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Nanobody Yeast Display Development Service

To meet the customized needs of customers, TekBiotech has developed a library high-throughput screening technology platform based on yeast surface display system of flow cytometry (FACS) to meet the development requirements of nanoantibodies for some customers who need to take into account affinity pre-grading screening. This technology platform can provide customers with camel-derived VHH nanoantibody development services, scFv discovery services of different species (rabbit, mouse, sheep and camel, etc.), and protein mutant evolution discovery services.

█ Development Services Based On Yeast Surface Display Technology

TekBiotech can provide customers with camel-derived VHH nanoantibody development services using yeast surface display technology. The VHH antibody gene from camel is inserted into the end of the lectin Aga2p gene through PCR technology for fusion expression. As shown in Figure 1, the Aga2p protein subunit binds to the Aga1p protein subunit fixed on the yeast cell wall through two disulfide bonds. Combined with flow cytometry technology, the specific antibodies targeting the antigen are screened out.

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Figure 1 VHH nanoantibody yeast display principle diagram

Based on yeast display technology, Tekbiotech can provide customers with high-quality VHH nanoantibody yeast display services, with a library capacity of 10^8, library diversity, insertion rate, and positive rate of more than 90%, to meet the quality requirements of various customers for antibody yeast display libraries.

█ Yeast Display Nanoantibody Development Service

Tekbiotech has 2 animal immunization bases to ensure that the background source of the immunized animals in the customer project is clear and healthy. VHH has a molecular weight of about 15kDa and is a variable region of the antibody heavy chain unique to camel-derived animals. It has natural advantages in the development of drug target antibodies and CAR-T/CAR-NK therapeutic antibodies. Compared with phage display library technology, yeast display library has a higher antibody activity level after trace antibody expression than the latter. Thanks to this foundation, antibodies with different affinities can be grouped during flow screening. The discovery path of VHH nanoantibodies displayed in yeast is shown in Figure 2:

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Figure 2 VHH nanoantibody discovery service based on yeast technology platform

█ Service Content and Cycle

Steps	Service Content	QC Standard	Cycle
Step 1: Antigen Preparation	*Antigen type: (1) Recombinant protein preparation; (2) Small molecule (modification) + coupling; (3) Peptide synthesis + coupling; (4) Customers provide inactivated viruses; (5) Customers provide packaged mRNA;	1) Recombinant protein (purity>85%); 2) Small molecule purity>90%; 3) Peptide purity>90%;	4-6 Weeks
Step 2: Animal Immunization	(1) Animals are immunized 4 times, with one booster shot, for a total of 5 shots; (2) Collect negative serum before immunization, and collect blood for ELISA to test serum titer at the 4th shot; (3) If the antibody titer of the 4th shot serum meets the requirements, another booster shot is performed 7 days before blood collection. If it does not meet the requirements, continue routine immunization; (4) If the titer is qualified, blood is collected to isolate monocytes;	1) Animals: clear background; 2) Immunization: protein/virus antigen titer detection; peptide/small molecule antigen titer detection;	10 Weeks
Step3: cDNA Preparation	(1) PBMC total RNA extraction; (2) High-fidelity RT-PCR to prepare cDNA;	1) PBMC cell quality control; 2) Total RNA quality control; 3) cDNA quality control;	1 Day
Step4: Antibody Yeast Display Library Construction	(1) Using library cDNA as template, two-round PCR amplification of VHH gene; (2) Yeast display vector construction and transformation: VHH gene splicing yeast display vector, electroporation transformation of yeast, construction of antibody library; (3) Identification: Randomly select clones, PCR identification of positive rate; Sequencing to calculate insertion accuracy and library diversity;	1) Library positive rate detection; 2) Library insertion rate detection; 3) Library capacity detection + sequence detection;	3-4 Weeks
Step5: Library Screening	(1) Fluorescent marker protein FACS screening; (2) NGS sequencing of different affinity groups is performed according to requirements; single clones are selected for induction expression + ELISA detection of positive clones; (3) Gene sequencing of positive clones;	1) Setting of double positive strains; 2) Setting of VHH sequence screening criteria	3-4 Weeks
Step 6: Antibody Verification	(1) Constructing a suitable expression vector for antibody sequence expression + affinity purification + antibody quantification; (2) ELISA verification of antibody binding to antigen (delivery of EC50 data); (3) BLI method verification of antibody affinity; (4) Cell function verification: flow blocking verification;	1) Quality control of recombinant antibody expression; 2) EC50 verification 3) Rapid affinity determination results; 4) Blocking verification results;	4-6 Weeks

After conducting preliminary drugability evaluation for customers, TekBiotech can also provide one-stop technical services such as antibody humanization, antibody affinity maturation, CAR-T/CAR-NK lead sequence design and cell killing verification, and conduct reasonable solution design and personalized customization according to customer needs to help customers' scientific research projects and drug antibody development.

█ Service Advantages

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Possessing immune base:

sufficient animal resources,

including camel, mouse,

rabbit and sheep sources,

etc.

