KMD Bioscience is one of the well-recognized experts who is professional in phage display technologies. With many years of research and development, we have established a solid platform for Fab and scFv libraries construction. Our scientists can offer high-quality phage display library construction and custom phage display library screening services to meet our clients’ demands precisely.
Human Fab Antibody Libraries
Obtaining sequence information of hybridoma monoclonal antibodies is the basis of antibody recombination and antibody drugs development. The Fab fragment is the major region of antibody recombination. This is because the Fab fragment is the antigen-binding region, which determines the affinity and specificity of an antibody. It is composed of one variable region (V) and one constant region (C1) from each heavy and light chain of the antibody .
The Fab antibody library refers to 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. In addition, we can offer a more advanced solution: after sensitizing human peripheral blood monocytes in vitro, high affinity monocytes were isolated by flow cytometry, and then monoclonalizayion was conducted. Using this method, highly consistent monocyte clones can be obtained, then constructed libraries.
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.
Service Procedure
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Steps
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Specification
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Timeline
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Deliverables
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Monoclonalization of peripheral blood mononuclear cells
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1. Blood mononuclear cells acquisition.
2. Monocyte sensitization.
3. Monoclonalization.
4. Monoclonal culture after flow cytometry.
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8-12 weeks
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* Experimental report: including detailed construction procedures and representative sequence information.
* Product delivery: including bacterial and phage antibody libraries.
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cDNA synthesis (Clients can provide cells)
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1. Total RNA extraction.
2. RT-PCR.
3. PCR amplification with Actin specific primers to identify the quality of cDNA.
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Library construction
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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.
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Note: 1. The client provides human peripheral blood lymphocytes, lymph node cells or spleen cells (preservation with RNA later or other solutions to avoid RNA degradation, and transported in dry ice), it takes only 8-9 weeks to complete the production of Fab antibody libraries from the second step.
2. Cell lysates were prepared using TriZol solution and transported in dry ice.
Service Highlights
*Extensive experience: Extensive experiences and strong track record in phage display platform.
*Large size of libraries: 107 - 1012 independent clones.
*High affinity: 10-7 - 10-13M.
*Various types of phage display systems: M13, T4, T7.
*Strict QC control.
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