High-affinity VNARs targeting human hemoglobin: Screening, stability and binding analysis.

Hemoglobin, composed of α- and β-chains, is essential for oxygen transport and is key in diagnosing and treating gastrointestinal and blood disorders. It also aids in detecting blood contamination and estimating transfusion volumes. Immunological methods, based on antigen-antibody interactions, are distinguished by their high sensitivity and accuracy. Consequently, it is necessary to develop hemoglobin-specific antibodies characterized by high specificity and affinity to enhance detection accuracy. The variable domain of the new antigen receptor (VNAR) from sharks, the smallest antigen-binding unit, is ideal for disease diagnosis and treatment due to its small size, stability, and high affinity. In this study, Chiloscyllium plagiosum was immunized with human hemoglobin protein. Nine VNAR immune libraries with sizes ranging from 1 × 10 to 1.82 × 10 colony-forming units (CFU) were constructed and biopanned using phage display, resulting in three hemoglobin-specific VNAR sequences (5-10C, 7-11 A, T-12-4D). These sequences were inserted into pTT5-TEV-Fc vectors and transfected into HEK 293F cells. The resulting VNAR-Fc fusion proteins were purified from the cell culture supernatants. Binding activity, cross-reactivity, physicochemical stability, and epitope competition were evaluated using non-competitive enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-12-4D-Fc exhibited the highest affinity with a K value of 7.59 nM and superior physicochemical stability. It maintained over 80 % binding activity at 90 °C, over 51 % in extreme pH conditions (pH 2 and 12), and above 65 % in urea concentrations up to 8 mol/L. Its binding activity remained largely unaffected after 6 h of incubation in human plasma-like medium (HPLM). The binding epitope competition results showed that 5-10C-Fc and T-12-4D-Fc targeted the same hemoglobin epitope. Molecular dynamics simulations revealed hydrogen bonds as the primary interaction force between VNARs and hemoglobin. Furthermore, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) method was established for the detection of human hemoglobin, utilizing T-12-4D-Fc as the coating antibody. This technique demonstrated high accuracy, reproducibility and specificity when applied to human whole blood samples. Hence, the identified VNARs, particularly T-12-4D, demonstrated good stability, specificity, and high affinity, filling the gap of hemoglobin-targeting shark-derived single-domain antibodies and offering a foundation for diagnosing and monitoring hemoglobin-related diseases.