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Research Article
Selection of high-affinity single-chain antibodies to human C3 by phage display
expand article infoRada Poryazova‡, Ginka Cholakova‡, Alexandra Kapogianni‡, Ivanka Tsacheva‡
‡ Sofia Universiy “St. Kliment Ohridski”, Sofia, Bulgaria

Corresponding author: Ivanka Tsacheva ( itsacheva@biofac.uni-sofia.bg )

Academic editor: Tsvetelina Velikova
© 2025 Rada Poryazova, Ginka Cholakova, Alexandra Kapogianni, Ivanka Tsacheva.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Poryazova R, Cholakova G, Kapogianni A, Tsacheva I (2025) Selection of high-affinity single-chain antibodies to human C3 by phage display. Pharmacia 72: 1-8. https://doi.org/10.3897/pharmacia.72.e150821
Open Access

Abstract

C3 is the key protein in the activation of the complement system, and it contributes to an effective immune response. However, C3 is also targeted by autoantibodies during the development of autoimmune diseases such as systemic lupus erythematosus, and the autoantigenicity of C3 is still poorly understood. In order to study the molecular aspects of C3 autoantigenicity and the localization of C3 autoepitopes, we selected high-affinity anti-C3 antibodies from the “Griffin 1” phage display library expressing human scFv antibodies. The rounds of phage selection were performed with a gradual decrease in the amount of the antigen C3, resulting in the selection of forty clones of recombinant anti-C3 scFv antibodies. Quantitative ELISA analysis determined four high-affinity monoclonal scFvs to C3, and their expression was optimized with IPTG induction and autoinduction methods. Dot blot analysis revealed that the selected high-affinity anti-C3 clones recognized C3 and its smaller fragments C3b and C3c, but not C3d.

Keywords

complement C3, recombinant human antibodies

Introduction

The complement system is a complex network of proteins that are essential for the immune system’s ability to recognize and destroy pathogens, to maintain homeostasis through clearance of apoptotic cells, and to bridge its innate and adaptive responses, synchronizing them in fighting infections. However, a considerable body of experimental evidence throughout the last thirty years has revealed the involvement of the complement system in human pathologies like autoimmune and neurodegenerative diseases, excessive inflammation, nephrologic conditions, and even cancer (Ricklin and Lambris 2013; Ricklin et al. 2016; Reis et al. 2018). This contemporary understanding of the complement system’s functionality, both defensive and offensive, has rekindled scientific research into the complement system with respect to various clinical applications.

The main therapeutic target is C3, the key component of the system—a protein that initiates and regulates complement activity regardless of the activation pathway (Palarasah et al. 2010; Geisbrecht et al. 2022; Lamers et al. 2022a). Structurally, C3 contains 8 macroglobulin-like (MG) domains, a linker region (LNK), an anaphylatoxin domain (ANA), a CUB domain composed of subunits C1r/s, Uegf, and B, and a thioester (TE) domain (Zarantonello et al. 2023). C3 is activated by a series of enzymatic reactions upon pathogen infection or cell damage, and each cleavage of C3 gives smaller biologically active fragments, such as C3a, C3b, iC3b, C3c, and C3d. Over a long period of extensive research on C3 activation and regulatory functions, an array of monoclonal antibodies to the intact protein or to its physiologically relevant fragments C3b, C3c, and C3d have been developed (Lachmann et al. 1980; Whitehead et al. 1981; Nilsson and Nilsson 1987; Garred et al. 1988; Thurman et al. 2013; Rasmussen et al. 2017; Geisbrecht et al. 2022). They were the molecular tools for the detailed structural and functional characterization of C3 (Alsenz et al. 1990; Geisbrecht et al. 2022) and for clinical quantification of complement activation. The therapeutic potential of C3 lies as well in finding potent C3 inhibitors. The monoclonal antibodies developed so far are full-format IgG molecules with a relatively high molecular weight of 150 kDa. Though highly effective for in vitro application, they would pose a threat as immunogens if introduced in vivo. To date, compstatin remains the only C3 inhibitor approved for clinical use (Janssen et al. 2007; Lamers et al. 2022b).

