Single Domain Antibody (SdAb) Functional Identification Service

Single domain antibodies (sdAbs, or V_HH) are the smallest antigen-binding units of antibodies, comprising either one variable domain or one engineered constant domain that solely facilitates target binding. They are popular as a novel class of proprietary therapeutic proteins containing unique structural and functional properties. At Creative Biolabs, we offer one-stop solutions for V_HH development and characterization to promote the identification of novel therapeutic V_HHs to meet your specific project demands.

Functions of Therapeutic V_HHs

As one of the novel antibody-based therapeutic agents, V_HHs display several advantages when compared to conventional antibodies. Firstly, as the smallest antigen-binding units of antibodies, V_HHs exhibit similar affinities as compared to full-sized antibodies. Due to their long, protruding, and highly variable CDR3 section, V_HHs can access epitopes normally hidden for conventional antibodies. Secondly, V_HHs are readily expressed in and purified from different host systems, including E. coli, yeast, plant, and mammalian cells. Thirdly, they have exhibited high solubility and thermal stability, high refolding capacity, and good in vivo tissue penetration. Moreover, V_HHs can be engineered into various formats by fusion with other proteins or peptides, thereby tailoring their utility for certain therapeutic applications. The above advantages make them ideally suited for various therapeutic applications.

Fig.1 Conventional IgG (IgG1), heavy chain only subtypes (IgG2 and IgG3), and V_HH^1,3

Potential targets for V_HH include cell surface proteins, cytokines, secreted proteins, intracellular proteins, etc. Depending on the targets, the therapeutic effects of V_HHs can be mediated by different mechanisms, including but not limited to:

V_HH Linked to IgG Fc Region

Thanks to their single domain nature, V_HHs can be readily equipped with various effector functions through molecular or biochemical engineering. These effector functions include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), thereby enhancing and broadening the efficacy of V_HHs. Moreover, the fusion has been also reported to result in a half-life extension.

V_HH Functional Assays Provided by Creative Biolabs

Given that therapeutic antibodies rely on different types of MOAs to achieve clinical efficacy, it is essential to apply diverse strategies to assess and identify the functions of these V_HH candidates. At Creative Biolabs, we are proud to present a comprehensive list of assays for screening and evaluating the functionality of V_HH therapeutics, including but not limited to:

Creative Biolabs is devoted to tailoring the best-fit proposal to screen and identify the desired V_HH candidates with expected functionalities to meet your objectives. If you are interested in learning more about our capacity for functional assays, please do not hesitate to contact us.

Published Data

The Development of a High-affinity V_HH Targeting CsuA/B against A. baumannii

This article details the development of a novel V_HH that exhibits specific recognition and binding to the cell surface protein CsuA/B of the bacterium A. baumannii. The research team initially utilized outer membrane vesicles (OMVs) derived from A. baumannii as an immunogen to generate V_HHs targeting bacterial cell surface antigens through the immunization of llamas. Following a series of screening and identification processes, the researchers successfully isolated a specific V_HH with a high affinity for CsuA/B, which was designated OMV81. As depicted in Fig. 3, they determined the binding kinetics between OMV81 and CsuA/B using SPR technology and verified its specific binding through competitive ELISA. Furthermore, OMV81 was shown to be capable of specifically binding to intact A. baumannii cells, indicating its potential in targeted therapy and diagnosis of A. baumannii infections. This study not only provides a new perspective for understanding the infection mechanism of A. baumannii but also paves the way for the development of novel antimicrobial treatment strategies and diagnostic tools.

FAQ

1. What challenges and solutions exist in identifying V_HH functions?

The identification process for single-domain antibodies (V_HHs) encounters several problems, and some challenges come from ensuring there is enough diversity in the library to find high-affinity binders. Additionally, functional screening methods do not always fully match the biological context. Various approaches are available to tackle these issues, such as high-throughput screening technology, and multiple rounds of selection can be used to enhance the pool of functional binders. Also, the links between sequence and function are explored by modern bioinformatics tools. Studies focused on structure might assist in better understanding binding mechanisms, and this could promote rational design to improve overall performance.

2. How does phage display facilitate the identification of V_HH antibodies?

Phage display is to put V_HHs on the surface of bacteriophages. An antigen of interest is employed to capture phages that carry V_HHs with specific binding properties. After multiple rounds of panning, phages with a strong affinity for V_HHs are identified. The DNA of these phages is sequenced to find the appropriate V_HH coding genes, which may then be tested for neutralizing activity.
Phage display means V_HHs are displayed on bacteriophages' surfaces. Then, a desired antigen is used to select those phages that carry V_HHs with specific binding characteristics. After several panning rounds, the phages showing a high binding strength for V_HHs are identified and picked. The DNA extracted from these phages is sequenced to find the genes coding for optimal V_HHs, which could be tested later for their neutralizing potential.

3. What role do functional assays play in V_HH discovery services?

Functional experiments are essential in V_HH discovery since they validate the biological activity of identified V_HHs, such as binding affinity tests like ELISA or SPR and neutralization assays against pathogens or cellular components. Functional characterization is commonly used to confirm the selected V_HHs not only bind to the target antigen with high affinity but also effectively neutralize or inhibit the pathogen's biological function.

4. What is the significance of epitope mapping in V_HH characterization?

Epitope mapping is used to find out which region of an antigen a V_HH attaches to. Knowing this location is important for gaining deeper insights into how neutralization works and also for confirming that the V_HH is attaching to the conserved areas across different strains of the pathogen. So, the process of epitope mapping could help in the development of more effective therapeutic options and vaccines by showing which parts of the antigen are significant.

Resources