Rabbit Single B Cell Screening Service
Based on the Single B cell sorting service, Alpha Lifetech has advantages in high specificity and high affinity antibody development.
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Rabbit Single B cell screening service
At the heart of Alpha Lifetch's service lies the meticulous immunization protocol designed to elicit a robust and diverse immune response in rabbits. Following immunization, we harness NGS to profile the antibody repertoire at the single-B-cell level, enabling the precise identification of antigen-specific clones. These clones are then isolated, cloned, and expressed to produce recombinant monoclonal antibodies with precise characteristics tailored to specific research needs.
Single B Cell Screening Background
Monoclonal antibodies, which is the core of the medicine and biology tools, has profound changes the disease diagnosis, treatment and targeted drug delivery system. However, the current mainstream technology such as hybridoma technology, although the efficiency of cell fusion is high, but at the expense of antibody diversity. Phage display technology is facing heavy chain and light chain of natural pairing challenge. These technical bottlenecks have led to an inefficient and lengthy antibody development process.Diverse and, therefore, to explore more efficient and accurate monoclonal antibody development strategy is particularly important.
In the past few decades, the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
Human monoclonal antibody: The variable and constant regions of the antibody are human, removing immunogenicity and toxic side effects. The relevant technologies for the preparation of whole human antibody mainly include human hybridoma technology, EBV transforming B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology and single B cell antibody preparation technology.
A single B cell antibody preparation technology compared with hybridoma technology and phage display technology, its significant advantage is the ability to directly capture and cloned from a single B cell innate immune response, so as to maintain the original pairing and the height of the diversity of antibody gene. This method not only reduce the inefficiency of traditional hybridoma technology of cell fusion and complexity, also avoid the phage display technology that may be encountered in heavy chain unnatural light chain matching problem. In addition, a single B cell antibody preparation technology can quickly and effectively screen out from a small amount of samples with high specificity and affinity antibodies, and is suitable for the development and production of antibody drugs, for immunology research and clinical application provides a strong support.
Rabbit single B cell Screening Service Process
The rabbit single B cell antibody production technology streamlines the process from animal immunization to antibody validation, incorporating advanced techniques like NGS sequencing and single B cell screening. By immunizing rabbits with the target antigen, specific B cells producing high-affinity antibodies are identified and isolated. These cells undergo NGS sequencing to decipher their antibody gene sequences, which are then cloned and expressed for antibody production. The resulting antibodies undergo rigorous antibody validation to ensure their specificity, affinity, and functionality, making them ideal for various research and clinical applications.The detailed process is shown in Figure 1.
Fig 1: Flow chart of rabbit monoclonal antibody service
single B cell Sorting Service Workflow
| Steps | Service Content | Timeline |
|---|---|---|
| Step 1: Animal immunization and ELISA detection |
(1)Animals were immunized 4-5 times, starting from the 4th dose, serum was collected for ELISA detection of antibody titer (protein antigen ELISA serum titer >10^5; Peptide antigen ELISA serum titer >10^4).
At the same time, 0.1ml of pre-immunization and post-immunization serum are sent to customers for testing; If the titer is not qualified, the immunization will continue or the client requests the termination of the project, and only the immunization part will be charged;
(2)If the titer was qualified, PBMC cells were isolated from whole blood.
|
10-12 weeks |
| Step 2: Single B cell sorting |
(1)Spleen was collected, single B cell suspension was prepared, peripheral blood was collected, and PBMC cells were isolated.
(2) Immunoantigen markers FITC and AF648.
(3) Double fluorescence flow sorting+ single b cell clone culture.
(4)ELISA identification + sequencing of positive clones.
|
2-3 weeks |
| Step 3: Antibody expression and validation |
(1) 6-10 sequence gene synthesis + mammalian expression test validation.
(2) ELISA identification.
|
3-4 weeks |
| Step 4: Delivery |
(1) Antibody sequences: 4-6 antibody sequences (unexpressed sequences are delivered together).
(2) Each expressed sequence delivered 0.2mg of antibody.
(3) Experimental report.
|
1 week |
Rabbit Monoclonal Antibody Advantage
High Specificity
The immune system of rabbits is more complex and robust than that of mice and other rodents, so rabbit mabs are able to produce antibodies with greater specificity.
High Affinity
B cells of the rabbit mature process to produce antibody affinity is 10-100 times of mice. High affinity for rabbit when single B cell antibodies in combination with the target antigen more closely and firmly.
High Stability
Compared with conventional mouse-produced mabs, rabbit IgG is more structurally stable and does not have other subclasses.
High Diversity
Rabbit has a rich B cell reaction library, and the antibodies produced by rabbit have significant advantages in epitope mutation, mutation, conformational change, and high diversity.
High Flux, High Efficiency
Single B cell technology, as a kind of technology for rapid production of monoclonal antibodies, has the advantages of high throughput and high efficiency.
Broad Prospect of Application
Rabbit single B cell antibody in the biological analysis, drug research and development, more and more extensive application prospect in the fields such as clinical diagnosis.
If you have any questions, please feel free to contact us at any time.
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2018-07-16
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