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Development of a highly thermostable immunoassay based on a nanobody-alkaline phosphatase fusion protein for carcinoembryonic antigen detection


Carcinoembryonic antigen–related cell adhesion molecule 5 (CEACAM-5) assays are employed in routine clinical settings to diagnose tumor. We selected two nanobodies with high-affinity to CEACAM-5, termed Nb11C12 and Nb2D5, using phage-display technology. The Nb2D5 fused with calf intestinal alkaline phosphatase (CAP), human placental alkaline phosphatase (HAP), or Pyrococcus abyssi alkaline phosphatase (PAP) were expressed in human embryonic kidney (HEK293) cells. The enzymatic activity of Nb2D5-HAP fusion protein was the best and remained stable at 60 °C for 7 days. The affinity of Nb2D5-HAP fusion protein to CEACAM-5 reached 42 pM. A chemiluminescent enzyme immunoassay (CLEIA) based on Nb2D5-HAP fusion protein was established for quantitative CEACAM-5 assay in clinical settings. The CLEIA exhibited a wide linear range of 0.31–640 ng/mL toward CEACAM-5, with a limit of detection (LOD) of 0.85 ng/mL. No cross-reactivity occurred with CEACAM-1, CEACAM-3, CEACAM-6, or CEACAM-8, and no interference was observed with rheumatoid factors. The CLEIA based on Nb2D5-HAP fusion protein was stable for 8 weeks at 37 °C and 50% relative humidity. The CLEIA developed from Nb2D5-HAP fusion protein had much better stability and linearity with similar reproducibility compared with the enzyme-linked immunosorbent assay developed from conventional monoclonal antibodies, which have been widely used in clinics over the past several decades.

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  1. 1.

    Graham RA, Wang S, Catalano PJ, Haller DG. Postsurgical surveillance of colon cancer - preliminary cost analysis of physician examination, carcinoembryonic antigen testing, chest x-ray, and colonoscopy. Ann Surg. 1998;228(1):59–63.

  2. 2.

    Muyldermans S, Cambillau C, Wyns L. Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci. 2001;26(4):230–5.

  3. 3.

    Huo J, Li Z, Wan D, Li D, Qi M, Barnych B, et al. Development of a highly sensitive direct competitive fluorescence enzyme immunoassay based on a nanobody-alkaline phosphatase fusion protein for detection of 3-phenoxybenzoic acid in urine. J Agric Food Chem. 2018;66(43):11284–90.

  4. 4.

    Liu X, Xu Y, Wan DB, Xiong YH, He ZY, Wang XX, et al. Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal. Anal Chem. 2015;87(2):1387–94.

  5. 5.

    Wang K, Liu Z, Ding G, Li J, Vasylieva N, Li QX, et al. Development of a one-step immunoassay for triazophos using camel single-domain antibody-alkaline phosphatase fusion protein. Anal Bioanal Chem. 2019;411(6):1287–95.

  6. 6.

    Swain MD, Anderson GP, Serrano-González J, Liu JL, Zabetakis D, Goldman ER. Immunodiagnostic reagents using llama single domain antibody-alkaline phosphatase fusion proteins. Anal Biochem. 2011;417(2):188–94.

  7. 7.

    Wang J, Majkova Z, Bever CR, Yang J, Gee SJ, Li J, et al. One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein. Anal Chem. 2015;87(9):4741–8.

  8. 8.

    Liu JL, Zabetakis D, Lee AB, Goldman ER, Anderson GP. Single domain antibody-alkaline phosphatase fusion proteins for antigen detection-analysis of affinity and thermal stability of single domain antibody. J Immunol Methods. 2013;393(1–2):1–7.

  9. 9.

    Knoll BJ, Rothblum KN, Longley M. Two gene duplication events in the evolution of the human heat-stable alkaline phosphatases. Gene. 1987;60(2–3):267–76.

  10. 10.

    Zappa S, Rolland JL, Flament D, Gueguen Y, Boudrant J, Dietrich J. Characterization of a highly thermostable alkaline phosphatase from the Euryarchaeon Pyrococcus abyssi. Appl Environ Microbiol. 2001;67(10):4504–11.

  11. 11.

    Millán JL. Mammalian Alkaline Phosphatases: From biology to applications in medicine and biotechnology. Weinheim: Wiley–VCH; 2006. p. 1–322.

  12. 12.

    Li D, Cui Y, Morisseau C, Gee SJ, Bever CS, Liu X, et al. Nanobody based immunoassay for human soluble epoxide hydrolase detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement: the rediscovery of PolyHRP? Anal Chem. 2017;89(11):6248–56.

  13. 13.

    Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken LG, et al. Single-domain antibody fragments with high conformational stability. Protein Sci. 2002;11(3):500–15.

  14. 14.

    Zhang J, Tanha J, Hirama T, Khieu NH, To R, Tong-Sevinc H, et al. Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents. J Mol Biol. 2004;335(1):49–56.

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This work was supported by Dongguan Social Science and Technology Development (Key) Project (201850715040330) and the National Natural Science Foundation of China (21676286).

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Correspondence to Rui Nian or Haipeng Song.

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The authors declare that they have no competing interests.

Research involving human participants

All procedures involving humans were conducted in compliance with the relevant Chinese laws and the Chinese Academy of Sciences regulations. This study received ethical approval from the Ethics Committee of Chinese Academy of Sciences and the local Ethical Board of Dongguan Dalang Hospital. Signed informed consent of all individual participant samples was obtained.

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Lin, J., Yu, J., Wang, H. et al. Development of a highly thermostable immunoassay based on a nanobody-alkaline phosphatase fusion protein for carcinoembryonic antigen detection. Anal Bioanal Chem (2020). https://doi.org/10.1007/s00216-020-02456-4

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  • Nanobody
  • Alkaline phosphatase
  • Thermostability
  • Chemiluminescent enzyme immunoassay
  • Carcinoembryonic antigen