antibodies (5 µL of a 16x concentration) were incubated with the conjugated GNRs (40 µL) for 50 minutes (35 µL of MES buffer was used to bring final volume to 80 µL), followed by full spectrum measurements with the SPECTROstar Omega. Using the MARS data analysis software and calculation templates, ratiometric analysis was easily done (ΔRu = λmax+80nm/λmax) and Kds were determined that correspond to other methods of analysis.

Fig. 3: This graph shows how the λmax red-shifts upon antibody binding (500 nM) to the hSA-GNRs. Using the λmax+80nm wavelength, ratiometric analysis is performed (λmax+80nm/λmax) which normalizes the data to the λmax.

Results and Discussion
Using a one-site binding model, dose response curves were produced for hSA-GNRs and its antibody (Figure 4) and for bSA- GNRs and its antibody (Figure 5). Using the opposite antibody for that system, cross-reactivity was shown not to occur. Signal to noise ratios (S:N) of more than 24 and Z'-factors greater than 0.80 prove this to be a robust and reproducible system. Furthermore, calculated Kds correspond with the expected results.

Fig. 4: Dose-response curve shows that hSA's antibody specifically binds to hSA conjugated GNRs, whereas the bSA antibody does not.

Fig. 5: Dose-response curve shows that bSA's antibody specifically binds to bSA conjugated GNRs, whereas the hSA antibody does not.

Conclusion
The label-free SoPRano™ assay from Pharma Diagnostics was performed on BMG LABTECH's SPECTROstar Omega microplate reader. This platform enables high-throughput, plate-based, homogeneous LSPR assays to be performed for the determination of protein-protein interactions, such as antibody binding to antigens, as presented here. Using the SPECTROstar Omega, 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. In addition, App-style buttons can be created to easily and quickly perform the SoPRano ™assay for a BMG LABTECH spectrometer-based instrument (see Figure 2).

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

Advances in surface plasmon resonance biosensor analysis. Rich RL, Myszka DG. Curr Opin Biotechnol. 2000 Feb;11(1):54-61. Review.
For more information on SoPRano™ label-free assays please refer to www.pharmadiagnostics.com.