Various antibody library

yeast display options: VHH

antibody library display,

scFv antibody library

display, etc.

Various target antibody

discovery services are

available: protein, peptide,

small molecule, virus,

membrane protein,

mRNA, etc.

Mature technology

platform: immune library

capacity 10^8-10^9,

affinity direct group

screening

Experimental records are

traceable: Chinese and

English experimental

reports, original

experimental records

Personalized solution

customization to meet

the needs of various

customer research

projects

█ Yeast Surface Display Technology Platform Service Advantages

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lent hydrophobic sequence display, suitable for

TCR surface display

Flexible configuration of single and double fluorescence

screening system, less antigen screening demand

Affinity groups can be distinguished during the

screening process

Nanobody Yeast Display Development Service Frequently Asked Questions

What is the basic principle of Nano Antibody Yeast Display Development Service?

Nano Antibody Yeast Display Development Service is a technology for screening and optimizing nanobodies through a yeast display platform. In this process, a library of nanobody genes is first cloned into a yeast expression vector so that a specific nanobody is displayed on the surface of each yeast cell. In this way, a diverse library of nanobodies can be constructed with broad specificity against different antigens. In the screening process, target antigens or target molecules are used to carry out binding reactions with the nanobodies displayed on the surface of the yeast cells, and only the yeast cells that bind with high affinity and specificity to the antigens can be screened. Ultimately, a series of nanobodies with high affinity and specificity are obtained through high-throughput screening and clonal isolation. The advantage of the yeast display platform lies in its ability to quickly find the nanobodies that meet the requirements through efficient sorting and screening techniques, as well as further optimization and validation.
What are the advantages of the Nanoantibody Yeast Showcase development service?

The nanobody yeast display development service has many significant advantages. First, yeast cells, as a eukaryotic expression system, are capable of folding and modifying nanobodies correctly, and in particular, they can undergo post-modifications such as glycosylation, which is crucial for the stability and affinity of certain nanobodies. In contrast, prokaryotic systems (e.g., E. coli) are unable to provide these complex post-modifications, and thus yeast display technology has a greater advantage in some demanding applications. Secondly, yeast display platforms have high-throughput screening capabilities and can screen a large number of high-affinity nanobodies in a relatively short period of time. In addition, the relatively high transformation efficiency of yeast cells enables the construction of larger libraries of nanoantibodies, providing customers with diverse options. Finally, yeast display technology can complete antibody screening at a lower cost, and the experimental operation is relatively simple, suitable for large-scale antibody development and optimization work.
How is the screening and optimization of nanoantibody yeast display development service carried out?

First, the customer provides the target antigen or target molecule, and the research team designs the appropriate screening conditions according to the characteristics of the antigen. The nanoantibody gene library is genetically engineered to be introduced into yeast cells and displayed on their surface, building a diverse library of nanoantibodies. The screening process is usually accomplished by high-throughput methods such as flow cytometry, which utilizes antigen binding to the nanobodies displayed on the surface of the yeast cells, and then sorts them by fluorescent labeling of the cells to screen the nanobodies that bind to the antigen. With multiple rounds of screening, it is possible to progressively improve the screening of nanobodies with higher antigen affinity and specificity. Meanwhile, the optimization process will also include affinity maturation, glycosylation modification and stability optimization as needed to further improve the functional properties of the nanobodies. Ultimately, the screened and optimized nanobodies are cloned and validated to ensure their high antigen-binding ability and application potential.
What are the application areas of nanobody yeast demonstration and development services?

Nanoantibody yeast demonstration and development services can be widely used in a variety of fields, especially in antibody discovery, vaccine development, targeted therapies and diagnostics. Firstly, in antibody discovery, yeast display technology can rapidly screen high-affinity nanobodies against specific antigens, which are widely used in the treatment of cancer, infectious diseases and immune disorders. Second, as carriers for vaccines and diagnostic reagents, nanobodies are able to efficiently recognize target molecules while avoiding the limitation of the larger size of conventional antibodies, thus providing unique advantages in the development of small molecule vaccines and highly sensitive diagnostic tools. Nanobodies are also used in biomarker detection, early disease diagnosis and therapeutic monitoring. In addition, nanoantibodies have a wide range of applications in protein engineering and enzyme catalysis due to their unique stability and small size, which allows them to remain active in harsh environments.
What are the limitations of Nano Antibody Yeast Display Development Services?

Despite the many advantages of nanobody yeast display development services, there are some limitations. Firstly, yeast display technology may be limited in displaying very large or complex antibody fragments, especially for those antibodies with more complex structures or requiring special folding, yeast may not be able to display their functions correctly, which may lead to unsatisfactory screening results. Second, yeast display technology has some limitations on the size of antibody libraries. Although yeast cells are capable of hosting larger libraries, the library capacity of yeast display is usually smaller compared to phage display technology, and more efficient screening techniques may be needed to compensate for this deficiency. In addition, the culture and screening process of yeast cells requires certain experimental conditions and time, especially when multiple rounds of screening are performed, which may involve more cumbersome operational steps and lead to a longer development cycle. Finally, although yeast demonstration is capable of certain post-modifications (e.g. glycosylation), yeast is still limited in certain post-modifications compared to mammalian cell systems, which may have an impact on the functionality of nanobodies for certain specific applications.

