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<p>The development of stable and reproducible calibrators is critical for the standardization of many serological assays, particularly as biological standards face limitations such as variable availability and batch-to-batch variability. Here, we present a proof of concept for producing recombinant calibrators based on nanobody-Fc (Nb-Fc) fusion proteins, designed as alternatives to biological samples in serology. Using a phage display library derived from a llama immunized with SARS-CoV-2 antigens, we selected an anti-receptor-binding domain (RBD) nanobody with broad reactivity against SARS-CoV-2 variants. This nanobody was fused to the Fc region of human IgG1 and expressed in HEK293 cells, achieving high yields with enhanced production efficiency due to the incorporation of a CMV Intron A sequence. Characterization of the resulting Nb-Fc confirmed its dimeric conformation, thermal stability, and strong RBD reactivity in enzyme-linked immunosorbent assay (ELISA), establishing it as a robust standard for COVID-19 serology. The versatility of the Nb-Fc expression system was further demonstrated by generating both an anti-nucleocapsid nanobody-Fc calibrator for alternative SARS-CoV-2 diagnostic tests and a nanobody-IgE-based calibrator for allergy diagnostics, underscoring the adaptability of this approach to different antibody isotypes. The capacity to replace nanobodies within the Nb-Fc system offers a flexible strategy to quickly adapt calibrators to emerging SARS-CoV-2 variants or new diagnostic targets. This work establishes Nb-Fc chimeras as a reproducible, scalable, and long-term alternative to biological standards, with promising potential for widespread application in both infectious and non-infectious serological diagnostics.</p>

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Nanobody-Fc Fusion Proteins as Calibrators for Serological Assays

  • 期刊论文

  • 发表日期：

    2025年10月17日

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  • Belén Márquez de los Santos,
  • Florencia Castagna-Arioli,
  • Macarena Pírez-Schirmer,
  • Paula Segovia-de los Santos,
  • María Belén Crampet,
  • Federico Carrión,
  • Martín Fló-Díaz,
  • Gualberto González-Sapienza,
  • Gabriel Lassabe

摘要

The development of stable and reproducible calibrators is critical for the standardization of many serological assays, particularly as biological standards face limitations such as variable availability and batch-to-batch variability. Here, we present a proof of concept for producing recombinant calibrators based on nanobody-Fc (Nb-Fc) fusion proteins, designed as alternatives to biological samples in serology. Using a phage display library derived from a llama immunized with SARS-CoV-2 antigens, we selected an anti-receptor-binding domain (RBD) nanobody with broad reactivity against SARS-CoV-2 variants. This nanobody was fused to the Fc region of human IgG1 and expressed in HEK293 cells, achieving high yields with enhanced production efficiency due to the incorporation of a CMV Intron A sequence. Characterization of the resulting Nb-Fc confirmed its dimeric conformation, thermal stability, and strong RBD reactivity in enzyme-linked immunosorbent assay (ELISA), establishing it as a robust standard for COVID-19 serology. The versatility of the Nb-Fc expression system was further demonstrated by generating both an anti-nucleocapsid nanobody-Fc calibrator for alternative SARS-CoV-2 diagnostic tests and a nanobody-IgE-based calibrator for allergy diagnostics, underscoring the adaptability of this approach to different antibody isotypes. The capacity to replace nanobodies within the Nb-Fc system offers a flexible strategy to quickly adapt calibrators to emerging SARS-CoV-2 variants or new diagnostic targets. This work establishes Nb-Fc chimeras as a reproducible, scalable, and long-term alternative to biological standards, with promising potential for widespread application in both infectious and non-infectious serological diagnostics.