CRP, a crucial inflammation biomarker, necessitates accurate quantification during infections and healing processes. Nanobodies, due to their cost-effective production and higher binder density on capture surfaces, emerge as promising alternatives to conventional antibodies in immunodiagnostics. By leveraging a pre-immune library, we compared two panning strategies: one solely employing phage display and the other integrating a yeast display round following phage display. The isolated anti-CRP nanobodies exhibited low nanomolar affinity, suitable for ELISA and immunoprecipitation, with one nanobody enabling CRP quantification using electrochemical impedance spectroscopy, achieving a remarkable sensitivity of 0.21 μg/mL.
Phage display technology was utilized for the initial selection of nanobodies specific to CRP from a llama pre-immune library. Through two panning rounds, a total of 90 clones were evaluated, with 36 exhibiting positive binding. Subsequent sequencing identified 14 unique sequences, with a few clones showing notably high ELISA scores, demonstrating their potential as effective binders for CRP. The selected nanobodies were further characterized, revealing conserved framework residues and heterogeneous CDR3 regions, underscoring their diverse binding characteristics and potential for customization.
Following the successful phage display selection, the nanobody sequences were sub-cloned into a yeast display vector for further evaluation. Flow cytometry-based sorting using CRP-labeled yeast cells led to the identification of promising clones, with some clones showing consistency with those isolated through phage display. Notably, the yeast display method allowed for the quantitative assessment of surface-bound nanobodies, providing a more efficient screening process compared to traditional phage display screening methods.
The isolated nanobodies were subsequently expressed and purified, with certain clones yielding between 5 to 11 mg nanobody per liter of culture. ELISA assays confirmed the specificity of the nanobodies towards CRP, with some clones exhibiting superior binding capacities under optimized coating protocols. Additionally, the nanobodies were engineered into fusion constructs for enhanced detection, showcasing their versatility in diagnostic applications.
Furthermore, the nanobodies were fused to SpyTag for immobilization on SpyCatcher-functionalized surfaces, facilitating the development of an electrochemical impedance biosensor for CRP detection. The biosensor demonstrated a detection limit of 0.21 μg/mL, with a linear range suitable for quantifying CRP concentrations relevant to predicting high-risk coronary events. This innovative biosensor design, leveraging nanobodies as specific capture elements, holds promise for enhancing CRP detection sensitivity and selectivity.
In conclusion, the integration of phage and yeast display platforms enabled the efficient selection of nanobodies targeting CRP, showcasing their potential for advanced immunodiagnostics. The characterized nanobodies exhibited strong binding affinity and specificity for CRP, paving the way for the development of novel diagnostic tools with improved sensitivity and cost-effectiveness. Future studies focusing on further optimization and engineering of these nanobodies could lead to enhanced CRP detection assays, addressing critical needs in inflammation monitoring and disease diagnostics.
Tags: secretion, biosensors, filtration, downstream, monoclonal antibodies, yeast, chromatography
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