Development of a highly sensitive immunoassay based on pentameric nanobodies for carcinoembryonic antigen detection
1CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189, Songling Road, Qingdao, 266101, China; University of Chinese Academy of Sciences, No 19(A), Yuquan Road, Beijing, 100049, China.
Background:
Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5) is a well-characterized biomarker for the clinical diagnosis of various cancers. Nanobodies, considered the smallest antibody fragments with intact antigen-binding capacity, have gained significant attention in disease diagnosis and therapy. Due to their peculiar properties, nanobodies have become promising alternative diagnostic reagents in immunoassay. However, nanobodies-based immunoassay is still hindered by small molecular size and low antigen capture efficacy. Therefore, there is a pressing need to develop novel nanobody-based immunoassays with superior performance.
Results:
A novel pentameric nanobodies-based immunoassay (PNIA) was developed with enhanced sensitivity and specificity for CEACAM-5 detection. The binding epitopes of three anti-CEACAM-5 nanobodies (Nb1, Nb2 and Nb3) were analyzed. To enhance the capture and detection efficacy of CEACAM-5 in the immunoassay, we engineered bispecific nanobodies (Nb1-Nb2-rFc) as the capture antibody, and developed the FITC-labeled pentameric nanobodies (Nb3-VT1B) as the detection antibody. The binding affinities of Nb1-Nb2-rFc (1.746 × 10-10) and Nb3-VT1B (1.279 × 10-11) were significantly higher than those of unmodified nanobodies (Nb1-rFc, 4.063 × 10-9; Nb2-rFc, 2.136 × 10-8; Nb3, 3.357 × 10-9). The PNIA showed a linear range of 0.625-160 ng mL-1 with a correlation coefficient R2 of 0.9985, and a limit of detection of 0.52 ng mL-1, which was 24-fold lower than the immunoassay using monomeric nanobody. The PNIA was validated with the spiked human serum. The average recoveries ranged from 91.8% to 102% and the coefficients of variation ranged from 0.026% to 0.082%.
Significance And Novelty:
The advantages of nanobodies offer a promising alternative to conventional antibodies in disease diagnosis. The novel PNIA demonstrated superior sensitivity and high specificity for the detection of CEACAM-5 antigen. This bispecific or multivalent nanobody design will provide some new insights into the design of immunoassays for clinical diagnosis.
Insights
A novel pentameric nanobody immunoassay (PNIA) enhances sensitivity and specificity for detecting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5). This improved assay offers a promising alternative for cancer diagnostics.
Area of Science:
- Biochemistry
- Immunotechnology
- Nanotechnology
Background:
- Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5) is a key biomarker in cancer diagnostics.
- Nanobodies offer unique advantages for immunoassays due to their small size and high affinity.
- Current nanobody immunoassays face limitations in sensitivity and antigen capture efficacy.
Purpose of the Study:
- To develop a novel pentameric nanobodies-based immunoassay (PNIA) for enhanced detection of CEACAM-5.
- To engineer bispecific nanobodies for improved capture and detection of the target antigen.
- To evaluate the performance of the PNIA in terms of sensitivity, specificity, and clinical applicability.
Main Methods:
- Engineered bispecific nanobodies (Nb1-Nb2-rFc) as capture antibodies.
- Developed FITC-labeled pentameric nanobodies (Nb3-VT1B) as detection antibodies.
- Analyzed binding affinities and validated the PNIA using spiked human serum samples.
Main Results:
- The PNIA demonstrated significantly higher binding affinities compared to unmodified nanobodies.
- Achieved a 24-fold lower limit of detection (0.52 ng/mL) compared to monomeric nanobody assays.
- Validated PNIA showed excellent performance in spiked human serum with high recovery rates (91.8%-102%) and low coefficients of variation (0.026%-0.082%).
Conclusions:
- The novel PNIA exhibits superior sensitivity and specificity for CEACAM-5 detection.
- Bispecific and multivalent nanobody designs represent a promising strategy for advancing immunoassay development.
- This approach offers new insights for designing improved immunoassays for clinical cancer diagnosis.
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