What is the basic principle of Nano Antibody Yeast Display Development Service?

Nano Antibody Yeast Display Development Service is a technology for screening and optimizing nanobodies through a yeast display platform. In this process, a library of nanobody genes is first cloned into a yeast expression vector so that a specific nanobody is displayed on the surface of each yeast cell. In this way, a diverse library of nanobodies can be constructed with broad specificity against different antigens. In the screening process, target antigens or target molecules are used to carry out binding reactions with the nanobodies displayed on the surface of the yeast cells, and only the yeast cells that bind with high affinity and specificity to the antigens can be screened. Ultimately, a series of nanobodies with high affinity and specificity are obtained through high-throughput screening and clonal isolation. The advantage of the yeast display platform lies in its ability to quickly find the nanobodies that meet the requirements through efficient sorting and screening techniques, as well as further optimization and validation.
What are the advantages of the Nanoantibody Yeast Showcase development service?

The nanobody yeast display development service has many significant advantages. First, yeast cells, as a eukaryotic expression system, are capable of folding and modifying nanobodies correctly, and in particular, they can undergo post-modifications such as glycosylation, which is crucial for the stability and affinity of certain nanobodies. In contrast, prokaryotic systems (e.g., E. coli) are unable to provide these complex post-modifications, and thus yeast display technology has a greater advantage in some demanding applications. Secondly, yeast display platforms have high-throughput screening capabilities and can screen a large number of high-affinity nanobodies in a relatively short period of time. In addition, the relatively high transformation efficiency of yeast cells enables the construction of larger libraries of nanoantibodies, providing customers with diverse options. Finally, yeast display technology can complete antibody screening at a lower cost, and the experimental operation is relatively simple, suitable for large-scale antibody development and optimization work.
How is the screening and optimization of nanoantibody yeast display development service carried out?

First, the customer provides the target antigen or target molecule, and the research team designs the appropriate screening conditions according to the characteristics of the antigen. The nanoantibody gene library is genetically engineered to be introduced into yeast cells and displayed on their surface, building a diverse library of nanoantibodies. The screening process is usually accomplished by high-throughput methods such as flow cytometry, which utilizes antigen binding to the nanobodies displayed on the surface of the yeast cells, and then sorts them by fluorescent labeling of the cells to screen the nanobodies that bind to the antigen. With multiple rounds of screening, it is possible to progressively improve the screening of nanobodies with higher antigen affinity and specificity. Meanwhile, the optimization process will also include affinity maturation, glycosylation modification and stability optimization as needed to further improve the functional properties of the nanobodies. Ultimately, the screened and optimized nanobodies are cloned and validated to ensure their high antigen-binding ability and application potential.
What are the application areas of nanobody yeast demonstration and development services?

Nanoantibody yeast demonstration and development services can be widely used in a variety of fields, especially in antibody discovery, vaccine development, targeted therapies and diagnostics. Firstly, in antibody discovery, yeast display technology can rapidly screen high-affinity nanobodies against specific antigens, which are widely used in the treatment of cancer, infectious diseases and immune disorders. Second, as carriers for vaccines and diagnostic reagents, nanobodies are able to efficiently recognize target molecules while avoiding the limitation of the larger size of conventional antibodies, thus providing unique advantages in the development of small molecule vaccines and highly sensitive diagnostic tools. Nanobodies are also used in biomarker detection, early disease diagnosis and therapeutic monitoring. In addition, nanoantibodies have a wide range of applications in protein engineering and enzyme catalysis due to their unique stability and small size, which allows them to remain active in harsh environments.
What are the limitations of Nano Antibody Yeast Display Development Services?

Despite the many advantages of nanobody yeast display development services, there are some limitations. Firstly, yeast display technology may be limited in displaying very large or complex antibody fragments, especially for those antibodies with more complex structures or requiring special folding, yeast may not be able to display their functions correctly, which may lead to unsatisfactory screening results. Second, yeast display technology has some limitations on the size of antibody libraries. Although yeast cells are capable of hosting larger libraries, the library capacity of yeast display is usually smaller compared to phage display technology, and more efficient screening techniques may be needed to compensate for this deficiency. In addition, the culture and screening process of yeast cells requires certain experimental conditions and time, especially when multiple rounds of screening are performed, which may involve more cumbersome operational steps and lead to a longer development cycle. Finally, although yeast demonstration is capable of certain post-modifications (e.g. glycosylation), yeast is still limited in certain post-modifications compared to mammalian cell systems, which may have an impact on the functionality of nanobodies for certain specific applications.

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