Synthetic Fab Library Construction Service

Introduction of Synthetic Fab Library Construction ServiceFab

Antibody libraries are usually divided into native, immune, or synthetic libraries. Antibody libraries include phage display antibody library, cell-free molecule display antibody library (ribosome display antibody library, mRNA display antibody library, DNA display antibody library), cell surface display antibody library (bacterial surface display antibody library, fungal surface display antibody library, yeast surface display antibody library, mammalian cell surface display antibody library).

Fab is a complete antibody fragment, consisting of VH and heavy chain constant region CH1, light chain VL and light chain constant region CL, which are connected by interchain disulfide bonds to form heterodimers, with only one complete antigen binding site, its molecular weight is about 1/3 of the complete IgG molecule, which can quickly screen out ideal antibodies with high affinity.

Fig.1 Structure of human IgG and fragments. (Reference source: Synthetic Antibodies in Infectious Disease - PubMed (nih.gov))

Antibody Phage Display

Antibodies are an integral part of the vertebrate immune system and function by specifically binding antigens with high affinity and specificity. With the development of technology, hybridoma technology occurred, and the world's first therapeutic antibody approved by FAD was produced using this technology.

However, monoclonal antibodies produced using hybridoma technology have certain limitations. When used as a therapeutic antibody to treat people, it can cause a strong facial reaction in the body. On this basis, chimeric and humanized antibodies with low immunogenicity were derived for treatment. Recently developed mouse and phage display techniques can produce fully human antibodies whose amino acid sequence is similar to human antibodies and therefore less immunogenic.

Antibody phage display produces target-specific antibodies and can overcome some of the limitations of antibody production methods based on animal immunity. Since 1990, different antibody formats have been used to construct phage libraries displaying antibodies, including heavy domain Human antibody fragments (VHs), heavy domain Moose and shark antibody fragments (VHHs), scFvs, Diabetes antibodies (bivalent scFvs), and full fragment antigenic binding (Fab) antibodies.

Alpha Lifetech Can Provide

Alpha Lifetech has been deeply engaged in antibody phage display technology for many years. It has built a perfect stable Phage display technology platform that saves time for scientific research time or project research and facilitates subsequent production. Alpha Lifetech can also provide customers with antibody phage display (nanobody phage display, scFv phage display, and Fab phage display).

Alpha Lifetech has a comprehensive M13/T7 phage display platform to ensure the construction and production of antibody libraries. We can also provide customized services, such as scFv phage display, Fab antibody production, and Fab antibody purification, to meet your needs.

A Case of Synthetic Fab Antibody Library Construction

Huang G established a new phage display Fab library method, which can be obtained after a single electroporation, and the library diversity is 10^9-10^10.

The experimental process includes 1) preparation of high-efficiency electro-competent cells; 2) Extraction of dU-ssDNA; 3) oligonucleotide-directed mutagenesis based on Kunkel method; 4) Electroporation and library size calculation; 5) Protein A/ L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity assessment; 6) Diversity DNA sequence analysis.

Fig.2 Phage titration to calculate library size. (Reference source: Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity - PubMed (nih.gov))

Kim YJ screened a Fab synthetic phage display library on the RBD of SARS-CoV-2 S protein and obtained a monoclonal antibody that could neutralize SARS-CoV-2, successfully preventing the invasion of the virus, and providing a basis for the antibody to be used as a therapeutic tool.

Fig.3 Panning of the phage-displayed synthetic Fab library on an immobilized SARS-2 receptor-binding domain (RBD). (Reference source: Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library - PubMed (nih.gov))

Synthetic Fab Antibody Library Construction Service

A recombinant phage plasmid was successfully constructed by combining antibody molecule Fab with single chain phage coat protein (PⅢ or PⅧ), then it was transformed into TG1 receptor cells, coated on the medium containing appropriate antibiotics, and then amplified and cultured after screening for proteus. After multiple rounds of culture, the phage replicates several times in the bacteria and successfully expresses the target protein or polypeptide. The process of antigen adsorption-elution-amplification is repeated to screen the desired Fab antibody, and then to Fab antibody purification.

Synthetic Fab Antibody Library Construction Workflow

Steps	Specification
Step1: Immune and ELISA Testing	1) Immune test animals (standard immunization 5 injections); 2)ELISA detection of serum antibody titer 4 and 5 titer detection respectively), if the antibody titer detection of the fourth dose meets the requirements (ELISA serum titer satisfying protein >10^5; Small molecule >10^4), then 7 days before blood collection, immunize again, if not meet the requirements, continue routine immunization (submit the titer test results and immune serum to the customer, according to the titer test results, select positive serum animals for follow-up operations) The titer was qualified and PBMC was isolated by blood sampling
Step2: Fab Monoclonal Antibody Screening Service	1) Using library cDNA as a template, the V gene was amplified by 3-wheel PCR 2) Construction and transformation of phage display vector: V gene splicing phage display vector, electric shock transformation of host bacteria, and construction of antibody library 3) Identification: 24 clones were randomly selected and the positive rate was determined by PCR 4) Default 3 rounds of screening: NGS sequencing of the third round of products; At the same time, 192 clones were selected for induction expression and ELISA detection. 5) All positive clones were selected for gene sequencing (>60 positive clones per screening, up to 192); 6) Delivery: Fab antibody sequence 5-10 (non-repeating sequence); Lab report, raw test data, phage library
Step3: Recombinant Expression of IgG Monoclonal Antibodies	1) According to the customer verification results, recommend the customer to reorganize the expression of 5 sequences; 2) Construct vector + cell expression + purification