As an evolutionarily conserved protein, the intact C3 is a challenge in the process of immunization due to its low immunogenicity. Antibody phage display is a technique that offers the advantage of omitting the step with the immunization (Marks et al. 1991), thus overcoming this problem. Moreover, this method generates recombinant antibodies in smaller formats, either as Fab or single-chain Fv (scFv) fragments, with molecular weights of 50 kDa or 20 kDa, respectively. Most importantly, they lack the Fc fragment, which confers most of the immunogenicity of mouse or rat monoclonal antibodies when used in vivo. Recombinant antibodies can be synthesized in large quantities for a short period of time, which is also cost-effective. The introduction of antibody phage display has led to the creation of many new pharmaceuticals, such as medications to counteract autoimmune, viral, inflammatory, and cancer diseases (Lu et al. 2020). This broad spectrum of application was acknowledged in 2018 when Sir Gregory P. Winter was awarded the Nobel Prize “for the phage display of peptides and antibodies.”

In the present work, we selected anti-C3 antibodies from the Griffin-1 phage library, which expresses human scFv antibodies, making them applicable in the human body for specific functions, such as inhibition of complement activation or targeting complement proteins to specific tissues. In addition, autoantibodies against C3 have been identified in several autoimmune diseases, including systemic lupus erythematosus, highlighting the importance of this protein in maintaining a well-functioning immune system. This represents the first successful attempt to produce recombinant human anti-C3 scFv antibodies.

Materials and methods

Buffers and stock solutions

The following buffers and stock solutions were used: PBS (Phosphate-buffered saline), pH 7.2 (10 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 27 mM KCl); TPBS (PBS with 0.05% Tween-20); TBS (Tris-buffered saline), pH 7.2 (20 mM Tris, 150 mM NaCl); AP (Alkaline phosphatase) buffer, pH 9.6 (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2); Phosphate buffer, pH 8.0 (10 mM NaH2PO4, 10 mM Na2HPO4, 500 mM NaCl); TB (transfer buffer), pH 8.3 (25 mM Tris, 192 mM Glycine); Carbonate buffer (100 mM NaHCO3, 100 mM Na2CO3), pH 9.6; AP substrate solution for immunoblotting (33 μL 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 50 mg/mL in 100% DMSO) mixed with 66 μL nitro-blue tetrazolium (NBT, 50 mg/mL in 70% DMSO) in 10 mL AP buffer); Coomassie Brilliant Blue G-250 (10% CH3COOH, 0.006% Coomassie G-250); 100 mM Triethylamine (C3H9N); PEG/NaCl (20% Polyethylene glycol-6000, 2.5 M NaCl); 10% Tween-20; 1 M Tris buffer, pH 7.4; 0.5 M IPTG (Isopropyl β-d-1-thiogalactopyranoside); 2 M Imidazole

Growth media and additives

The following growth media were used: 2xTY medium (1.6% tryptone; 1% yeast extract; 0.5% NaCl, pH 7.20) supplemented with 1% glucose, 1mM MgSO4, 100 μg/mL ampicillin (Amp), 50 μg/mL kanamycin (Kan); TYE (1% tryptone; 0.5% yeast extract; 0.8% NaCl; 1.5% agar) supplemented with 1% glucose, 100 μg/mL ampicillin, 50 × 5052 (25% glycerol, 2.5% glucose, 10% α-lactose); 20xNPS (66 g/L (NH4)2SO4; 136 g/L KH2PO4; 142 g/L Na2HPO4 monobasic anhydrous); ZYP medium (1% tryptone; 0.5% yeast extract; 1 mМ MgSO4; 2% 50 × 5052; 5% 20xNPS; 100 μg/mL ampicillin, pH 7.2); LB medium (1% tryptone; 0.5% yeast extract; 1% NaCl) supplemented with 1% glucose, 1 mM MgSO4, 100 μg/mL ampicillin.

