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scFv Antibody Development Services

TekBiotech is committed to providing customers with high-affinity and high-specificity antibody drug early discovery technology services, and providing strong support for customers' subsequent CAR-T/CAR-NK lead sequence design, antibody humanization, drug target antibody development, bispecific antibody development, and efficient blocking neutralizing antibody development and other downstream R&D work. TekBiotech has 10 years of project development experience and experience in drug antibody discovery, and can provide customers with high-quality scFv discovery services for targets including but not limited to proteins, peptides, small molecules, viruses, membrane proteins, and mRNA from different species (rabbit, mouse, human, etc.).

█ scFv Antibody Development Technology Services

TekBiotech can provide customers with scFv antibody library construction and screening services. The primer combination PCR technology is used to obtain the germline gene fragments of the antibody VH region and VL region (Kappa and lambda chain) in different species. After obtaining the scFv antibody by overlapping PCR method, it is inserted into the N-terminus of the P3 protein of the M13 phage, so that the scFv antibody gene is displayed on the phage surface along with the expression of the P3 protein. Finally, the scFv antibody binding to the target antigen is screened out by a screening method similar to "fishing". Compared with yeast antibody display technology, the scFv format has high requirements for library capacity and diversity display due to its high diversity. The library capacity of the scFv antibody library based on phage display technology can easily reach 10^9-10^10, and the library diversity is easily >90%, which is a more suitable display system. At the screening level, the phage display scFv library is also easier to screen with target objects such as cell lines and VLPs. The phage display discovery path of scFv antibody discovery service is shown in Figure 1:

scFv Antibody Development Services-tekbiotech.png

Figure 1 scFv antibody discovery service based on phage technology platform

█ scFv Technology Antibody Development Service Host Type

TekBiotech provides customers with scFv antibody discovery services based on the M13 phage display system, as shown in Figure 2 or Table 1:

Table 1 ScFv antibody discovery type

Antibody Form	Species Source	Library Type
scFv Antibody Form	Human	Natural
	Mouse	Immune/Natural
	Rabbit	Immune/Natural
	Sheep	Immune/Natural
Fab Form Antibody	Human	Natural
	Mouse	Immune/Natural
	Rabbit	Immune/Natural

scFv Antibody Development Services-tekbiotech1.png

Figure2 ScFv antibody discovery type

█ scFv Antibody Development Service Content and Cycle

Steps

Service Content

QC Testing

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 the 4th shot to test

serum titer by ELISA;

(3) If the serum antibody titer of the 4th shot meets the requirements, another booster shot

is performed 7 days before blood collection. If it does not meet the requirements, continue

with routine immunization;

(4) If the titer is qualified, blood is collected to separate monocytes;

1) Animal: clear background;

2) Immunity: protein/virus antigen titer detection;

peptide/small molecule antigen titer detection;

8-10 Weeks

Step3: Template 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: Phage Display

Library Construction

(1) Using cDNA as a template, combine primers for multiple rounds of PCR to amplify

VH and VL genes;

(2) Phagemid construction and transformation: VH-VL gene splicing phagemid vector,

electroporation transformation of TG1 host bacteria, and construction of antibody library;

(3) Identification: Randomly select clones, PCR identification of positive rate

+ insertion rate;

(4) Auxiliary phage preparation: M13 phage amplification + purification;

(5) scFv display library rescue;

1) Library positive rate detection;

2) Library insertion rate detection;

3) Library capacity detection + sequence detection;

2-3 Weeks

Step5: Library Screening

(1) Antigen coating (single protein screening, the default is solid phase screening or

magnetic bead sorting);

(2) Default 3-5 rounds of screening: pressure screening, maximally removing

non-specific antibodies;

(3) Pick a single clone to amplify phage + induce expression + ELISA to

detect positive clones;

(4) Pick all positive clones for gene sequencing;

1) ELISA positive standard setting;

2) VHH screening standard setting;

2-3 Weeks

Step6: Drugability

Evaluation

(1) Construct a suitable expression vector for expression + affinity purification + antibody

protein quantification of the obtained antibody sequence;

(2) ELISA verification of antibody-antigen binding;

(3) BLI method to verify antibody affinity;

(4) Cell function verification: flow blocking verification;

1) Recombinant antibody expression quality

control;

2) EC50 Verification;

3) Rapid affinity determination results;

4) Blocking verification results;

4-6 Weeks

Note: Customers can choose the above service steps according to their needs.

