The cDNA sequence encoding the Nb specific to human CRP (31) and the cDNA sequence for ALP of Escherichia coli, which exhibited a 35-fold increase in specific activity after substituting the serine for the completely invariant Asp101 using site-directed mutagenesis, were previously prepared (32). Sangon Biotech Co., Ltd., provided the E. coli strain BL21 (DE3) used in the present study. The PCL expression plasmids were synthesized based on the sequence of pIG6 (33,34), with the Amp resistance gene replaced by the kanamycin resistance gene in the new vector. Our laboratory (Dalian Key Laboratory of Oligosaccharide Recombination and Recombinant Protein Modification, Medical College, Dalian University, Dalian, Liaoning, China) maintained the PCL-PelB-anti VHHs plasmid.

The cDNA sequences for anti-CRPNb-VTB-AP-histidine (H), CRPNb-C4bp-H, CRPNb-H and CRPNb-RHCC-H were obtained from Sangon Biotech Co., Ltd., and inserted into the PCL-PelB-antiGFP VHHs vector using the restriction enzymes BamHI and XhoI. The cDNA sequences for VTB (35), RHCC self-assembled peptide (36) and C4bp (17) were synthesized based on the published peptides sequences (Table SI). Restriction enzymes, DNA ligation kit reagents and various enzymes (such as LA Taq and T4 Polynucleotide Kinase) were purchased from Takara Biotechnology Co., Ltd., whilst isopropyl thio-β-D-galactoside (IPTG), kanamycin and additional reagents (such as NaCl, NaH₂PO₄ and KH₂PO₄) were sourced from Sangon Biotech Co., Ltd.

The PCL-PelB-CRPNb-VTB-AP-H and PCL-PelB-CRPNb-H vectors were subsequently digested with the restriction enzymes BamHI and EcoRI, yielding the fragments PCL-PelB-CRPNb, AP-H and PCL-PelB-CRPNb-H, respectively. T4 DNA Ligase was used to ligate the CRPNb and AP-H fragments, forming CRPNb-AP-H. Finally, the CRPNb-AP-H fragment was inserted into the PCL-PelB-anti-GFP VHHs vector using the restriction enzymes BamHI and XhoI to form PCL-PelB-CRPNb-AP-H. The recombinant plasmid was then transformed into E. coli strain BL21 (DE3) and positive clones were selected on Luria-Bertani (LB) plates containing 50 µg/ml kanamycin from Sangon Biotech Co., Ltd. Monoclonal clones were picked for digestion analysis, before the positive clones were sent to Sangon Biotech Co., Ltd., for DNA sequencing identification using Sanger sequencing.

The protocols for the expression and purification of CRPNb-H, CRPNb-AP-H, CRPNb-RHCC-AP-H, CRPNb-VTB-AP-H and CRPNb-C4bp-AP-H were largely identical. BL21 (DE3) cells harboring the PCL-PelB vectors containing CRPNb-H, PCL-PelB-CRPNb-RHCC-AP-H, PCL-PelB-CRPNb-VTB-AP-H, PCL-PelB-CRPNb-C4bp-AP-H and PCL-PelB-CRPNb-AP-H were grown overnight in LB medium (Sangon Biotech Co., Ltd.), supplemented with the appropriate antibiotics such as kanamycin (50 µg/ml), at 37˚C with shaking at 220 rpm. The following day, the culture was inoculated at a ratio of 1:100 into a medium containing 500 ml LB liquid medium and 50 µg/ml kanamycin and incubated at 37˚C with shaking until the cell optical density (OD) detected using the multifunctional microplate detector SpectraMax^® Plus 384 (Molecular Devices, LLC) at 600 nm reached ~0.6. IPTG was then added to a final concentration of 0.2 mM, before 200 ml culture was harvested after 6 h of induction at 25˚C. The pellet was resuspended in PBS (0.02 M KH₂PO₄ and 0.02 M Na₂HPO₄; pH=7.4) and then lysed by sonication on ice for 15 min of 3-sec pulses with 2-sec intervals at 36 W. The lysate was collected and centrifuged at 12,000 x g at 4˚C for 10 min. The supernatant was recovered as the ‘soluble’ fraction. After washing once with ice-cold PBS, the pellets were re-suspended in 80 µl ice-cold PBS and designated as the ‘insoluble’ fraction. An equal volume of 40 mM imidazole was added to the supernatant to a final concentration of 20 mM before filter sterilization. Tween-20 was then added into the supernatant to a final concentration of 3%. The HisTrap Excel (Sigma-Aldrich; Merck KGaA) and injection tubing were washed with ultrapure water filtered through a 0.45-µm membrane, and then equilibrated with binding buffer (0.02 M PBS, 0.5 M NaCl; pH 7.4) at a flow rate of 1 ml/min for a total volume of 20 ml. The supernatant was loaded onto the column, followed by washing with 20 mM imidazole at a flow rate of 2 ml/min for a volume of 20 ml. An elution buffer with different concentrations of imidazole (20, 40 and 80 mM) was then added to the column to elute the target proteins. Protein concentrations were measured using the BCA assay with BSA (Thermo Fisher Scientific, Inc.) as standard.

