Nanobody Yeast Display Development Service-Tekbiotech-Yeast Display Service,Phage display technology

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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