█ Advantages of scFv Antibody Development Service


Possessing immune base: sufficient animal resources, including but not limited to camel, mouse, rabbit and sheep sources	Short development cycle: after obtaining PBMC, it takes 4-6 weeks from library construction to screening to obtain antibody sequences	Multiple target antibody discovery services are available: protein, peptide, small molecule, virus, membrane protein, mRNA, etc.	Mature technology platform: immune library capacity 10^9-10^10, natural library capacity 10^10-10^12

Diversity of library screening methods: design the best screening method according to customer project requirements, such as solid phase screening, liquid phase screening, cell screening, Magnetic bead screening, etc.	Experimental records are traceable: QC quality control standards (immunity titer, PBMC quality control, library quality control and screening verification quality control), Chinese and English experimental reports, original experimental records	One-to-one personalized program customization (including immunization program, library construction program, screening program and subsequent in vitro expression verification program, etc.) to meet the scientific research project needs of various customers	A series of supporting downstream drug antibody development services can be provided, including antibody in vitro expression verification, antibody humanization, antibody affinity maturation, bispecific antibody development, CAR-T lead sequence molecular design, etc.

scFv Antibody Development Services Frequently Asked Questions

Advantages of scFv antibody discovery service

scfv antibody has many advantages over monoclonal antibody, firstly scfv antibody is stable and can be stored at 4°C for several years. Meanwhile the gene of scFv antibody is easy to manipulate, scFv antibody can bypass hybridoma and immunization to produce. In addition, higher affinity mutants of scFv can be generated by targeted mutagenesis, which is easier and simpler to perform. scFv is the smallest functional unit of an antibody and has shown promise in cancer radioimmunoscintigraphy due to its specific properties, including higher tumor penetration and faster clearance compared to the parent Ig. After scFv antibody design, the antibody is customized so that the antibody can be demonstrated on phage. scFv single-chain antibodies play an important role in cancer therapy and are used to develop targeting vectors for curable gene delivery. scFv single-chain antibodies can also be used as other detection reagents. Over the past few years, antibodies produced by scFv antibody customization have become an alternative to many common reagents that are not available.
What are scFv antibodies?

Single-chain antibodies are produced primarily by hybridomas, murine spleens, and human B cells. scFv (single-chain fragment variable region) is a non-covalent heterodimer consisting of the VH and VL structural domains, of which can be used to construct recombinant scFv (single-chain fragment variable region) antibodies. scFv is a modification of the variable regions of conventional antibodies, which are linked together by a junction that can be used in E.coli expressed single-chain variable region antibodies, enabling protein engineering for improved characterization. Tektronix Biologics has an extensive recombinant antibody customization program that offers a wide range of different antibody customization methods. At the same time, Tektronix Biologics provides scFv and Fab antibody customization services and antibody humanization services. Tektronix Biologics also provides a full chain of services including antigen preparation, scFv antibody design, phage library construction, and recombinant antibody purification.
What are the main components of scFv antibody discovery services?

