Antibody Yeast Display Service-Tekbiotech-Yeast Display Service,Phage display technology

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Antibody Yeast Display Service

Tek Biotech is committed to providing high-affinity and high-specificity antibody drug discovery services for our clients, which will provide strong support for their downstream research and development work, such as CAR-T/CAR-NK lead sequence design, antibody humanization, drug target antibody development, antibody coupled drug (ADC) development, bispecific antibody development, and highly efficient blocking neutralizing antibody development. With 10 years of experience in drug antibody discovery, Tek Biotech is able to provide high-quality camel-derived VHH discovery services for targets including but not limited to proteins, peptides, small molecules, viruses, membrane proteins, and mRNAs, as well as scFv discovery services for different species (rabbit, mouse, sheep, camel, etc.).

Yeast display technology refers to the fusion expression of the antibody sequence variable region with the lectin Aga2p. The Aga2p protein subunit binds to the Aga1p protein subunit immobilized in the yeast cell wall through two disulfide bonds, and combined with the flow sorting technology, the specific antibody targeting the antigen will be screened out. Based on yeast technology, Tek Biotech is able to provide high-quality yeast display services for single-chain antibodies, including but not limited to VHH, scFv, etc. The library capacity of yeast libraries can be up to 10^8, and the library diversity, insertion rate, and positivity rate can be more than 90%, which can satisfy the quality requirements of all kinds of customers for yeast display libraries of antibodies. At the same time, Tek Biotech can also provide one-stop technical services such as downstream antibody in vitro validation (including, but not limited to, affinity validation, antibody blocking validation, cross-reactivity validation, and other downstream validation experiments), antibody humanization, antibody affinity maturation, CAR-T/CAR-NK lead sequence design, and cell-killing validation, etc., which are complementary to the antibody-yeast demonstration. Customers only need to provide specific experimental requirements and target information, and Tek Biotech's scientists will be able to design and customize the solutions according to customers' needs, which will be helpful for customers' scientific research projects and drug antibody development.

█ Yeast Display Antibody Library Construction Service

Tek Biotech has 2 animal immunization bases to ensure that the animals immunized in our clients' projects are of clear and healthy background. VHH molecular weight is about 15kDa, which is a kind of antibody heavy chain variable region unique to camelid animals, with low immunogenicity and high stability, and it is easy to obtain neutralizing antibodies with high affinity, high specificity and effective blocking, which has a natural advantage in the areas of antibody development for drug targets, CAR-T/CAR-NK therapy ( CAR-T/CAR-NK therapeutic antibodies, which are currently widely used in CAR-T/CAR-NK therapeutic antibodies, mainly camel-derived VHH antibodies (also known as nanobodies)) and other aspects of the natural advantages. Yeast display libraries are more active than phage display libraries in terms of antibody activity after micro-antibody expression, which enables the differentiation of antibody affinity during flow-through screening.The discovery pathway of VHH/scFv antibody yeast display is shown in Figure 1:

Antibody Yeast Display Service-Tekbiotech.jpg

Figure 1 VHH/scFv antibody discovery service based on yeast technology platform

█ Content and Periodicity of Services

Step

Service Content

QC Standards

Period

Step1: Antigen preparation

*Antigen type:

(1) Recombinant protein preparation

(2) Small molecule (modified) + coupling

(3) Peptide synthesis + coupling

(4) Inactivated virus provided by customer

(5) Customer provides encapsulated mRNA

Antigen QC standard:

Recombinant protein 3-3.5mg (purity >85%);

Small molecule purity >90%;

Peptide purity >90%;

4-6weeks

Step2: Animal immunization

(1) Animals were immunized 4 times, with one booster shot, for a total of 5 shots;

(2) Negative serum is collected before immunization, and blood is collected in the 4th injection for ELISA detection of serum potency;

(3) If the serum antibody potency of the 4th needle meets the requirements, then the blood collection 7 days before the booster immunization of 1 shot, if not meet the requirements, then continue the routine immunization;

(4) If the antibody potency meets the requirements, blood will be collected to isolate monocytes;

Animals: clear background (age, sex, immunization status);

Immunity: protein/viral antigen potency >10^5; peptide/small molecule antigen potency >10^4;

10 weeks

Step3: Template cDNA preparation

(1) PBMC total RNA extraction (RNA extraction kit)

(2) High-fidelity RT-PCR for cDNA preparation (reverse transcription kit)

cDNA: Uniform distribution of the gum map;

1 day

Step4: Antibody yeast display library construction

(1) Amplification of VHH gene by two rounds of PCR, using library cDNA as template.

(2) Yeast display vector construction and transformation: VHH gene was spliced into yeast display vector, yeast was transformed by electroshock, and antibody library was constructed.

(3) Identification: 48 clones were randomly selected, and PCR was used to identify the positive rate; sequencing was used to calculate the correct insertion rate and library diversity.