The samples corresponding to purified CRPNb-H, CRPNb-AP-H, CRPNb-RHCC-AP-H, CRPNb-VTB-AP-H and CRPNb-C4bp-AP-H were mixed with 20 µl 5X loading buffer separately and boiled for 10 min. After cooling to room temperature, 8-µl samples were separated on a 15% polyacrylamide gel and transferred onto a PVDF membrane. For the control (CRPNb-VTB-AP-H), 20 µl (240 µg/ml) was loaded. Following blocking of non-specific binding sites with 5% skim milk in TBS with Tween-20 (TBST) buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20) for 2 h at room temperature, the PVDF membrane was further incubated with anti-His antibody (MA1-21315; Thermo Fisher Scientific, Inc.) diluted 1:4,000 in TBST buffer for 1 h at room temperature. The membrane was then washed eight times (5 min each) in TBST buffer prior to incubation with the secondary antibody conjugated to horseradish peroxidase (31430; Thermo Fisher Scientific, Inc.) diluted 1:4,000 in TBST buffer for 40 min at room temperature. An enhanced chemiluminescence kit (Shanghai Epizyme Biopharmaceutical Technology Co., Ltd.; Ipsen Pharma) and a system with Image Lab^™ image capture software (version 5.2.1; Bio-Rad Laboratories, Inc.) were used for signal detection. Semi-quantitative gray analysis was performed with ImageJ software (v1.8.0; National Institutes of Health). These data are derived from at least three replicate experiments.

A direct ELISA was used to assess the activity of the CRPNb-RHCC-AP tetramers, CRPNb-C4bp-AP heptamers and CRPNb-VTB-AP pentamers. CRP antigen at 5 µg/ml was added to 96-well microplates and the plates were incubated at 4˚C for 24 h. Following this, serial dilutions of the multimers (100 µl/well) were added and the plates were incubated for 2 h at 37˚C. After washing the plates with PBS containing 0.05% Tween 20 (PBST), the microplates were incubated with a polyclonal anti-His-tag antibody (100 µl/well; diluted 1:3,000 in PBS; MA1-21315; Thermo Fisher Scientific, Inc.) at 37˚C for 1 h. Subsequently, the plates were washed again with PBST, before an HRP-conjugated goat anti-mouse secondary antibody (diluted 1:5,000 in PBST; 31430; Thermo Fisher Scientific, Inc.) was added and plates were incubated at 37˚C for 1 h.

After rinsing the plates with PBST, 100 µl 3,3',5,5'-tetramethylbenzidine substrate solution was added to each well and plates were incubated at 37˚C for 10 min. The chromogenic reaction was immediately stopped by adding 50 µl 2 M H₂SO₄ to each well. The OD of the final solution was measured at 450 nm using a multifunctional microplate reader (SpectraMax^®Plus 384; Molecular Devices, LLC).

