Antigen-binding Capacity Research Articles

Development of piezoelectric immunosensor for the rapid detection of marine derived pathogenic bacteria, Vibrio vulnificus

Biosensors consist of biochemical recognition agents like antibodies immobilized on the surfaces of transducers that change the recognition into a measurable electronic signal. Here we report a piezoelectric immunosensor made to detect Vibrio vulnificus. A 9MHz AT-cut piezoelectric wafer attached with two gold electrodes of 5mm diameter was used as the transducer of the QCM biosensor with a reproducibility of <TEX>${\pm}0.1Hz$</TEX> in frequency response. We have tried different approaches to immobilize antibody on the sensor chip. Concerning the orientation of antibody for the best antigen binding capacity, the antibody was immobilized by specific binding to protein G or by cross-linking through hydrazine. In addition, protein G was cross-linked on glutaraldehyde activated immine layer (PEI) or EDC/NHS activated sulfide monolayer (MPA). PEI was found to be more effective to immobilize protein G following glutaraldehyde activation than MPA. However, hydrazine chip showed a better capability to immobilize more IgG than protein G chip and a higher sensitivity. The sensor system was able to detect V. vulnificus in dose dependent manner and was able to detect bacterial cells within 5 minutes by monitoring frequency shifts in real time. The detection limit can be improved by preincubation to enrich the bacterial cell number.

Fabrication of antibody microarrays by light-induced covalent and oriented immobilization.

Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.

Asymmetric peptide dendrimers are effective linkers for antibody-mediated delivery of diverse payloads to b cells in vitro and in vivo.

Safe, targeted delivery of therapeutics remains a focus of drug/gene delivery, the aim being to achieve optimal efficacy while minimising off-target delivery. Dendrimers have a vast array of potential applications and have great potential as gene and drug delivery tools. We previously reported the development of peptide dendrimers that effectively complexed DNA and that have distinct advantages over conventional spherical dendrimers. Here, to expand the application of peptide-based low generation dendrimers we tested their capacity to be transformed into linkers for antibody-based targeting of diverse payloads. Peptide-based low-generation asymmetric dendrimers were generated and conjugated to partially-reduced antibodies specific for B cell surface antigens or an irrelevant antigen. Preservation of antigen binding by the antibodies and targeting of the conjugated dendrimers carrying a small molecule (biotin) or plasmid DNA payloads was tested. Peptide-based low generation dendrimers were efficiently and site-specifically conjugated to antibodies with retention of antigen-binding capacity. Altering the branching termini of dendrimers facilitated delivery of diverse payloads in vitro and in vivo. We propose that safe, non-toxic peptide dendrimers, which are readily synthesised and modifiable for a variety of applications, form the basis of a new family of biocompatible "linkers" with substantial potential for targeted delivery applications.

Interfacial structure of immobilized antibodies and perdeuterated HSA in model pregnancy tests measured with neutron reflectivity.

Experimental studies of antibody adsorption and antigen binding that mimicked pregnancy test immunoassays have been performed using neutron reflectivity studies of a model antibody/antigen system immobilized on the silica/water interface. The study revealed the nature of the antibody/antigen interaction and also the importance of a blocking protein, in this case human serum albumin (HSA), that enhances the immunoassay's specificity and efficiency. Of central importance to this study has been the use of a perdeuterated human serum albumin (d-HSA), providing contrast that highlights the orientation and position of the blocking agent within the adsorbed layer. It was found that the adsorbed HSA filled the gaps between the preadsorbed antibodies on the substrate, with decreased adsorption occurring as a function of increased antibody surface coverage. In addition, the antigen binding capacity of the adsorbed antibodies was investigated as a function of antibody surface coverage. The amount of specifically bound antigen was found to saturate at approximately 0.17 mg/m(2) and became independent of the antibody surface coverage. The ratio of bound antigen to immobilized antibody decreased with increased antibody surface coverage. These results are of importance for a full understanding of immunoassay systems that are widely used in clinical tests and in the detection of environmental contaminants.

Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease

Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals.

Influence of temperature and pH on the antigen-binding capacity of immunoglobulin G in cheese whey derived from hyper-immune milk

Sweet whey containing immunoglobulin G (IgG), active in vitro against Campylobacter jejuni, was submitted to different treatments (pH 5.0, 6.5 and 8.0; temperatures between 68 and 80 °C and time between 30 and 3600 s) to evaluate the influence of these conditions on the IgG antigen-binding capacity (ABC). ABC losses followed a first-order kinetic reaction at each temperature and pH studied. Kinetic (k and z) and thermodynamic data (EA, ΔH#, ΔG# and ΔS#) were obtained. ABC was not affected by pasteurisation treatments (72 °C, 16 s), which may suggest the possibility of using hyper-immune sweet whey as a source of dairy products with activity against human pathogens.