Selection of anti-C3 phages

A Griffin-1 naïve scFv phage library was screened for anti-C3 phages following the protocol described in Todorova et al. (2021). The panning procedure of four rounds of selection was carried out with gradually decreasing amounts of C3 in each consecutive round. The first two rounds were performed with 40 µg of immobilized human C3 (1 μg/µL, Quidel, A401) diluted in carbonate buffer. The next two rounds of selection were performed with 12 μg and 10 μg immobilized C3, respectively. The immobilization of C3 was overnight onto immunotubes (Nunc-Immuno Tubes, Thermo Scientific), which were washed three times with distilled water and once with carbonate buffer (pH = 9.6). Next, the tube was blocked with 0.5% Tween-20 for 2 h, then incubated with 1 mL (approximately 1013 PFU) of phage scFv in 0.5% Tween-20 for 30 min with rotation, followed by a stationary incubation for an additional 90 min. All incubations were performed at room temperature. After the incubation, the tube was washed 10 times with TPBS and 10 times with PBS. Bound phage-displayed scFv were eluted with 1 mL of 100 mM triethylamine and gentle rotating of the tube for 10 min. The eluted suspension was immediately neutralized with 0.5 mL of 1 M Tris (pH 7.4). Half of the eluted phage suspension was used to infect 10 mL of log-phase E. coli TG1 cells (OD600nm ~ 0.4) at 37°C for 30 min and then plated on TYE-Amp plates for an overnight cultivation. The infected cells were transferred to 100 mL of 2xTY-Amp supplemented with 0.2% glucose, and upon reaching log phase, the culture was co-infected with VCS-M13 helper phage (Stratagene) and grown in 2xTY-Amp-Kan overnight at 30°C. The bacterial culture was centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant (SN), containing the rescued phages, was precipitated with 1/5 volume of SN using PEG/NaCl for 1 h on ice. The PEG-precipitated phages were harvested by centrifugation at 10,000 rpm for 30 min at 4°C, and the pellet was resuspended in 2 mL sterile PBS. Half of the amplified polyclonal anti-C3 phage-displayed scFv were used for the next round of selection, and the other half was kept frozen at -70°C. Serial dilutions of the polyclonal anti-C3 phages from each round of selection were plated on TYE-Amp for titer determination.

Titration of anti-C3 phages

The titer of eluted anti-C3 phages and PEG-precipitated anti-C3 phages was determined by serial dilutions of 10−2, 10-4, 10-6, and 10−8 in LB, and 10 μL of each dilution was used to infect 100 μL of log-phase E. coli TG1 cells at 37°C for 30 min. The cells for every dilution were plated on TYE-Amp agar plates containing 1% glucose and incubated at 37°C overnight. The colonies from each dilution were counted, and the phage titer was calculated using the formula: phage titer (PFU/mL) = (number of colonies) × 100× (dilution factor).

Selection of monoclonal scFv

The plates with serial dilutions of the polyclonal phage from the last two rounds were used for selecting random monoclonal scFv phages. The selection was on the basis of colony morphology. The selected clones were inoculated into 100 µL/well 2xTY-Amp in 96-well plates (Cell Well, Corning) and grown with shaking (190 revs/min) overnight at 37°C. The overnight cultures were transferred into a new 96-well plate by inoculation of 2 µL in 200 µL fresh 2xTY-Amp and cultivated for 1 h. Then, 50 µL of 2xTY-Amp containing 1010 PFU of VCS-M13 was added to each well, and the plate was incubated at 37°C for 30 min without shaking. The cells were spun down by centrifugation at 3000 rev/min at 4°C, resuspended in 200 µL/well 2xTY-Amp-Kan, and grown overnight at 30°C 120 rev/min with shaking. The SN containing rescued monoclonal phage was used to transfect log-phase non-suppressor strain E. coli HB2151 for 30 min stationary at 37°C. Next, 5 μL of E. coli HB2151 infected with each rescued anti-C3 phage clone was transferred to a new 96-well plate with 100 μL/well 2xTY-Amp supplemented with 1% glucose and incubated at 37°С/120 rpm shaker speed overnight. Next, 50 μL of culture from each E. coli HB2151 clone was transferred into a 24-well plate containing 1 mL 2xTY-Amp supplemented with 1% glucose and incubated at 37°C for 2 h at 250 rpm shaker speed. The expression of soluble monoclonal anti-C3 scFv was induced with 1 μL/well of 0.5 M IPTG overnight at 25°C/120 rpm shaker speed. After the induction, the 24-well plate was centrifuged at 3000 rpm at 4°C for 10 minutes, supernatants were collected, and assayed for the presence of soluble anti-C3 scFv antibodies.

