Naïve Fab Antibody Library Screening Service

Introduction of Naïve Fab Antibody Library Screening ServiceFab

The key region in the antibody structure that can bind to antigens is the Fab fragment. A portion of the light and heavy chains of the antibody are connected through disulfide bonds to form Fab fragments, where the light chain has a variable region (VL) and a constant region (CL), while the heavy chain has a variable region (VH) and a portion of the constant region (CH1). VL and VH form antigen binding sites (CDRs) through hydrophobic interactions and hydrogen bonding. One characteristic of Fab fragments is their relatively small molecular weight, typically between 47-55 kDa, which gives them good tissue penetration and low immunogenicity. Fab fragments retain their complete antigen binding ability and can bind with specific antigens with high affinity. In addition, it lacks the Fc segment, so the immune response mediated by the Fc segment will not be triggered, and the probability of hypersensitivity reactions is also reduced. The smaller molecular weight allows Fab fragments to more easily penetrate tissue barriers and reach target sites. The half-life of Fab fragments in vivo is relatively short, which helps to reduce potential side effects of drugs. Fab antibody production can be achieved through genetic engineering technology and phage display technology.
Fab antibody-Alpha Lifetech

Alpha Lifetech Can Provide

Alpha Lifetech has many years of experience in phage antibody library construction and screening, and is committed to producing ideal recombinant antibodies for our customers. We have specialized Fab phage screening technology and are happy to provide efficient Fab antibody screening strategies for our customers. We can construct natural and immunized Fab phage antibody libraries for our clients, which are large in capacity and rich in diversity. We clone Fab genes into phage vectors and transform them to obtain Fab phage display libraries. After 2-3 rounds of elution, we can isolate Fab antibodies with strong binding and affinity in about 10 days, which greatly shortens the experimental cycle and facilitates the overall progress of customer projects. We can screen the pre-prepared Fab natural libraries directly without immunization of animals, which is conducive to accelerating the progress of Fab preparation. We can screen a variety of phage libraries based on M13, T4, T7 and λ from human, mouse, rabbit, chicken, sheep and camel species according to the needs of our clients. We generally select Fab antibodies with an affinity of 10^8 after 4 rounds of library screening. Alpha Lifetech has a variety of fab antibody purification instruments and equipment, which can provide antibody purification services from various sources such as rabbit, sheep, chicken, and mouse monoclonal antibodies, as well as Protein A/G affinity purification services and antibody separation and purification services. And we will choose the purification method based on the specific purpose of the customer. Based on the comprehensive platform system construction of antibody platform, protein platform, etc., we cover the upstream and downstream services of fab antibody production, and can provide technical services from antibody preparation, fab antibody purification and antibody separation and purification, antibody sequencing, antibody validation, etc.

Naïve Fab Antibody Library Screening Service Process

The Naïve Fab library is constructed by cloning antibody gene fragments from non immunized B lymphocytes, making it a natural antibody library. The naïve libraries contain a large number of antibody sequences that have not been exposed to antigens or screened by the immune system, thus possessing broad antigen binding potential and diversity. The antibody gene fragments contained in the library are mainly VH-CH and VL-CL fragments, which are linked together by disulfide bonds to form Fab fragments.

Firstly, B lymphocytes are isolated from the peripheral blood of non immunized animals, and the antibody gene sequences contained in these non immunized B lymphocytes are diverse. The gene fragments encoding antibodies VH-CH and VL-CL were amplified from B lymphocytes by PCR, and these gene fragments contain the variable regions of the antibodies. The amplified antibody gene fragment is fused with the phage capsid protein gene, expressed, and displayed on the surface of the phage. Each bacteriophage can carry a specific antibody sequence and can be extensively amplified and screened in vitro, resulting in the construction of a na ï ve library containing a large number of different antibody sequences.

There are multiple methods for naïve libraries Screening, such as yeast library screening and phage display screening. Among them, yeast library screening is mainly used to study the interactions between proteins. Through the construction of yeast hybrid libraries, yeast two hybrid systems are used to screen proteins that interact with specific proteins. Known target protein genes are fused with reporter genes within yeast cells to construct a yeast hybrid library and yeast library screening.

Yeast cells in the library were co-cultured with cells expressing the target protein, and proteins that interact with the target protein were screened by reporting gene expression levels. The positive clones selected were validated by methods such as immunoprecipitation and Western Blot to confirm protein-protein interactions. Phage display screening uses bacteriophages as carriers to express exogenous genes on the surface of bacteriophages and screen them through antigen antibody interactions. The antibody gene was cloned into a phage vector to construct a phage library. The antigen is encapsulated on a solid-phase carrier and incubated with a phage library. After multiple rounds of washing and amplification, phages that specifically bind to the antigen are selected.

Naïve Fab Antibody Library Screening Service Workflow

Steps	Specification	Timeline
Step1: Revival of the Fab antibody library	The Fab primary antibody library was resuscitated with 2×YT-AG medium	1 day
Step2: Assisted phage infestation	Infestation of TG1 Escherichia coli using M13KO7 helper phage	1 day
Step3: Preparation of phage supernatants	Collection of phage supernatant	1 day
Step4: Phage precipitation and titration	Phage precipitation using PEG/NaCl	1 day
Step5: Fab screening and titration	Conduct 3-5 rounds of screening to calculate library capacity	7 days
Step5: PCR identification of positive clones and sequencing	PCR identification of positive clones and sequencing	7 days