Screening and Identification of Anti-CEA Nanobody and Construction of a Double-nanobody ELISA for CEA Detection

doi:10.13523/j.cb.2307014

China Biotechnology

2024, Vol. 44

Issue (2/3): 48-58 DOI: 10.13523/j.cb.2307014

Screening and Identification of Anti-CEA Nanobody and Construction of a Double-nanobody ELISA for CEA Detection

WANG Xinting,HU Qianqian,LOU Chu,YANG Tianning,LI Jiangwei^*(

)

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China

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Abstract

Objective: To screen nanobodies that bind to carcino-embryonic antigen (CEA) with high affinity for the development of rapid detection methods for CEA in the future. Methods: Using human CEA protein to immunize Bactrian camels, lymphocyte cDNA was used as a template, and nested PCR was used to amplify the variable domain of heavy chain of heavy-chain antibody (VHH) gene, ligated to pMECS vector, and electroporated into TG1 E. coli to construct a VHH cDNA library. After infection with helper phage M13K07, a phage display library was generated, and VHH clones bound to CEA were enriched by biopanning in ELISA, nanobodies with high binding to CEA were screened by periplasmic soluble ELISA (PE-ELISA), and IPTG-induced expression and affinity purification were performed in WK6. The binding specificity of the nanobodies to CEA was by measured by ELISA,and the epitope overlap was detected by competitive ELISA to screen paired nanobodies for the construction of sandwich ELISAs. Results: A VHH library with a size of 2.8×10⁸ cfu was constructed. Colony PCR showed that VHH insertion was about 100%. A total of 90 clones enriched in the 2nd-3rd round were randomly selected for PE-ELISA detection, and 45 clones were positive. Sequence analysis showed that they encoded 7 nanobody sequences. In WK6, these nanobodies are expressed in a soluble form, and recombinant nanobodies with a purity close to 90% are obtained by Ni affinity chromatography. Indirect ELISA showed that all 7 Nbs specifically bound CEA, and 2 nanobodies 3G2 and 3F4 had high affinity and concentration-dependent binding to CEA, and did not bind to the unrelated protein lysozyme even at high concentrations (5 μg/mL), showing high specificity. Competitive ELISA results showed that 3G2 and 3F4 did not compete in binding to CEA, indicating that 3G2 and 3F4 bind to different epitopes of CEA antigens. Using 3F4 as the capture antibody and 3G2 as the detection antibody, the double nanobody sandwich ELISA method (referred to as 2Nbs-ELISA) was established, and its limit of detection (LOD) for CEA in solution was 1.8 ng/mL. Conclusion: The two CEA-specific Nbs obtained in this study were used to establish a sandwich ELISA method with higher sensitivity and specificity for the detection of CEA.

Key words： CEA Nanobodies Phage display Epitope binning Double nanobody sandwich ELISA

Received: 11 July 2023 Published: 03 April 2024

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WANG Xinting, HU Qianqian, LOU Chu, YANG Tianning, LI Jiangwei. Screening and Identification of Anti-CEA Nanobody and Construction of a Double-nanobody ELISA for CEA Detection. China Biotechnology, 2024, 44(2/3): 48-58.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2307014 OR https://manu60.magtech.com.cn/biotech/Y2024/V44/I2/3/48

Table 1 Primers used for nested PCR cloning of VHH genes

Fig.1 ELISA detection the tite of CEA-specifc antibody in camel immune serum

Fig.2 Construction of VHH library A: PCR amplification of VH and VHH gene fragments from cDNA of PBMC.Left: First round of PCR;Right: Second round of PCR B: The size of VHH library was determined by the number of diluted TG1-VHH clones grown on plate C:Detecting the VHH inserts in TG1-VHH library with colony PCR. Lane 1-30: Randomly selected 30 TG1-VHH clones; -:Negative control

Table 2 Results of input and output of library panning

Fig.3 Enrichment of each round of biopanning

Fig.4 Sequence analysis of seven CEA specific nanobodies A:Different amino acids of CDR3 among seven CEA nanobodies B: Phylogenetic analysis of seven CEA nanobodies

Fig.5 SDS-PAGE analysis of IMAC purified 7 CEA specific nanobodies B: Periplasmic lysate of transformed WK6 binding on Ni - agarose resin; F: Flow-through; E: Elution of nanobodies with 500 imidazole

Fig.6 Evaluation of nanobodies binding with CEA A:Evaluation of nanobodies binding with CEA B:Detection of 7 Nbs binding with CEA at three concentrations C: Binding curves of Nbs 3F4 and 3G2 with CEA and Lysozyme

Fig.7 Analysis of epitope overlap on CEA bound with 3G2 and 3F4 by competitive ELISA A: Schematic view of epitope binning by competitive ELISA B:The binding values of M13-3F4 in the presence and absence of 3G2. Bar values represented three repeats

Fig.8 Establishment of standard curve for 2Nbs-ELISA A:Schematic view of detecting CEA with 2Nbs-ELISA B: 2Nbs-ELISA calibration curve C: Standard curve of 2Nbs-ELISA method


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