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Camelid VHH Antibody Library Services

KMD BioscienceTM is able to provide high quality, M13 phage display-based camel antibody screening services, including bactrian camel and dromedary (alpaca)-derived nanobody phage display technology services. Our scientists have many years of heavy chain antibody (VHH antibody) library display construction and screening experience and can obtain high quality nanobodies in a short period of time.

 

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 about 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, that is, a VHH region sequence, which is also called a nano-antibody 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 has 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

 

Content

Camel VHH Antibody Library

Alpaca VHH Antibody Library

Antigenic Tolerance

High, multiple antigens mixture, high doze immunization

High, multiple antigens mixture, medium doze immunization

Antiserum Titer

High

High

Neutralizing Antibody Titer

High

Medium

Antibody Affinity

Easier to obtain high affinity antibodies

Able to obtain high affinity antibodies

Antigen Amount

4-6mg, purity>85%

2-3mg, purity >85%

Blood Amount

~200ml

~20ml

PBMC Cell Amount

High

Common

Library Capacity

High, Immune library 108-109

High, Immune library 107-108

Cost of Immunization

Lower

High

Small Molecule Antigen Reactivity

High

Medium

 

 

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

 

KMD BioscienceTM is able to provide high quality, camel-derived VHH antibody phage display library construction services. We insist on using the QbD (Quality by Design) design principles of human vaccines and biopharmaceuticals, based on the full understanding and respect of ICH principles. The Camel VHH antibody phage display library was designed and prepared with a traceable product traceability system and document support system. An excellent VHH antibody phage display library includes antigen design, animal immunization, library construction and screening.

 

---Antigen Design of VHH Antibody Phage Display Library

 

For the camel VHH antibody library, 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 are able to 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 BioscienceTM 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. The protein antigen was >105 standard according to ELISA serum titer, the small molecule antigen immunization ELISA serum titer >104.

 

---Library Construction and Screening of VHH Antibody Phage Display Library

 

KMD BioscienceTM uses the pIII protein of M13 phage for VHH antibody (nanobody) surface display. Our scientists constructed a phage vector with Flag and 6*His tag double tags, and prepared TG1 and XL1-Blue host competent cells with 108 transfection efficiency, after 2-3 transfections and amplification, it is able to provide customers with a high quality VHH antibody display library with an effective library capacity of 108-109 and a packaging capacity of 1013-1016/ml. Up to 20-30 high-affinity phage strains can be obtained by three-round nanobody library screening by antigen-limited pressure screening. Through simple IPTG induction, a large number of antibody expression products can be obtained within 1-2 weeks to meet the research needs of customers such as FACS and cell activity testing.

 

Camelid Antibody Library Construction Service—Service Procedure:

 

Steps

Specification

Timeline

Deliverables

Step1: Animal Immunization

1. A healthy, full-aged Bactrian camels (28-36 months old)
2. Camels were immunized 4-7 times.

3. Serum titers was measured by ELISA and collected peripheral blood. 

6-8 weeks

1.Experimental report: including detailed construction procedures and representative sequence information.

2. VHH antibody library 2ml, >1⨯1013 /ml phage particles

3. 10-15 positive clones were expressed to prepare VHH antibody supernatant.

4. 10-15 expressing strains of positive clone.

Step2: cDNA synthesis (Clients can provide cells)

1. Total RNA extraction.
2. RT-PCR

3. PCR amplification with Actin specific primers to identify the quality of cDNA.

3-4 weeks

Step3: Library construction

1. Primer design & synthesis.

2. PCR amplification of variable region genes of heavy and light chains using cDNA as a template.

3. scFv gene splicing.

4. Plasmid construction & transformation: after enzyme digestion, scFv and phagemid vector were ligated and transformed into TG1 host bacteria by electric shock to construct antibody libraries.

5. Identification: 20-50 clones were randomly selected, PCR identification, sequencing and analysis of antibody sequences.

6. Packaging: >1*1013 phage particles /ml.

Step4: Library Screening (3 Rounds)

1.The antigenic limit pressure method is used for 3 rounds of panning

2. Identification: 96 clones were randomly selected for ELISA titer determination

3. Up to 96 positive clones were sequenced, and the optimal 10-15 clones were selected for prokaryotic expression transformation.

4. 10-15 positive clones were expressed to construct VHH antibody supernatant.

3-4 weeks

 

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 VHH antibody library, only 3-4 weeks is needed.

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

 

Camelid Antibody Library Construction Service----Service Advantages

 

---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: effective 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

 

Camelid Antibody Library Construction Service---Experiment Procedure

 

How to Order?

 

If you are interested in our services, please call the hotline at +86-022-8216-4980/156-0119-0182 or send an email to info@kmdbioscience.com