Selection of Nano-antibody Library-Nano Antibody Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Selection of Nano-antibody Library

Antibody Library

Use polymerase chain reaction and other means to obtain a complete set of antibody variable region light and heavy chain genes in vitro, recombinant these genes into a certain vector, and transfer them into appropriate receptor cells or phages. The collection of these antibody gene clones is called an antibody library. The required monoclonal antibodies can be expressed and screened from this library.

Nano-antibody Library

Nano antibody library is to replace the antibody sequence in the process of antibody library construction with the sequence of nano antibodies, and transfer them into appropriate receptor cells or phages to obtain the corresponding library.

The classification of nano antibody library is consistent with that of traditional antibody library, which can be divided into immune library, natural library and synthetic library. Let's introduce them one by one below:

Immune nanoantibody Library

The immune nanoantibody library is to immunize camelids with specific antigens. It usually takes 5 immunizations, which takes about 6-8 weeks. After immunization, jugular vein blood is collected, PBMC/lymphocytes are separated to extract RNA, reverse transcription is performed to obtain cDNA, VHH gene sequences are amplified, and gene sequences are cloned into phage display vectors. Escherichia coli is transformed to obtain immune libraries. The library capacity is determined by the number of transformants grown. The display library is then used to screen phages that express the target antibody, remove phages that do not express the target antibody, and detect the antibody gene insertion rate and library diversity of the library through PCR amplification and monoclonal sequencing.

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Figure 1 Immune nanoantibody library

Natural nanoantibody Library

In some cases where antigens cannot be immunized (for example, antigens are not immunogenic or antigens are toxic), natural nanoantibody libraries are more advantageous.

Natural libraries do not require the preparation of antigens to immunize animals. They only need to collect jugular blood from young and healthy camelids to separate lymphocytes to extract RNA or extract RNA from the spleen for reverse transcription to obtain cDNA, PCR amplify the gene fragments of VHH antibodies, clone the gene fragments into phagemid vectors, and transform TG1 to obtain natural nanoantibody libraries. Because phagemid vectors are used, superstaining with helper phages (M13KO7, R408 and VCSM13) is required to display the inserted genes on the phage surface. After that, the antibody gene insertion rate and library diversity of the library are determined, screened, and identified in accordance with the methods of immune nanoantibody libraries mentioned earlier.

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Figure 2 Natural nanoantibody library

Synthetic nanoantibody Library

However, in order to obtain high-affinity antibodies, it is usually necessary to construct a library with a capacity of 10^9-10^10 and more than 80% of the clones should be able to encode nanoantibodies. In order to construct a natural nanoantibody library with a large capacity and rich library diversity, a large amount of jugular vein blood needs to be drawn from different camelids at the same time (if you want to obtain 10^10 different VHH clones, you will need more than 10 liters of blood), which also increases the workload. Therefore, it is more meaningful to construct a synthetic nanoantibody library with rich diversity.

The core of the construction of a synthetic nanobody library is to artificially create VHH fragments with diversity. The general process is as follows:

(1) Select the protein framework: Unlike each nanobody from an animal host, which has its own framework region, all nanobodies from a synthetic nanobody library need to share the same framework region sequence. The shared framework needs to be stable, easy to express, and highly versatile. The basic framework of the synthetic nanobody library is consistent. A natural nanobody called cAbBCII10 has been tested as a good choice for the framework of the synthetic nanobody library.

(2) Template determination;

(3) Random mutation in CDR through primer design: The diversity of the library is reflected in the CDR. The number of mutated CDR regions and the length of each CDR region must be determined. Generally, the CDR3 region is the main region that determines affinity and specificity. Only the CDR3 region can be mutated, and the CDR3 region can be designed with multiple lengths. In order to prevent the insertion of stop codons or frameshift mutations, triple nucleotide mutation technology is currently commonly used to synthesize primers.

(4) Synthetic templates and primers: Considering the effectiveness of the synthetic library, it is necessary to retain important amino acid sites, such as the Cys site for forming disulfide bonds. The designed primers should contain the diverse CDR regions and the overlapping sequences between the fragments during overlapping extension.

(5) Synthesize diverse VHH fragments using asymmetric and overlapping PCR: When synthesizing random primers, the primer synthesis company will add mixed bases to each specific site, and each mixed base pool can be customized in a certain proportion. For CDR regions of different lengths, each length can be prepared into a mixed pool, which can be further mixed in a specific ratio later and the VHH fragments can be recombined using PCR technology.

The subsequent library screening and verification are also consistent with the two libraries mentioned earlier.

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Figure 3 Construction and verification of synthetic nanoantibody library

Comparison of three nanoantibody libraries

	Immune nanoantibody library	Natural nanoantibody library	Synthetic nanoantibody library
Is immunization required?	Yes	No	No
Is blood sampling required?	Yes	Yes	No
Library size	10^6-10^8	10^9-10^11	10^9-10^15
Others	Affinity-matured Nbs will be obtained (relatively) high titer of target-specific binders	Can be used for non-immunogenic targets One library can be used for multiple projects Nbs may need to improve stability/affinity	Can be used for non-immunogenic targets One library can be used for multiple projects Nbs may need to improve stability/affinity

Tekbiotech has been committed to the production of alpaca VHH antibodies for more than 9 years and has an experienced technical team. According to our experimental project data statistics, hundreds of alpaca VHH antibody construction services are successfully delivered every year.

Tekbiotech has established a complete and mature phage antibody display technology platform. Based on the phage display technology platform, Tekbiotech can provide major experimental links including antigen design, alpaca immunization, library construction and screening, and active function verification, and provide highly specific and high-affinity camelid VHH antibodies to scientists around the world.

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