VHH Antibody Library Construction Service

Camelid VHH Antibody Library Service-Heavy Chain Antibody Background:

There are four IgG subclasses (IgG1, 2, 3, and 4) in traditional serum antibodies, but only three IgG subclasses (IgG1, 2, 3) in camelid. The structure of camelid IgG1 is similar to traditional IgG: two heavy chains and two light chains; IgG2 and IgG3 lack the CH1 and light chain domains and are therefore also referred to as heavy-chain antibodies (hcAb), as shown in Figure 1.

Figure 1 Classical antibody and heavy chain antibody structure

*(a) Conventional antibodies and their Fab/scFv antibody forms (b) Heavy-chain antibodies and their VHH antibody forms

The distribution of heavy-chain antibodies in alpaca and camel serum is uneven. The ratio of heavy chain antibodies in camel serum antibodies is about 60-75%, and the proportion of heavy chain antibodies in alpaca serum antibodies is approximately 40-50%. This significant difference led to the innate advantages of the constructed camel VHH antibody library, as shown in Table 1. The camel source antibody library mainly refers to a VHH antibody library constructed by using the variable region of a camel-derived heavy chain antibody, a VHH region sequence, which is also called a nanoantibody library. The VHH antibody is characterized by an antigen-binding region containing only the heavy chain antibody. As shown in Fig. 2, the amino acid length is about 110 AA, and the SDS-PAGE shows a molecular weight of about 15 kDa (much smaller than the molecular weight of the conventional antibody 150 kDa) and has excellent stability. It is resistant to extreme pH environments. Meanwhile, VHH antibodies also have good adaptability, and it is easy to meet the requirements of transformation and sorting experiments of immune cells such as CAR-T.

Fig. 2 Alpaca heavy chain antibody and VHH antibody

Comparison of Camel and Alpaca VHH Antibody Library:

Camelid VHH Antibody Library Service -Camel VHH Antibody Library Construction Service:

Antigen Design of VHH Antibody Phage Display Library:

For camelid antibody production service, we designed a series of human immune checkpoint proteins from the fusion expression of camelid antibody Fc fragment. Combined with our mature Freestyle 293F and Expi CHO cell suspension expression platform, we can provide gene expression, vector construction and gram-level protein expression in a short period of time. For potential service needs, please consult our protein expression platform. At the same time, based on our experimental results, we found that the immune effect of small molecule protein directly immunized with the human Fc fragment is much better than the target protein fusion alpaca or camel Fc fragment (Please contact our technical support if you are interested in our service).

Animal Immunization of VHH Antibody Phage Display Library:

KMD Bioscience provides an unimmunized, age-old (28-36-month-old) camel or alpaca for antigen immunization through collaboration with camel and alpaca farming communities. According to standard immunization methods, 4-7 needles are immunized. According to the ELISA serum titer, the protein antigen was >10⁵ standard, and the small molecule antigen immunization ELISA serum titer was >10⁴.

Library Construction and Screening of VHH Antibody Phage Display Library:

KMD Bioscience harnesses the pIII protein of the M13 phage as the cornerstone for VHH antibody (nanobody) surface display, ensuring robust and efficient presentation of target-specific antibodies. Our dedicated team of scientists has meticulously crafted a phage vector equipped with Flag and 6*His tag double tags, enhancing the recognition and purification capabilities of the resulting antibodies. In tandem, our proprietary TG1 and XL1-Blue host competent cells boast an impressive transfection efficiency of 10⁸, ensuring optimal expression and display of the VHH antibody library. Through a streamlined process involving 2-3 transfections and subsequent amplification steps, we proudly deliver high-quality VHH antibody display libraries with ample capacity ranging from 10⁸ to 10⁹, alongside packaging capabilities reaching 10¹³-10¹⁶/ml. Furthermore, our innovative approach to nanobody library screening employs antigen-limited pressure screening, facilitating the isolation of 20-30 high-affinity phage strains following just three rounds of screening. This efficient methodology ensures rapid identification of potent antibodies tailored to meet specific research objectives. With a simple induction protocol using IPTG, our clients can swiftly access a vast array of antibody expression products within a mere 1-2 weeks. These products are readily amenable to diverse research applications, including Fluorescence-Activated Cell Sorting (FACS) and cell activity testing, empowering researchers with the tools they need to advance their scientific inquiries with confidence.

VHH Antibody Library Construction Service Procedure:

Note: 1. If the client provides camel peripheral blood lymphocytes, lymph node cells or spleen cells (preserved in RNA later and other solutions to avoid RNA degradation, blue ice transport), the service can start from the second step to construct the VHH antibody library, only 3-4 weeks are needed.

2. Cell lysates were prepared using Trizol solution and transported on dry ice.

Service Process:

VHH Antibody Library Construction Service Highlights:

---Provide immune library construction services and natural camel/alpaca antibody library screening services

---Short cycle, high-quality library and monoclonal antibody cell line can be obtained in about 16-20 weeks

---High library capacity: adequate library capacity 10⁸ – 10⁹/immune library, 10⁹ – 10¹¹/natural library

---High-affinity antibody construction

---Personalized panning strategy

---Complete document traceability system and QC quality control file

How to Order?

If you have any questions regarding our services or products, please feel free to contact us by E-mail: info@kmdbioscience.com or Tel: +86-400-621-6806