In order to obtain scFv, messenger ribonucleic acid is first isolated and then reverse transcribed as an amplification template. The heavy chain (VH) and light chain (VL) genes of the antibody are then amplified separately by RT-PCR. The heavy chain and light chain genes, through gene technology, form the scFv gene fragments we need, and then ligated to the vector, using this method, can be completed with a different range of scfv gene library construction. Then it will be transformed into different host cells (such as E. coli, yeast, etc.) to construct libraries containing the target antibody fragments. Then Tektronix conducts repeated rounds of elution and screening to enrich the scfv phage library, and finally completes the construction of the scfv antibody library. From the constructed antibody library, we screen out the customer's target scFv antibody fragments, and then sequence and analyze the sequence of the scfv antibody to confirm its sequence and characteristics. Teckbio purifies the antibody according to the customer's requirements and then delivers it to the customer.
What are the structural peculiarities of scFv antibody and the role of the junction?

ScFv, as a small molecule genetically engineered antibody, has a molecular weight of only about one-sixth of that of a complete antibody, conferring it with outstanding penetration ability and low immunogenicity. This scFv antibody, consists of a fusion of VH and VL via a flexible peptide junction segment. This junction allows for single-chain variable region antibodies expressed in E. coli, enabling protein engineering to improve scFv properties such as increased affinity and altered specificity. the length of the flexible DNA junction connecting the two V structural domains in scFv antibodies is critical for the correct folding of the antibody. It has been analyzed that scFv antibody peptide junctions must span 3.5 nm (35 A) between the carboxyl terminus of the variable region and the amino terminus of the other structural domain, and must not have an effect on the folding of the structural domains nor interfere with the ability to form a complete antigen-binding site. In addition to the scratch-designed junction peptide on the antibody, we can apply a peptide sequence derived from the protein structure, which is used to provide a compatible length and conformation in the variable region of the bridging scFv (single-chain fragment variable region), thus stabilizing the antibody spatial structure so that it does not change.
In scFv antibody discovery service we often encounter antibodies with low affinity, poor stability and so on, how do we solve it?

The affinity of scFv antibody is low compared with other antibodies due to its own structural limitations. Meanwhile, scFv antibody is easy to be degraded in the body and has poor stability. Teckbio has optimized the sequence of scFv antibody through genetic engineering to help increase the affinity of scFv antibody. At the same time, Teckbio has constructed scFv multimers (e.g. Diabodies, Tribodies) to increase the binding valence between the antibody and the antigen, thus improving the affinity. TeckBio also humanizes scFv to genetically reduce its immunogenicity while potentially increasing the affinity of the antibody. Selection of appropriate Linkers by Tektronix Biologics can help to increase the stability of scFv antibodies, as the length and composition of the Linkers have a significant impact on the stability of scFv. Optimize the expression system: Meanwhile, the expression of scFv antibody is not the same in different expression systems, and the expression may be too low. Teckbio finds the most suitable conditions for stable expression by trying to express scFv in prokaryotic systems (e.g. E. coli), yeast systems or mammalian cell systems. We also help to improve the stability of the antibody in the organism by adding protective groups, such as PEG (polyethylene glycol) modification, to both ends of the antibody.

Advantages of scFv antibody discovery service

scfv antibody has many advantages over monoclonal antibody, firstly scfv antibody is stable and can be stored at 4°C for several years. Meanwhile the gene of scFv antibody is easy to manipulate, scFv antibody can bypass hybridoma and immunization to produce. In addition, higher affinity mutants of scFv can be generated by targeted mutagenesis, which is easier and simpler to perform. scFv is the smallest functional unit of an antibody and has shown promise in cancer radioimmunoscintigraphy due to its specific properties, including higher tumor penetration and faster clearance compared to the parent Ig. After scFv antibody design, the antibody is customized so that the antibody can be demonstrated on phage. scFv single-chain antibodies play an important role in cancer therapy and are used to develop targeting vectors for curable gene delivery. scFv single-chain antibodies can also be used as other detection reagents. Over the past few years, antibodies produced by scFv antibody customization have become an alternative to many common reagents that are not available.
What are scFv antibodies?