The AP activity of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers was assessed using a calorimetric assay with p-nitrophenyl phosphate (pNPP; Dalian Meilun Biology Technology Co., Ltd.). Briefly, various concentrations (0, 0.312, 0.625, 1.25, 2.5, 5.0 and 10 µM) of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers were added to 96-well microplates at 100 µl/well. Subsequently, 50 µl/well of a 2.7-mM pNPP solution (pH 9.8), containing 1 M diethanolamine and 0.5 mM MgCl₂, was added. After incubation at 37˚C for 10 min, the chromogenic reaction was terminated by adding 3 M NaOH to each well at 50 µl/well. The OD of the solution was then measured at 405 nm using a multifunctional microplate reader.

The thermal stability of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers was evaluated using two different heat treatment methods. Initially, eight aliquots of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers were subjected to heating at 25, 30, 40, 50, 60, 70, 80 and 90˚C for 5 min each. In total, 10 aliquots of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers were heated at 80˚C for 0, 5, 15, 25, 35, 45, 55 and 65 min. Subsequently, the activity of the Nb was assessed as aforementioned. The remaining Nb activity was calculated using the following equation: Remaining Nb activity (%)=[(OD450 of heat-treated multimers)/(OD450 of untreated multimers)] x100%.

A direct ELISA was conducted to evaluate the impact of pH and PBS concentration on the Nb activity. A 5 µg/ml solution of CRP antigen was added to a 96-well microplate (442404; Thermo Fisher Scientific, Inc.), which was then incubated at 4˚C for 24 h. Following this, serial concentrations of multimers (100 µl/well) were added, with varying pH (4, 5, 6, 7, 7.4, 8, 9 and 10) and PBS concentrations (2X, 1X, 0.5X and 0.25X). The procedure outlined in the Nb activity subsection was followed for the remaining steps. The remaining Nb activity was calculated using the following equation: Remaining Nb activity (%)=[(OD450 of treated multimers)/(OD450 of untreated multimers)] x100%.

A competitive ELISA was conducted according to previously established methods (15). A checkerboard titration was utilized to determine the optimal concentration of the coating antigen and multimers. CRP (0, 0.25, 0.50, 1.0, 2.0, 5.0 and 10.0 µg/ml) and multimers (0.25, 0.5, 1.0, 2.0 and 5.0 µg/ml) were diluted at different concentrations for ELISAs to determine the optimal concentration of the coating antigen and multimers. Subsequently, the reaction conditions were optimized, including the antigen and antibody concentration (5, 2.5 or 1.25 µg/ml CRP; 3, 1.5 or 0.75 µg/ml pentamers), blocking solution (3% BSA, 1.5% BSA, 5% skim milk powder or 2.5% skim milk powder), competitive reaction duration (30, 60 or 120 min), pH value (6.0, 7.0, 7.4, 8.0 or 9.0) and PBS concentration (2X, 1X or 0.5X).

After overnight coating of the plates (442404; Thermo Fisher Scientific, Inc.) at 4˚C with 100 µl CRP coating antigen (2.5 µg/ml), the plate was blocked with 5% skimmed milk at 37˚C for 2 h. The plates were washed five times with PBST. Subsequently, 50 µl CRP and 50 µl fusion proteins, namely CRPNb-AP, CRPNb-C4bp-AP, CRPNb-VTB-AP and CRPNb-RHCC-AP, were added to each well. After incubation at room temperature for 1 h, the plates were rinsed three times with PBST, and 100 µl pNPP substrate (5 µg/ml in ethanol) was added and plates were incubated at 37˚C for 15 min. The reaction was stopped by adding 50 µl NaOH, and the absorbance was measured at 405 nm using a SpectraMax^®Plus 384 microplate reader (Molecular Devices, LLC).

