With years of research and development, KMD Bioscience has become a world-leading service provider and research institution in phage display techniques. We can offer human antibody libraries that can derive highly specific, high affinity antibodies, such as human scFv libraries, human Fab libraries.
Human scFv Antibody Libraries
Phage display antibody libraries are widely used in screening high affinity antibodies, such as screening of angiogenesis markers fibronectin, tumor-specific B3/B4 antigen and EGFR. Antibody libraries constructed by phage display techniques are usually derived from RT-PCR amplification of peripheral blood mononuclear cells, spleen cells, bone marrow cells and tonsil cells mRNAs to form cDNA fragments. These gene fragments were spliced into phagemid vectors, and then transferred into phages to form covalent structural protein and display in vitro. The heavy and light chains of the antibody are expressed independently and then randomly combined to form a large antibody library with phage clone numbers of 1011-1012. The scFv antibody library refers to the variable region (VH and VL), especially the CDR region genes of the antibody, forms a large number of different clones by different strategies, such as amino acid mutations, frameshift mutations. And high affinity single chain Fv antibodies against the target antigen were screened from these clones. Since the scFv antibody has a smaller molecular weight (25 kDa), the scFV antibody is more stable and does not easily form a multimer compared with the Fab antibody library.
Types of Phage Display System
KMD Bioscience has extensive experience in phage display platform by employing various types of phage in various project objectives. In general, M13 phage system is the most popular vehicle choice for phage display and extensively used in many types of research. We also provide phage display related services via either T4 or T7 phage systems, which can provide different promising alternatives by their special merits. Meanwhile, clients can also provide antigens, then higher affinity and specificity of monoclonal antibodies can be obtained by sensitizing peripheral blood lymphocytes in vitro.
Service Procedure:
|
Steps
|
Specification
|
Timeline
|
Deliverables
|
|
cDNA synthesis
|
1. Total RNA extraction.
2. RT-PCR.
3. PCR amplification with Actin specific primers to identify the quality of cDNA.
|
8-9 weeks
|
* Experimental report: including detailed construction procedures and representative sequence information.
* Product delivery: including bacterial and phage antibody libraries.
|
|
Library construction
|
1. Primer design & synthesis
2. PCR amplification of variable region genes of heavy and light chains using cDNA as a template.
3. scFv gene splicing.
4. Plasmid construction & transformation: after enzyme digestion, scFv and phagemid vector were ligated and transformed into TG1 host bacteria by electric shock to construct antibody libraries.
5. Identification: 20-50 clones were randomly selected, PCR identification, sequencing and analysis of antibody sequences.
6. Packaging: >1*1013 phage particles /ml.
|
Service Highlights
*Extensive experience: We have many years’ experience and a strong track record in phage display platform.
*Large size of libraries: 107 - 1012 independent clones.
*High affinity: 10-7 - 10-13M.
*Multiple phage display systems: M13, T4, T7.
*Strict QC control.
中文