Fabrication of a Detection Platform with Boronic-Acid-Containing Zwitterionic Polymer Brush

Development of technologies for biomedical detection platform is critical to meet the global challenges of various disease diagnoses, especially for point-of-use applications. Because of its natural simplicity, effectiveness, and easy repeatability, random covalent-binding technique is widely adopted in antibody immobilization. However, its antigen-binding capacity is relatively low when compared to site-specific immobilization of antibody. Herein, we report that a detection platform modified with boronic acid (BA)-containing sulfobetaine-based polymer brush. Mainly because of the advantage of oriented immobilization of antibody endowed with BA-containing three-dimensional polymer brush architecture, the platform had a high antigen-binding capacity. Notably, nonspecific protein adsorption was also suppressed by the zwitterionic pendants, thus greatly enhanced signal-to-noise (S/N) values for antigen recognition. Furthermore, antibodies captured by BA pendants could be released in dissociation media. This new platform is promising for potential applications in immunoassays.

The Higher Frequency of Blood Group B in a Brazilian Population with HIV Infection

Objective:To analyze the frequency of and odds for and against HIV infection based on ABO blood type in a large sample of blood donors.Background:Coevolution between pathogens and hosts may explain the ABO system of polymorphisms. HIV-infected cells add ABO(H) blood group antigens to the viral envelope. Naturally occurring antibodies against ABO(H) antigens that are present in normal human sera are able to neutralize ABO-expressing HIV in vitro. Blood donors are ideal for studying blood groups and HIV infection in vivo because all donors are routinely typed and tested.Methods:All blood donors who donated blood between 1994 and 2010 were tested for HIV (ELISA antibody tests and Western blot test or immunofluorescence testing) and were ABO typed (direct and reverse grouping tests). HIV infection based on the ABO blood group was analyzed using the chi-square test and game theory.Results:The total number of examined blood donors during this period was 271,410, of whom 389 were infected with HIV. B-group donors were more infected than non-B donors (p= 0.006).Conclusions:A more restricted antigen recognition capacity of anti-Galα1-3Gal in blood groups AB and B and a weaker antigen-binding capacity of anti-A antibodies may contribute to a higher frequency of HIV infection in blood group B.

Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies

Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

Platelet glycoprotein Ibα ectodomain shedding and non-surgical bleeding in heart failure patients supported by continuous-flow left ventricular assist devices

Non-surgical bleeding (NSB) is a major complication among heart failure (HF) patients supported by continuous-flow left ventricular assist devices (CF-LVADs). Understanding the hemostatic defects contributing to NSB after CF-LVAD implantation is crucial for prevention of this adverse event. The aim of this study was to examine the link between platelet glycoprotein Ibα (GPIbα) ectodomain shedding and NSB in CF-LVAD recipients and to identify a potential biomarker of NSB. Serial blood samples were collected from 35 HF patients supported with CF-LVADs. Platelet function was evaluated by a platelet function analysis device and thromboelastography (TEG). Platelet GPIbα shedding, von Willebrand factor (vWF) antigen and vWF collagen binding capacity were determined using enzyme-linked immunosorbent assays (ELISAs). The structural analysis of vWF was performed by gel electrophoresis. These platelet function measures with vWF parameters of the patients who had NSB between 4 and 32 days after CF-LVAD implantation (bleeder) were analyzed against those without NSB (non-bleeder). Blood samples from 7 healthy individuals were collected to obtain healthy reference values for the laboratory assays. Elevated GPIbα shedding was found to be a pre-existing condition in all HF patients prior to CF-LVAD implantation. Post-operative level of GPIbα shedding increased and remained elevated in the bleeder group, whereas a consistent decrease was found in the non-bleeder group. A receiver operating characteristic (ROC) analysis indicated that the level of GPIbα shedding had a predictive power of NSB in patients on CF-LVAD support. Platelet GPIbα ectodomain shedding which attenuates platelet reactivity is associated with NSB. Plasma GPIbα level may potentially be used to refine bleeding risk stratification in CF-LVAD patients.