The cDNA sequences for anti-CRPNb-VTB-AP-H, CRPNb-C4bp-H, CRPNb-H and CRPNb-RHCC-H were obtained from Sangon Biotech Co., Ltd., and inserted into the PCL-PelB-antiGFP VHHs vector using the restriction enzymes BamHI and XhoI. The recombinant plasmids PCL-CRPNb-RHCC-AP, PCL-CRPNb-VTB-AP and PCL-CRPNb-C4bp-AP were successfully introduced into E. coli BL21 (DE3) for the expression of tetramers, pentamers and heptamers of CRPNb-RHCC-AP, CRPNb-VTB-AP and CRPNb-C4bp-AP, respectively. The expression and purification of these multimers were then analyzed using western blotting. In the present study, CRPNb, which was derived from llama and specifically targets CRP, was used (24). The linear schematic of the proposed CRPNb and CRPNb-AP peptides is shown in Fig. 1A. Using self-assembling peptides RHCC, VTB or C4bp, CRPNb and AP were then fused to obtain recombinant constructs CRPNb-RHCC-AP, CRPNb-VTB-AP and CRPNb-C4bp-AP, respectively. The linear peptide schematics of these recombinant multimers are shown in Fig. 1B. In addition, linker 1 (APKADNKGGGGS) and linker 2 (APKADNKEFGGGGS) are derived from protein A, and can separate the different domains. Based on their amino acid composition, the calculated molecular weights of CRPNb, CRPNb-AP, CRPNb-RHCC-AP, CRPNb-VTB-AP and CRPNb-C4bp-AP are 12.85, 65.29, 72.38, 74.47 and 73.15 kDa, respectively.

Western blotting was performed using the mouse anti-His tag antibody and a precipitated ECL substrate solution. The predicted thick bands, corresponding to ~12, 65, 72, 74 and 73 kDa, matching the molecular weights of CRPNb, CRPNb-AP, CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers, respectively, could be observed (Fig. 2A). Based on a previous study, it can be inferred that β-mercaptoethanol and heating lead to the complete degradation of the CRPNb-RHCC-AP multimers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers into monomers (37). Although native PAGE was not performed in the present study, previous studies have confirmed that these recombinant constructs can efficiently self-assemble into specific oligomers: Tetramers mediated by RHCC, pentamers mediated by VTB and heptamers mediated by C4bp (15-18,35,37-41). Correspondingly, the genes fused with RHCC, VTB and C4bp can successfully express the tetrameric, pentameric and heptameric target proteins, respectively.

The recombinant proteins (CRPNb-AP, CRPNb-VTB-AP and CRPNb-C4bp-AP) were found to be expressed in both soluble and insoluble forms (Fig. 2B). CRPNb-RHCC-AP was found to be expressed in the soluble form (Fig. 2B). The soluble fraction was sonicated and the supernatant was subsequently purified. The purification process involved elution with imidazole using a HisTrap Excel column, followed by BCA protein quantification. In the present study, the relative expression levels of the multimers were determined using purified CRPNb-VTB-AP as a control (Fig. 2B). The relative protein expression levels for the soluble fraction normalized to the control were as follows: 2.1 for the CRPNb-RHCC-AP tetramers, 3.0 for the CRPNb-VTB-AP pentamers, 0.5 for the CRPNb-C4bp-AP heptamers and 2.7 for CRPNb-AP. The proportions of soluble to insoluble components for CRPNb-VTB-AP, CRPNb-C4bp-AP and CRPNb-AP were found to be 3.06, 0.63 and 2.16, respectively. The proportion of soluble to insoluble components could not be calculated for CRPNb-RHCC-AP because the level of the insoluble components was too low.

In the present study, CRPNb-VTB-AP exhibited the highest level of soluble components among the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers.

The protein levels of CRPNb-RHCC-AP tetramers and CRPNb-C4bp-AP heptamers aligned with previous findings, which reported successful expression and proper folding of tetrameric EG2 [an anti-epidermal growth factor receptor single-domain antibody (sdAb)]-RHCC and heptameric EG2-C4bp in prokaryotic expression systems such as BL21 and E. coli TG1 (12,38). Zhang et al (39) previously developed a multimerization strategy and used size-exclusion chromatography to characterize the formation of pentamers.

