Customer Breakthrough: Advancing Immunotherapy with SpectraMax Microplate Readers

The Challenge

The field of HLA research plays a critical role in immunotherapy target discovery, peptide validation, TCRm antibody generation, and TCR screening. At Pure Protein, we specialize in high-accuracy and precision IC50 determination to validate HLA peptide epitope targets before advancing to drug development. Our work extends to epitope mapping of oncogenic proteins and viruses like Influenza and HIV.

To achieve this, we rely on state-of-the-art peptide binding assays for HLA Class I molecules using Fluorescence Polarization (FP). This technology allows us to measure the binding affinity of peptides to HLA molecules in real time, providing crucial insights into molecular interactions without the need for separation steps. The assay works by incubating a reference fluorescent-labeled peptide with activated soluble HLA (sHLA) in the presence of a competitor peptide. A successful binding event results in an increase in polarization values due to the slower molecular rotation of the bound probe. The effectiveness of the peptide is then quantified through logIC50 values derived from dose-response curves.

When we started Pure Protein over 20 years ago, suitable equipment for executing an assay like ours was limited. That’s when Molecular Devices first stepped in, providing us with the Analyst AD instrument, which offered the fine-tuned optimization required for our assays. This instrument delivered outstanding performance and operated flawlessly for over two decades. However, when it reached the end of its service life, we faced a major challenge: the Xenon Arc lamp technology that enabled its extreme sensitivity was no longer in use. Finding a replacement capable of matching its performance became our top priority.

The Solution

Molecular Devices stepped in once again, offering expert guidance and access to their latest instrumentation. Understanding our stringent requirements, they facilitated on-site testing of multiple systems to ensure compatibility with our well-established FP assay protocols. After extensive evaluation, we identified the SpectraMax Paradigm as the perfect successor.

The transition was seamless—our assay outputs from the SpectraMax Paradigm were virtually identical to those from the Analyst AD, demonstrating negligible variation. This meant that all historical data remained relevant, eliminating the need for system re-qualification. The Paradigm’s advanced optical system provided the sensitivity and precision required for our FP assays while also expanding our capabilities in fluorescence absorbance, OD measurements, and quality control testing for fluorophore-labeled HLA tetramers.

SpectraMax Paradigm Multi-Mode Microplate Reader

Details

Products Used

The SpectraMax Paradigm is a versatile multi-functional platform that extends beyond FP. It also supports fluorescence absorbance, OD measurements, and more. This flexibility allowed us to integrate additional quality control assays, such as fluorophore-labeled HLA tetramer validation across multiple wavelengths, further enhancing our research capabilities.

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The Results

Extensive testing followed to ensure that the new instrument could match the performance of our previous system. We conducted a series of comparative assays, confirming that the data generated remained fully aligned with our historical results. This meant that past and future data sets would be consistent, preserving the integrity of our research and ensuring continuity for ongoing projects.

The transition to the SpectraMax Paradigm also enabled us to expand the scope of our work beyond initial expectations. Today, we manufacture 128 different HLA alleles, with 58 successfully converted into assays for FP-based peptide binding validation—an achievement that sets a world record in our field.

Figure 1

Figure 1: The key experiment run during the validation process. The figure illustrates the comparative assay results between the Analyst AD and the SpectraMax Paradigm, demonstrating that logIC50 values, R² values, and Hill slopes remained in high-confidence alignment, validating the seamless transition.

Figure 2

Figure 2: The expansion of our capabilities using Molecular Devices' equipment. This figure highlights our ability to develop FP-based peptide binding assays for 58 HLA alleles, setting a new benchmark in the industry.

References

Pure Protein Website: www.hlaprotein.com

1. Development and validation of a fluorescence polarization-based competitive peptide-binding assay for HLA-A*0201--a new tool for epitope discovery. Buchli R, et al. Biochemistry. 2005;44(37):12491-507.
2. Real-time measurement of in vitro peptide binding to soluble HLA-A*0201 by fluorescence polarization. Buchli R, et al. Biochemistry. 2004;43(46):14852-63.
3. Critical factors in the development of fluorescence polarization-based peptide binding assays: an equilibrium study monitoring specific peptide binding to soluble HLA-A*0201. Buchli R, et al. J Immunol Methods. 2006;314(1-2):38-53.
4. Computational prediction of MHC anchor locations guides neoantigen identification and prioritization. Xia H, et al. Sci Immunol. 2023;8(82):2200.

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