Site‐selective immobilization of anti‐CD34 antibodies to poly(l‐lactide) for endovascular implant surfaces

Aiming at a speed up of the re-endothelialization process of biodegradable endovascular implants, novel approaches for the functionalization of poly(l-lactide) (PLLA) with anti-CD34 antibodies were established. We propose a three-step process involving PLLA surface activation with functional amino groups, attachment of a protein repelling peptide spacer, and covalent random or site-selective immobilization of the antibodies. Obtainable antibody surface densities and antigen binding capacities were thoroughly evaluated by means of enzyme-linked immunosorbent assay. Results indicate that a lower amount of anchoring sites on the antibody favors high coupling efficiency, while localization of the anchoring sites, facing the antigen binding moiety, strongly enhances the antigen capture capacity of the support. Besides minimization of physisorption and cell adhesion exemplarily shown with bovine serum albumin, avidin, and human umbilical vein endothelial cells, respectively, the inclusion of the protein-repelling spacer strengthened this effect, yielding antigen capture capacities exceeding values so far reported in literature. In contrast, the number of amino groups on the PLLA surfaces, which is indeed highly dependent on the applied activation procedure, does not seem to influence antibody coupling efficiency and antigen capture capacity considerably. This allows the choice of surface activation treatment, plasma or wet-chemical, regarding other processing parameters as for instance sterilizability or favored modification depth.

Characterization of Asia 1 sdAb from Camels Bactrianus (C. bactrianus) and Conjugation with Quantum Dots for Imaging FMDV in BHK-21 Cells

Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious viral disease affecting cloven-hoofed animals. Camelids have a unique immunoglobulin profile, with the smallest functional heavy-chain antibodies (sdAb or VHH) naturally devoid of light chains with antigen-binding capacity. We screened and characterized five sdAbs against FMDV by immunized library from C. bactrianus with Asia 1 virus-like particles (VLPs). Three of five recombinant sdAbs were stably expressed in E.coli, remained highly soluble, and were serotype-specific for VP1 protein of FMDV Asia 1 by ELISA. These failed to completely neutralize the Asia 1 virus. According to the KD value of binding affinity to three sdAbs, which ranged from 0.44 to 0.71 nm by SPR, sdAb-C6 was selected and conjugated with Zn/CdSe quantum dots (QDs) to form a QDs-C6 probe, which was used to trace and image the subcellular location of FMDV in BHK-21 cells. The results show that FMD virions were observed from 3 h.p.i., and most of virions were distributed on one side of the nucleus in the cytoplasm. We demonstrate the utility of sdAbs as functionalized QDs are powerful tools for FMDV research.

Abstract 5624: Anti-c-Met Nanobody®: A potential new drug in cancer treatment.

Abstract c-Met is the tyrosine kinase receptor of the hepatocyte growth factor (HGF). HGF-c-Met signaling is involved in a wide variety of human malignancies including colon, gastric, bladder, breast, ovarian, pancreatic, kidney, liver, lung, head and neck, thyroid, and prostate cancers, as well as sarcomas, hematological malignancies, melanoma and central nervous system tumors. In recent years, multiple agents have been developed directed to interfere at different levels in the HGF-c-Met signaling pathway, and some are currently being tested in Phase II and/or III clinical trials. Nanobodies® are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies, occurring in the Camelidae family. Importantly, they retain the full antigen-binding capacity of the original heavy chain only antibodies and are highly stable. In this study, we examined the anti-cancer effects of an anti-c-Met Nanobody in vitro using human multiple myeloma as a model system. The anti-c-Met Nanobody comprises of two Nanobody moieties, one targeting c-Met and one binding to human serum albumin for half-life extension. HGF is thought to contribute to the pathogenesis of multiple myeloma in different ways, as HGF is a pivotal growth and pro-migratory factor and inhibits osteoblastogenesis in vitro suggesting that HGF may contribute to the development of myeloma bone disease. Elevated levels of HGF in serum of multiple myeloma patients are correlated with a poor prognosis. We show that the anti-c-Met Nanobody effectively inhibited the proliferation of ANBL-6 human multiple myeloma cells via inhibition of an HGF autocrine growth loop, as well as the proliferation of INA-6 cells induced by exogenously added HGF. In addition, the HGF-induced migration of INA-6 cells was completely and specifically blocked following treatment with the Nanobody at a concentration of 1 μM. The Nanobody also inhibited the HGF-induced adhesion of multiple myeloma cells to fibronectin, but did not affect stromal cell-derived factor-1 alpha-induced adhesion. Furthermore, the Nanobody abolished the inhibiting effect of HGF on bone morphogenetic protein-2-induced ALP-activity and the mineralization of human mesenchymal stem cells. Finally, we show that the Nanobody reduced the HGF mediated phosphorylation of the c-Met Tyrosine (Y) residues Y1349, Y1234/1235 and Y1003 and the phosphorylation of the downstream proteins MAPK and Akt in INA-6 cells. In conclusion, the anti-c-Met Nanobody inhibited c-Met signaling with high specificity and potency resulting in inhibition of multiple myeloma cell migration, proliferation and adhesion, and in blocking of the HGF mediated inhibition of osteoblastogenesis. Given the potential of Nanobodies to surpass drawbacks of antibodies, this anti-c-Met Nanobody might represent a potential novel therapeutic agent in the treatment of multiple myeloma and other cancers driven by HGF-c-Met signaling. Citation Format: Tobias S. Slørdahl, Tinneke Denayer, Siv Helen Moen, Therese Standal, Magne Børset, Cedric Ververken, Torstein B. Rø. Anti-c-Met Nanobody®: A potential new drug in cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5624. doi:10.1158/1538-7445.AM2013-5624

A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10−2μgmL−1, superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10−3mgg−1 of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food.