Induction of soluble scFv and affinity purification

The soluble scFv was induced by two alternative induction protocols (Nikolova et al. 2021). Briefly, for IPTG induction, E. coli HB2151 cultures of the selected clones (OD600nm ~ 0.9) were induced with 0.5 mM IPTG for 5 h at 25°C. For autoinduction, overgrown E. coli HB2151 daily cultures of the selected clones were inoculated 1:100 in ZYP-5052 autoinduction medium and grown for 16 h at 25°C. The induced cells were lysed in 1/20th of the culture volume in ice-cold 100 mM Tris, pH 8.0, containing 20% sucrose and 1 mM EDTA for 1 h on ice and then centrifuged at 9000 rpm for 45 min at 4°C. The yielded supernatant (SN1), containing soluble scFv, was kept on ice and later pooled with SN2, obtained after the subsequent lysis of the cell pellet in the same volume of ice-cold 5 mM MgSO4 for 15 min on ice and then centrifuged at 9000 rpm for 45 min at 4°C. The pooled supernatants were dialyzed against phosphate buffer, pH 8.0, containing 10 mM imidazole and applied on a HIS-Select® Ni-affinity gel column (Sigma) at a flow rate of 0.5 mL/min. The scFv antibodies were eluted with phosphate buffer, pH 8.0, containing 250 mM imidazole. The eluted protein samples were dialyzed against PBS, pH 7.2.

SDS-PAGE analysis

Human C3 and its smaller fragments were run in 15% SDS-PAGE. PageRuller™ unstained or prestained protein ladder (Thermo Fisher Scientific™ Inc.), was used as a molecular weight standard. The gel was fixed in a buffer containing 25% i-propanol and 10% CH3COOH for 15 min and then stained with 0.006% Coomassie Brilliant Blue G-250 in 10% CH3COOH for 15 min. The protein molecular weights were estimated with ImageJ software.

Immunoblotting

Western blot: Human C3 and its smaller fragments were run in 12% SDS-PAGE in reducing (using dithiothreitol-containing sample buffer) and non-reducing conditions, then transferred to nitrocellulose membrane (NC) by semi-dry electro transfer in TB (40 V/100 mA for 90 min). The complete transfer of proteins was estimated with MemCode™ Reversible Protein Stain (Thermo Fisher Scientific), and then the signal was erased with MemCode™ Stain Eraser (Thermo Fisher Scientific). The membrane was blocked for 1 h with 0.5% Tween-20 in PBS, and it was incubated with a supernatant of a single clone of the selected anti-C3 scFv antibodies. The membrane was incubated with mouse anti-c-Myc clone 9E10 (Merck Millipore, 1:2000 in TPBS) for 1 h with shaking at room temperature. Next, the membrane was incubated with goat anti-mouse IgG-AP (Agrisera, 1:2000 in TPBS) for 1 h with shaking at room temperature. The membrane was incubated with AP substrate solution for immunoblotting. The membrane was washed three times with TPBS after each immobilization of a reagent. The molecular weight of detected proteins was estimated with PageRuler™ Prestained Protein Ladder (ThermoFisher Scientific) using ImageJ software.

