Initial Development of SARS-CoV-2 Antigen Assay Using BAMS Technology and MALDI-TOF

Significance of the topic

The rapid and reliable detection of SARS-CoV-2 antigens remains essential for controlling the COVID-19 pandemic. While RT-PCR tests dominate diagnostics, antigen assays offer faster turnaround and simpler workflows. The integration of Bead Assisted Mass Spectrometry (BAMS) with MALDI-TOF mass spectrometry provides a novel route to capture and identify viral proteins directly, leveraging the specificity of engineered Affimer reagents. This approach promises high throughput, multiplex capability and compatibility with benchtop instruments, addressing the need for scalable clinical testing platforms.

Objectives and overview

This study aimed to develop and validate a SARS-CoV-2 spike glycoprotein subunit 1 (S1) antigen assay using BAMS technology combined with MALDI-TOF detection. Key goals included:

• Immobilize S1-specific Affimer molecules on magnetic beads for selective antigen capture.
• Demonstrate detection of both intact S1 protein and its tryptic peptides.
• Establish assay performance on a high-performance rapifleX TOF/TOF system and transfer the method to microflex benchtop MALDI-TOF instruments.

Used instrumentation

• BAMS high-binding magnetic beads functionalized with covalently linked Affimer reagents.
• Bruker rapifleX TOF/TOF system (10 kHz smartbeam 3D laser) for initial development.
• Bruker microflex smart LS (200 Hz smartbeam laser) and microflex LT (60 Hz nitrogen laser) for benchtop analysis.
• Disposable steel MALDI plates and MSP 96 ground steel plates for sample deposition.
• Software: flexAnalysis for peak processing; BioPharma Compass and BioTools for sequence coverage and MS/MS interpretation.

Methodology

• Preparation of beads: Covalent coupling of S1-specific Affimer to BAMS beads.
• Affinity capture: Incubation of beads with recombinant SARS-CoV-2 S1 glycoprotein in PBS containing digested BSA, followed by washing.
• Intact protein analysis: Elution with DHAP matrix directly onto MALDI plates; MALDI-TOF acquisition in linear mode (10–300 kDa range).
• Bottom-up workflow: On-bead tryptic digestion (1 h at 37 °C), elution with CHCA matrix; MALDI-TOF acquisition in reflector mode (700–7000 Da range) and TOF/TOF MS/MS.
• Data processing: Peak picking, mass calibration, and sequence coverage calculations using dedicated software.

Main results and discussion

• Intact S1 detection: Broadened [M+H]+ envelope observed due to multiple glycoforms; comparable signals on rapifleX and microflex smart LS.
• Peptide-level detection: On-bead digest yielded distinct S1 peptide peaks absent in Affimer controls; butterfly plots confirmed specificity across m/z 800–5000.
• Sequence coverage: On rapifleX, 65.0 % MS and 22.5 % MS/MS coverage; on microflex platforms, MS coverage ranged from 28.5 % to 39.4 % for elutions from 1 and 5 beads.
• Instrument transfer: Although benchtop sequence coverage was lower than high-performance system, data quality supports method translation to clinical MALDI-TOF instruments.

Benefits and practical applications

• High specificity capture using Affimer reagents offers an alternative to antibodies, with potential for multiplex assays.
• Dual detection modes (intact and peptide) enhance assay flexibility and sensitivity.
• Compatibility with benchtop MALDI-TOF devices enables deployment in clinical and routine laboratory settings.
• Rapid sample preparation and MS readout support high-throughput screening of patient samples.

Future trends and potential applications

• Optimization of detection limits through sample enrichment and advanced data processing algorithms.
• Extension of BAMS-MALDI workflows to detect emerging SARS-CoV-2 variants or co-capture multiple viral targets.
• Integration with automated liquid handling and MALDI staging for fully automated clinical assays.
• Adaptation to portable or point-of-care MALDI devices for decentralized testing solutions.

Conclusion

The presented BAMS-based MALDI-TOF assay demonstrates selective capture and detection of SARS-CoV-2 S1 glycoprotein using Affimer-functionalized beads. Both intact protein and bottom-up peptide workflows deliver reliable mass spectrometric signatures, and successful transfer from a high-performance TOF/TOF system to microflex benchtop instruments underlines the method’s clinical applicability. This platform offers a promising avenue for rapid, multiplexed antigen diagnostics in pandemic response.

References

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4. Adeptrix Corporation. SARS-CoV-2 S1 Glycoprotein product information.