Improving immunosensor performance through oriented immobilization of antibodies on carbon nanotube composite surfaces

We report the straightforward oriented covalent attachment of antibodies (Abs) on the surface of carboxylated multiwalled carbon nanotube-polystyrene (MWCNT-PS) materials. The combination of this composite material, applied as a robust electrochemical transducer platform, and its covalent functionalization with Abs in a controlled way by means of a two-step process, could contribute to the development of highly sensitive immunosensor devices. Using the simple and versatile carbodiimide chemistry, Abs were attached to the carboxylic groups of the MWCNT-PS composite surfaces via their superficial amine groups. By taking into account the Ab isoelectric point and the net charge of the composite surface, we engineered an immobilization process to achieve the oriented binding of the Ab molecules by favoring an ionic pre-adsorption step before covalent binding occurred. Thus, the antigen binding capacity of the attached Abs was enhanced by up to 10 times with respect to the capacity estimated for a random spatial distribution of these molecules. The proposed strategy would also serve as a model for the efficient biofunctionalization of other carboxylated carbon-based polymer composite materials with potential applications in the biosensor field.

Nano-visualization of oriented-immobilized IgGs on immunosensors by high-speed atomic force microscopy

Oriented immobilization of sensing molecules on solid phases is an important issue in biosensing. In case of immunosensors, it is essential to scrutinize not only the direction and shape of immunoglobulin G (IgG) in solution but also the real-time movement of IgGs, which cannot be achieved by conventional techniques. Recently, we developed bio-nanocapsules (BNCs) displaying a tandem form of the IgG Fc-binding Z domain derived from Staphylococcus aureus protein A (ZZ-BNC) to enhance the sensitivity and antigen-binding capacity of IgG via oriented-immobilization. Here, we used high-speed atomic force microscopy (HS-AFM) to reveal the fine surface structure of ZZ-BNC and observe the movement of mouse IgG3 molecules tethered onto ZZ-BNC in solution. ZZ-BNC was shown to act as a scaffold for oriented immobilization of IgG, enabling its Fv regions to undergo rotational Brownian motion. Thus, HS-AFM could decipher real-time movement of sensing molecules on biosensors at the single molecule level.

Open-Sandwich Molecular Imprinting: Making a Recognition Matrix with Antigen-Imprinted Antibody Fragments

A novel antibody-polymer conjugation method termed open-sandwich molecular imprinting (OS-MIP) has been proposed to produce a specific recognition matrix in the presence of a target antigen. The resultant carboxymethyldextran matrix conjugated with two separate antibody variable region fragments imprinted with the cognate antigen showed higher antigen-binding capacity than non-imprinted ones and was successfully used to sensitively monitor multiple antigen binding/desorption events by a surface plasmon resonance biosensor. Furthermore, when each fragment was labeled with different fluorophores before conjugation, the fluorescence signals of the matrix made by OS-MIP clearly showed an antigen concentration dependent increase in Förster resonance energy transfer between the two dyes. By using a combination of various methods for detecting interaction, OS-MIP will be a useful platform for detecting various targets from small molecules to proteins with high sensitivity and specificity.

Improved stability of multivalent antibodies containing the human collagen XV trimerization domain

We recently described the in vitro and in vivo properties of an engineered homotrimeric antibody made by fusing the N-terminal trimerization region of collagen XVIII NC1 domain to the C-terminus of a scFv fragment [trimerbody (scFv-NC1)3; 110 kDa]. Here, we demonstrated the utility of the N-terminal trimerization region of collagen XV NC1 domain in the engineering of trivalent antibodies. We constructed several scFv-based trimerbodies containing the human type XV trimerization domain and demonstrated that all the purified trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Importantly, type XV trimerbodies demonstrated substantially greater thermal and serum stability and resistance to protease digestion than type XVIII trimerbodies. In summary, the small size, high expression level, solubility and stability of the trimerization domain of type XV collagen make it the ideal choice for engineering homotrimeric antibodies for cancer detection and therapy.

ScFv antibody: principles and clinical application.

To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.

Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments

The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.