Generation and evaluation of multimers of anti C-reactive protein Nb and alkaline phosphatase based on three...

Nanobodies (Nbs) have been extensively utilized in medical diagnosis and therapies due to their advantages, such as ease of genetic manipulation and efficient soluble expression in prokaryotic systems. However, enhancing their binding affinity to antigens to match or even exceed that of polyclonal antibodies for high-sensitivity bioassays remains a challenge. Therefore, in the present study, a novel strategy to enhance the binding affinity of antibodies to antigens was developed by fusing an anti-C-reactive protein Nb (CRPNb) to the N-terminus of self-assembling peptides such as right-handed coiled coil [RHCC; derived from a right-handed coiled-coil peptide of an archaebacterium (Staphylothermus marinus)], verotoxin (VTB; the B-subunit of Escherichia coli verotoxin) and C4-binding protein (C4bp; derived from human plasma C4-binding protein α-chain). For functional detection, alkaline phosphatase (AP) was further fused to the C-terminus of RHCC, VTB and C4bp, respectively. This approach enabled the formation of CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers. These were then expressed as soluble cytoplasmic proteins in the E. coli strain BL21 (DE3) and purified by imidazole elution. The protein expression was assessed by western blotting. Additionally, the stability of the multimeric constructs was evaluated using a direct ELISA, whilst their sensitivity was assessed using a competitive ELISA. The CRPNb-VTB-AP pentamers and CRPNb-RHCC-AP tetramers demonstrated antigen-specific recognition and enhanced affinity compared with the CRP-AP monomer based on a direct ELISA. Furthermore, they exhibited good thermal stability based on a direct ELISA. At 80˚C, CRPNb-VTB-AP, CRPNb-AP and CRPNb-RHCC-AP retained ~85, 75 and 60% activity, while CRPNb-H only retained ~40%. After incubation at 80˚C for 55 min, the two multimers still maintained ~40% activity. The results suggested them to be suitable for storage and transportation at ambient temperatures. In conclusion, a novel nanobody-fusion protein platform was established in the present study. The fusion proteins functioned as high-affinity binders for target antigens and heat-stable signal tracers for immunoassay detection and quantitative analysis, providing a novel type of thermostable immunoreagent.