Single-chain antibodies are produced primarily by hybridomas, murine spleens, and human B cells. scFv (single-chain fragment variable region) is a non-covalent heterodimer consisting of the VH and VL structural domains, of which can be used to construct recombinant scFv (single-chain fragment variable region) antibodies. scFv is a modification of the variable regions of conventional antibodies, which are linked together by a junction that can be used in E.coli expressed single-chain variable region antibodies, enabling protein engineering for improved characterization. Tektronix Biologics has an extensive recombinant antibody customization program that offers a wide range of different antibody customization methods. At the same time, Tektronix Biologics provides scFv and Fab antibody customization services and antibody humanization services. Tektronix Biologics also provides a full chain of services including antigen preparation, scFv antibody design, phage library construction, and recombinant antibody purification.
What are the main components of scFv antibody discovery services?

In order to obtain scFv, messenger ribonucleic acid is first isolated and then reverse transcribed as an amplification template. The heavy chain (VH) and light chain (VL) genes of the antibody are then amplified separately by RT-PCR. The heavy chain and light chain genes, through gene technology, form the scFv gene fragments we need, and then ligated to the vector, using this method, can be completed with a different range of scfv gene library construction. Then it will be transformed into different host cells (such as E. coli, yeast, etc.) to construct libraries containing the target antibody fragments. Then Tektronix conducts repeated rounds of elution and screening to enrich the scfv phage library, and finally completes the construction of the scfv antibody library. From the constructed antibody library, we screen out the customer's target scFv antibody fragments, and then sequence and analyze the sequence of the scfv antibody to confirm its sequence and characteristics. Teckbio purifies the antibody according to the customer's requirements and then delivers it to the customer.
What are the structural peculiarities of scFv antibody and the role of the junction?

ScFv, as a small molecule genetically engineered antibody, has a molecular weight of only about one-sixth of that of a complete antibody, conferring it with outstanding penetration ability and low immunogenicity. This scFv antibody, consists of a fusion of VH and VL via a flexible peptide junction segment. This junction allows for single-chain variable region antibodies expressed in E. coli, enabling protein engineering to improve scFv properties such as increased affinity and altered specificity. the length of the flexible DNA junction connecting the two V structural domains in scFv antibodies is critical for the correct folding of the antibody. It has been analyzed that scFv antibody peptide junctions must span 3.5 nm (35 A) between the carboxyl terminus of the variable region and the amino terminus of the other structural domain, and must not have an effect on the folding of the structural domains nor interfere with the ability to form a complete antigen-binding site. In addition to the scratch-designed junction peptide on the antibody, we can apply a peptide sequence derived from the protein structure, which is used to provide a compatible length and conformation in the variable region of the bridging scFv (single-chain fragment variable region), thus stabilizing the antibody spatial structure so that it does not change.
In scFv antibody discovery service we often encounter antibodies with low affinity, poor stability and so on, how do we solve it?

The affinity of scFv antibody is low compared with other antibodies due to its own structural limitations. Meanwhile, scFv antibody is easy to be degraded in the body and has poor stability. Teckbio has optimized the sequence of scFv antibody through genetic engineering to help increase the affinity of scFv antibody. At the same time, Teckbio has constructed scFv multimers (e.g. Diabodies, Tribodies) to increase the binding valence between the antibody and the antigen, thus improving the affinity. TeckBio also humanizes scFv to genetically reduce its immunogenicity while potentially increasing the affinity of the antibody. Selection of appropriate Linkers by Tektronix Biologics can help to increase the stability of scFv antibodies, as the length and composition of the Linkers have a significant impact on the stability of scFv. Optimize the expression system: Meanwhile, the expression of scFv antibody is not the same in different expression systems, and the expression may be too low. Teckbio finds the most suitable conditions for stable expression by trying to express scFv in prokaryotic systems (e.g. E. coli), yeast systems or mammalian cell systems. We also help to improve the stability of the antibody in the organism by adding protective groups, such as PEG (polyethylene glycol) modification, to both ends of the antibody.

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