Dot blot: 1 μL of C3 (1 μg/μL) was immobilized in duplicate on NC. The membrane was blocked with 0.5% Tween-20 for 1 h at room temperature, washed three times with TPBS, and incubated with 1 mL SN of a single clone of the selected anti-C3 scFv antibodies at 25°C and 90 rpm shaker speed for 2 h. Bound scFv antibodies were detected by incubation with mouse anti-c-Myc IgG (Sigma, diluted 1:2000 in TPBS) at 25°C at 90 rpm shaker speed for 1 h and then with goat anti-mouse IgG-AP (AgriSera, diluted 1:2000 in TPBS) for 1 h at RT. The dots were visualized with AP substrate solution for immunoblotting. After each incubation, the membrane was washed three times with TPBS. Dot blot analysis was performed using ImageJ software, with background subtraction and measuring the integrated density of each dot. The mean of integrated density was plotted and expressed in arbitrary units (a.u.).

Enzyme-linked immunosorbent assay (ELISA)

Microtiter 96-flat-bottom plates were coated with human C3 protein (Quidel) at 1 μg/well in carbonate buffer overnight at 4°C and blocked with 0.5% Tween-20 (300 μL/well) for 1 h at 37°C. Next, 150 μL of SN from a single anti-C3 scFv clone, mixed with 50 μL of PBS containing 0.2% Tween-20, was added to each well and incubated overnight at 4°C. Subsequently, the wells were incubated with mouse anti-c-Myc IgG (Sigma, diluted 1:2000 in TPBS) for 1 h at 37°C and then with goat anti-mouse IgG-AP (AgriSera, diluted 1:2000 in TPBS) for 1 h at 37°C. The bound complexes were detected by p-Nitrophenylphosphate (Acros Organics) at a working concentration of 0.5 mg/mL in AP buffer at 100 μL/well. The absorbance was read at 405 nm with a microplate reader (DR-200B, Hiwell Diatek Instruments, Wuxi, China). After each incubation, the wells were washed three times with TPBS (300 μL/well). All samples were analyzed in triplicate, and their standard deviation (SD) was calculated.

Results

We used the human complement protein C3 as a target antigen for the screening of the phage library “Griffin.1,” expressing human synthetic V(H) + V(L) scFv antibodies cloned in the pHEN2 phagemid vector as fusion proteins with the capsid pIII of the filamentous M13 phage. According to the standard protocol, the amount of antigen for one round of selection is 40 µg to 400 µg. We performed four rounds of panning, aiming at the selection of anti-C3 phages displaying scFv antibodies with high affinity. In order to steer the selection to phages with increasing affinities, we started with the minimum required amount of 40 µg C3 and decreased its amount to 12 µg for the third round and 10 µg for the fourth round, respectively. The polyclonal phage suspensions after each round of panning were titrated in E. coli TG1 cells, and the titer was expressed in PFU/mL (plaque-forming units per milliliter). The titer of eluted anti-C3 phages after the third round was 1.26 × 105 PFU/mL and 2.76 × 104 PFU/mL after the fourth round. The titration was performed on TYE plates, which we used to select 40 E. coli TG1 colonies (20 colonies from the third round named B and C, and 20 from the fourth round named E and F) as a source of phage-displayed monoclonal anti-C3 scFv antibodies. The selected 40 monoclonal scFv-displaying phages were rescued and used to transfect the non-suppressor strain E. coli HB2151 for the induction of soluble scFv antibodies. The successful expression of soluble anti-C3 scFv antibodies was assessed by Dot Blot assay with immobilized human C3. All 40 analyzed monoclonal scFvs specifically bound human complement protein C3 (Fig. 1).

Figure 1. 

Dot blot assay for detection of positive anti-C3 scFv antibodies selected in round III (clones B2-C11) and in round IV (clones E2-F11). 1 dot = mean value of duplicate measurement of integrated density of a sample normalized to a control. Statistical analysis was done with a paired Student t-test, error bars: mean ± SD, **** (p ≤ 0.0001).