The antibody activities of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers were next examined and compared with those of CRPNb-H. Fig. 3 shows that CRPNb-H exhibited a markedly higher OD450 signal compared with CRPNb-VTB-AP, CRPNb-RHCC-AP tetramers and CRPNb-C4bp-AP heptamers, suggesting a superior antigen-binding ability. Additionally, the OD450 for CRPNb-VTB-AP was slightly higher compared with that of CRPNb-RHCC-AP, suggesting that CRPNb-VTB-AP had a stronger antigen-binding capacity compared with CRPNb-RHCC-AP. By contrast, CRPNb-C4bp-AP exhibited no antigen-binding capacity, which is inconsistent with previous studies (15-17). The enzymatic activities of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers were next examined and compared with those of CRPNb-AP. Fig. 4 shows that CRPNb-RHCC-AP tetramers and CRPNb-VTB-AP pentamers exhibited higher enzyme activity compared with CRPNb-AP. The CRPNb-C4bp-AP heptamers exhibited no AP activity, contradicting previous studies (15,17). Due to experimental constraints, the control group in Fig. 3 was CRPNb-H. However CRPNb-AP should have been employed as the control to align with Fig. 4, which would have made the experiment more scientifically sound and reasonable. Therefore, the results, such as Fig. 3 showing that CRPNb-H exhibited a markedly higher OD450 signal compared with CRPNb-VTB-AP, CRPNb-RHCC-AP tetramers and CRPNb-C4bp-AP heptamers, have certain limitations. If the control group were replaced with CRPNb-AP, the results might have been different.

As shown in Fig. 4, the AP activity of CRPNb-RHCC-AP tetramers and CRPNb-VTB-AP pentamers was increased compared with that of CRPNb-AP. This indicated that AP fusion with the multimeric domain did not impair AP activity in the CRPNb-RHCC-AP tetramers and CRPNb-VTB-AP pentamers. Compared with findings from previous studies (29,36), the present study revealed that CRPNb-C4bp-AP heptamers exhibited both a lack of antigen-binding capacity and no AP activity. There are a number of possible reasons for these observations. The position of CRPNb and AP at either the N- or C-terminus of C4bp may have impacted their activity (18). In addition, the spatial structure of the CRPNb-C4bp-AP fusion protein may have affected the key amino acid sites responsible for CRP recognition in the heptamers. Furthermore, the flexible linker may not have adequately separated the functional regions of CRPNb and AP, potentially impacting the key amino acid sites involved in CRP recognition due to interference from the fusion expression with AP. In summary, the pentameric CRPNb-VTB-AP demonstrated superior AP activity and CRP binding ability compared with the other multimers tested.

Two distinct heat treatment methods were used for the thermal stability testing of CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers. As shown in Fig. 5A, the Nb activity of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers decreased with increasing temperature. As the temperature increased, ~85% of the activity of the CRPNb-VTB-AP pentamers, ~75% of the activity of CRPNb-AP and ~60% of the activity of the CRPNb-RHCC-AP tetramers was maintained even at 80˚C, whilst the activity of CRPNb-H dropped to ~40%. Based on these results, the impact of incubation time at 80˚C on the binding activity between the antigen and antibody was further examined. Fig. 5B shows that after 55 min of incubation at 80˚C, ~40% of the activity was retained for the CRPNb-RHCC-AP tetramers and CRPNb-VTB-AP pentamers. This suggested that the multimeric Nbs were thermostable, suggesting that they may be suitable for long-term storage at room temperature.