Library positivity rate: >90%;

Library insertion rate: >90%;

Library diversity: >90%

Library capacity: 10^7-10^8

3-4weeks

Step5: Library Screening

(1) Default 3 rounds of screening: fluorescent tagged protein FACS screening;

(2) The products of the third round were subjected to NGS sequencing; at the same time, the positive clones were picked for monoclonal induced expression + ELISA detection;

(3) Pick all positive clones for gene sequencing;

Positive criteria: FACS and ELISA double positive strains;

VHH screening criteria: different CDR amino acid sequences

3-4weeks

Step6: Antibody Validation

(1) Antibody sequence construction of suitable expression vector for expression + affinity purification + antibody quantification;

(2) ELISA to verify antibody-antigen binding (delivery of EC50 data);

(3) BLI method to verify antibody affinity;

(4) Cell function verification: flow blocking verification;

Recombinant antibody 1mg, 90% purity;

Rapid affinity assay results;

Blocking validation results;

4-6weeks

█ Service Advantages

-- A wide range of camel sources available for immunization

-- Immunization bases: sufficient animal resources, including but not limited to camel, mouse, rabbit and sheep.

-- Various antibody libraries for yeast display: VHH antibody library display, scFv antibody library display, etc.

-- A variety of target antibody discovery services are available: protein, peptide, small molecule, virus, membrane protein, mRNA, etc.

-- Mature technology platform: library capacity up to 10^7-10^8, insertion rate >95%, and affinity of antibodies obtained through screening are generally at nM-pM level.

-- High standard of delivery: validity assurance, library quality assurance, high affinity and high specificity antibody assurance, providing strong support for customers' downstream experiments.

-- Traceability of experimental records: QC standards (immuno potency, PBMC QC, library QC and screening validation QC), Chinese and English lab reports, original lab records.

-- Provide a series of complementary downstream antibody development services, including in vitro antibody expression validation, antibody humanization, antibody affinity maturation, bispecific antibody development, CRA-T lead sequence molecular design, and antibody coupled drug (ADC) development.

-- One-on-one customized solutions to meet the needs of various customers' research projects.

Antibody Yeast Display Service Frequently Asked Questions

Q: Yeast may not be able to effectively express antibodies or have low expression levels, which can affect the production and activity of antibodies.

A: Optimize yeast transformation conditions, select efficient expression vectors, adjust culture conditions (such as temperature, pH, medium composition, etc.), optimize signal peptides and antibody gene sequences, etc., to improve antibody expression levels.
Q: During the construction of yeast display antibody libraries, there may be contamination issues caused by hybridization or non-specific binding of antibodies.

A: Strictly control the construction process of the antibody library, use high-quality antibody genes, and eliminate contaminated antibodies through specific screening and identification to ensure the purity and quality of the library.
Q: There may be a problem of insufficient coverage in the antibody library, that is, not covering all the antigens of interest.

A: By designing a reasonable library construction strategy, including optimizing antigen selection, increasing library capacity and diversity, and using multiple rounds of screening, the coverage of the library can be improved.

Q: Yeast may not be able to effectively express antibodies or have low expression levels, which can affect the production and activity of antibodies.

A: Optimize yeast transformation conditions, select efficient expression vectors, adjust culture conditions (such as temperature, pH, medium composition, etc.), optimize signal peptides and antibody gene sequences, etc., to improve antibody expression levels.
Q: During the construction of yeast display antibody libraries, there may be contamination issues caused by hybridization or non-specific binding of antibodies.

A: Strictly control the construction process of the antibody library, use high-quality antibody genes, and eliminate contaminated antibodies through specific screening and identification to ensure the purity and quality of the library.
Q: There may be a problem of insufficient coverage in the antibody library, that is, not covering all the antigens of interest.

A: By designing a reasonable library construction strategy, including optimizing antigen selection, increasing library capacity and diversity, and using multiple rounds of screening, the coverage of the library can be improved.

Consult Now Antibody Yeast Display Service

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Antibody Yeast Display Service Frequently Asked Questions

Q: Yeast may not be able to effectively express antibodies or have low expression levels, which can affect the production and activity of antibodies.

Q: During the construction of yeast display antibody libraries, there may be contamination issues caused by hybridization or non-specific binding of antibodies.

Q: There may be a problem of insufficient coverage in the antibody library, that is, not covering all the antigens of interest.

Q: Yeast may not be able to effectively express antibodies or have low expression levels, which can affect the production and activity of antibodies.

Q: During the construction of yeast display antibody libraries, there may be contamination issues caused by hybridization or non-specific binding of antibodies.

Q: There may be a problem of insufficient coverage in the antibody library, that is, not covering all the antigens of interest.

Consult Now Antibody Yeast Display Service