The affinity of interaction of the selected anti-C3 scFv antibodies with human C3 was analyzed by ELISA (Fig. 2). One scFv clone from the B series, namely B11 from round III, exhibited strong binding affinity to C3. In comparison, clones F8, F10, and F11 from round IV showed higher affinity to human C3. Clones C2-C11 (Fig. 2, middle panel) and clones E2-E11 (data not shown) showed negligible affinity to C3, and they were excluded from further analysis. The highest affinity was registered for clone F11.

Figure 2. 

Comparative ELISA analysis of soluble monoclonal anti-C3 scFv antibodies from clones B2-B11, clones C2-C11 (selection round III), and clones F2-F11 (selection round IV).

The next step of the analysis was the localization of the epitopes of the selected clones—B11, F8, F9, F10, and F11—by the use of intact C3 and its fragments under reducing and non-reducing conditions. The five clones were induced in a soluble form for analysis by both immunoblot and dot blot. Of note, the immunoblot analysis of C3 and its fragments after denaturing SDS-PAGE, which may not allow detection of epitopes maintained by the native protein structure, whereas immobilization of the antigens in the dot blot assay preserves their native state. This may be essential to determine their biological activity and functional characteristics. Clone F9 failed to induce a detectable amount of soluble scFv, and consequently it was also excluded from further analysis. The four clones B11, F8, F10, and F11 recognized the intact C3, and its fragment C3b. This was observed in both the Western blot analysis when the proteins were transferred after non-reducing SDS PAGE (Fig. 3B) and the dot blot (Fig. 3C). This confirmed that the selected recombinant antibodies have preserved their specificity for C3 and also recognized C3b.

Figure 3. 

Immunoblotting of the selected high-affinity scFv clones for the recognition of the intact C3 and its smaller fragments C3b, C3c, and C3d A NC with blotted proteins after non-reducing 15% SDS PAGE. Western blot B and dot blot C of antigens with selected anti-C3 scFv clones D NC with blotted proteins after reducing 15% SDS PAGE E Western blot of reduced antigens with selected anti-C3 scFv clones.

Western blot analysis was also performed under reducing conditions. This resulted in some differences (Fig. 3E) compared to the non-reducing conditions, the main one being the binding of the C3c fragment as well. The reason for this binding is most likely due to a conformational change as a consequence of proteolysis and the detection of a hidden epitope that only becomes accessible for recognition after reduction.

As clones B11, F8, F10, and F11 were found to be suitable for modulating the activity of complement protein C3, the next stage of our work was aimed at optimizing the conditions for their induction in larger quantities for preparative application. Cell cultures of E. coli HB2151 of all four clones were subjected to inductions of scFv synthesis by the alternative approaches—IPTG induction and autoinduction. Autoinduction resulted in significantly more induced cells compared to IPTG induction for all analyzed clones, and this effect was particularly pronounced in clones F8 and F11 (Fig. 4A, C). For clone F8, autoinduction was the most efficient approach, resulting in 5.840 g of induced cells, considerably more than that achieved with IPTG induction (1.861 g). This suggested that autoinduction was a more appropriate strategy for this clone and may be the preferred method for future large-scale production. Upon IPTG induction, clones F8 and F11 showed the highest amount of induced cells (1.861 g and 1.953 g, respectively), while F10 resulted in the lowest amount (1.153 g). The same trend was maintained in autoinduction, where F8 and F11 again generated the highest amount of induced cells (5.840 g and 4.314 g), while F10 remained with the lowest (1.247 g). This may indicate that clone F10 is less adaptive to autoinduction conditions, necessitating further optimization of expression conditions for this clone.

Figure 4. 