Effect of pH and PBS on Nb activity. To evaluate the influence of pH on the analysis performance, the effect of pH on the assay was evaluated over a range of pH 4 to 10 using the optimized assay buffer by direct ELISA. The binding signal of the antibody at pH 7.4 was defined as 100%, and the binding activity at other pH conditions was expressed as the percentage relative to this value, denoted as % binding activity at room temperature (RT). As shown in Fig. 6, the % RT binding of the CRPNb-RHCC-AP tetramers and CRPNb-VTB-AP pentamers was increased from 69 to 105% and 70.5 to 102% as the pH of the assay buffer increased from pH 4 to 8, before decreasing from pH 9 onwards. By contrast, the binding of CRPNb-H, CRPNb-C4bp-AP and CRPNb-AP was increased from 50, 53 and 62 to 100% as the pH of the assay buffer increased from 4 to 7.4, before decreasing from pH 7.4 onwards, suggesting that pH affected Nb binding activity. Therefore, pH 7.4 was chosen as the optimal pH for the assay buffer. Similarly, different concentrations of PBS were next optimized (Fig. 7). The OD450 absorbance of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-AP was slightly increased as the concentration of PBS decreased from 2X to 1X PBS, then decreased from 1X to 0.25X PBS. Therefore, 1X PBS was selected as the optimal PBS concentration.

To assess the detection performance of the monomers and multimers, a competitive ELISA was used to evaluate the inhibition curves of the CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers. The optimal concentrations of the coating antigen and antibody were determined using checkerboard titration (Fig. S2). The optimal antigen concentration was 2 µg/ml and the optimal antibody concentration was 1 µg/ml. At these concentrations, the absorbance value approached 1. Fig. S3 presents an experimental study on conditional optimization using CRPN-VTB-AP pentamers as a case study. Appropriate experimental conditions were selected for subsequent competitive ELISAs. As shown in Fig. 8, the CRPNb-VTB-AP pentamers exhibited the highest sensitivity, with an IC₅₀ of 11.86 ng/ml, compared with CRPNb-AP and CRPNb-RHCC-AP, which had IC₅₀ values of 45.47 and 23.86 ng/ml, respectively. Compared with the CRPNb-AP monomer, these values were 4X lower for CRPNb-VTB-AP and 2X lower for CRPNb-RHCC-AP. This suggested that multimerization strategies could effectively enhance the affinity of Nbs to antigens. The increased affinity of Nbs is essential for improving the sensitivity of immunodetection. Nb multimers from previous studies have demonstrated improved antigen affinity and detection sensitivity compared with monomers (40,41), which is consistent with the results observed in the present study.

In the present study, recombinant CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers were successfully constructed using the three self-associating peptides RHCC, VTB and C4bp. Within the cytoplasm, these peptides are likely to adopt the right conformation and are purified as soluble proteins, thus avoiding laborious denaturation and refolding procedures, making the process both cost-effective and environmentally friendly (10,33).

In future studies, non-denaturing PAGE should be used to verify the multimer products. Additionally, the CRPNb-VTB-AP pentamers and CRPNb-RHCC tetramers demonstrated antigen-specific recognition and enhanced affinity compared with the CRP-AP monomer. Furthermore, all three multimers exhibited thermal stability, making them suitable for storage and transportation at ambient temperatures. This platform enables the fusion of Nbs with various detection tags, allowing for the generation of multimers with different combinations of Nbs and different tags. By selecting compatible Nbs and self-assembling peptides, Nb activity, enzyme activity, binding affinity and detection performance can be optimized. This approach may lead to the development of bio-functional immune reagents that serve as both high-affinity binders and sensitive tracers.

Although previous studies have studied tetramers, pentamers and heptamers based on RHCC, VTB and C4bp using other Nbs such as EG2-sdAb (13) with AP or other tags, to the best of our knowledge, the present study is the first to form tetramers, pentamers and heptamers using CRPNb and AP based on the three self-assembled peptides and to assess the activity of CRP and AP. The present study aimed to establish a platform for identifying the most effective multimers with superior antigen-binding capabilities and increased AP activity. This will facilitate the development of sensitive and reliable detection methods for CRP.