E. coli cell yield obtained after IPTG induction and autoinduction of A Clones F8 and F10 and C Clones B11 and F11 B Non-reducing 15% SDS-PAGE of anti-C3 scFv eluates of clones F8 and F10. Lanes: M, molecular marker; 2 and 3: clone F8 eluates from IPTG induction; 4 and 5: clone F8 eluates from autoinduction; 6 and 7: clone F10 eluates from IPTG induction; 8 and 9: clone F10 eluates from autoinduction; D Non-reducing 15% SDS-PAGE of anti-C3 scFv eluates from clones B11 and F11. Lanes: M, molecular marker; 2, 3, 4: clone F11 eluates from IPTG induction; 5, 6, 7: clone F11 eluates from autoinduction; 8 and 9: eluates from culture growth medium of clone B11; 10 - cell lysate from IPTG induction of clone B11; 11 - cell lysate from autoinduction of clone B11. The relative protein molecular weight (kDa) was estimated with ImageJ software.

Induced cells of the four anti-C3 scFv clones by the two alternative approaches were lysed and purified by affinity chromatography, and the resulting eluates were subjected to further electrophoretic analysis to verify protein presence and quality. Along with the induced cells, the growth medium (GM) from bacterial cultures subjected to both types of induction was also analyzed for the presence of significant amounts of extracellularly released scFv antibodies (Fig. 4B, D). Only clone B11 showed the presence of scFv in the GM. However, the presence of high molecular weight impurities in the preparation isolated from the GM compromised its quality, making it unsuitable for the purpose of the study. For the remaining clones (F8, F10, and F11), neither induction method detected scFv in the culture medium. Clone B11 showed low expression of scFv synthesis, and its presence in the cell lysate was similar whether IPTG or autoinduction was used. The F8 clone showed a greater amount of protein in the IPTG cell lysate compared to autoinduction. This suggests that autoinduction is a less efficient method for this clone. Clone F11 was characterized by the highest amount of induced protein by IPTG and slightly lower by autoinduction. This makes it most suitable for the production of large quantities of anti-C3 scFv antibodies due to its high expression and optimal yield after chromatographic purification. Although autoinduction provided many more induced cells, the efficiency of the method in terms of protein yield varied from clone to clone.

Discussion

In the present study, recombinant human scFv antibodies against the complement protein C3 were generated and characterized for the first time, given that C3 is an evolutionarily conserved protein and a weak immunogen, making it difficult to produce high-affinity antibodies against it. Our modification of the standard protocol by gradually decreasing the amount of antigen for selection resulted in increased affinity of the eluted recombinant antibodies after four rounds. Clone F11 was capable of binding C3 in complexes at least twice as effectively as the other selected clones.

The transition from phage-displayed to soluble scFv did not alter the conformation of the selected scFv clones, nor their affinity. Clone F11 seems most promising for large-scale expression and for an analysis of its potential role in modulating the immune response via complement activation for therapeutic application.

Conclusion

The gradual decrease of the amount of antigen in the successive rounds resulted in the selection of high-affinity antibodies to C3. Comparative quantitative ELISA analysis highlighted four monoclonal antibodies suitable for further experimental work—B11, F8, F10, and F11. Four clones, B11, F8, F10, and F11, recognized C3 and C3b but not C3c and C3d.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statements

The authors declared that no clinical trials were used in the present study.

The authors declared that no experiments on humans or human tissues were performed for the present study.

The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study.

The authors declared that no experiments on animals were performed for the present study.

The authors declared that no commercially available immortalised human and animal cell lines were used in the present study.

Funding

This study was financed by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project № BG-RRP-2.004-0008-C01, and by grant KP-06-M51/1 of the Bulgarian NSF.

Author contributions

All authors have contributed equally.

Author ORCIDs

Ginka Cholakova https://orcid.org/0000-0003-4375-5115

Alexandra Kapogianni https://orcid.org/0009-0003-4419-1603

Ivanka Tsacheva https://orcid.org/0000-0002-3308-4154

Data availability

All of the data that support the findings of this study are available in the main text.

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