Human Single B Sorting Service
Based on the single b cell screening platform, Alpha Lifetech has advantages in screening time and obtaining high-quality antibodies.
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Human Single B Sorting Service
Based on the single b cell screening platform, Alpha Lifetech has advantages in screening time and obtaining high-quality antibodies. We can provide one-stop technical services from antigen design, synthesis and modification to animal immunity, single-cell enrichment screening, high throughput screening, expression and purification, functional verification, etc., to meet the different needs of customers. Alpha Lifetech has been dedicated to antibody preparation research for many years. We have an experienced antibody preparation team, accumulated mature technical experience, and complete antibody production equipment, which can provide customers with high-quality recombinant monoclonal antibody preparation services, including rabbit source, mouse source, camel source, sheep source, chicken source, etc.
Single B cells are single B lymphocytes, which are white blood cells that play a role in the immune system. Single B cells can differentiate into plasma cells under antigen stimulation and produce antibodies to combat pathogens. Each B cell contains only one functional heavy chain variable region DNA sequence and one light chain variable region DNA sequence, and can only produce one specific antibody. In 1996, Babcook et al. first reported a new method for antibody preparation, which utilized complementary DNA of immunoglobulin variable regions for molecular cloning and antibody expression. First, a specific hemolytic plaque assay is used to screen individual B cells that produce specific antibodies, and variable region cDNA is extracted from them. Then, the heavy chain variable region and light chain variable region genes were amplified and recovered using PCR, and connected to the human Ig constant region gene for expression, ultimately obtaining specific antibodies.
The principle of single b cell screening preparation is mainly based on the characteristic that each B cell contains only one functional heavy chain variable region and one light chain variable region, and each B cell produces only one specific antibody. After being stimulated by an antigen, each B cell will specifically produce antibodies against that antigen. By using single-cell PCR technology, variable region genes of heavy and light chains can be amplified from single antibody secreting B cells. The amplified heavy and light chain genes are linked to appropriate expression vectors and expressed in mammalian cells. After high throughput screening, monoclonal antibodies with biological activity can be obtained.
introduction to Single B cell Technology
Single B cell technology is currently one of the most widely used methods for preparing monoclonal antibodies. Compared with other techniques for preparing monoclonal antibodies, it has advantages such as high efficiency and rich genetic diversity.
Table 1. Comparison of Advantages and Disadvantages of Antibody Development Technologies
| Monoclonal Antibody Production Technology | Advantage | Disadvantage |
|---|---|---|
| Single B Cell Antibody Production technology | The preparation of whole human antibodies has a short cycle, high efficiency, and mature technology | The quality requirements for adversarial agents are high, and the cost is high |
| Hybridoma Technology | Mature technology and high purity of monoclonal antibodies prepared | The operation steps are cumbersome, the cycle is long, and the efficiency is low. Antibodies cannot be directly applied in human clinical research |
| EBV Immortalization Technology | Preparation of human antibodies | EBV operation needs to be carried out in a biosafety level three laboratory |
| Phage Display Technology | Large storage capacity, short screening cycle, and high mass production | Non naturally occurring antibodies are easily cleared in the body and difficult to apply for treatment |
human single B cell Screening Service Process
The samples for human single-cell sorting are usually samples containing B cells such as peripheral blood, spleen, or lymph nodes. If the sample contains a large number of red blood cells, red blood cell lysis treatment should be performed first to avoid affecting the subsequent steps. After appropriate digestion of the tissue sample, a single-cell suspension can be obtained. Suitable antigens are selected to immunize animals, and peripheral blood isolation is performed on animals that have already been immunized. If the animal's body size is small, spleen isolation can be chosen. Enrich B cells in peripheral blood or spleen to improve sorting efficiency. By using facs cell sorting to selectively sort enriched B cells, based on the expression patterns of cell surface markers, facs cell sorting can efficiently identify B cells. The sorted individual B cells are lysed to release their nucleic acids. The mRNA inside the cell is reverse transcribed, and primers are designed based on the genes of the variable heavy chain and variable light chain of the target antibody. The variable region sequence is then amplified by PCR. The variable region sequence was inserted into a vector plasmid containing a constant region gene, and a plasmid expressing monoclonal antibodies was obtained. Select the appropriate expression system according to the requirements to obtain antibodies, and perform antibody validation on the obtained antibodies. Throughout the entire sorting process, the activity of cells needs to be maintained, avoiding the use of reagents and conditions that are harmful to cells, and ensuring that the entire sorting process is carried out under sterile conditions to avoid cell contamination.
Fig 1: Steps of single B cell antibody validation technology
single B cell Sorting Service Workflow
| Steps | Service Content | Timeline |
|---|---|---|
| Step 1: Single B Cell Sorting |
(1) The samples are usually peripheral blood, spleen, or lymph nodes. If the sample contains a large number of red blood cells, red blood cell lysis should be performed first to avoid affecting subsequent steps. 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 2: Amplification and Cloning | PCR amplification, vector construction |
1 day |
| Step 3: Antibody expression and validation | Antibody expression test, ELISA detection, facs cell detection |
1 day |
| Step 4: Positive Clone Sequencing | Screening and sequencing of positive clones
Recombinant antibody expression and purification (optional)
|
1 week |
| Step 5: Recombinant Plasmid Transfected Cells (HEK293, CHO) | Transient expression, SDS-PAGE and WB detection, purification of recombinant antibody |
1 week |
| Step 6: 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 |
single B Cell Sorting Service Advantage
Mammalian Cell Expression Platform
independently designed high expression vectors, optimized proprietary culture media, and proprietary transfection reagents.
Professional Technical Team
We have a professional technical team that can customize personalized one-on-one antibody discovery solutions.
Rich experience
Rich experience accumulation and perfect operational SOP.
Related Service
Antibody stable expression cell line construction service can be provided.
High Serum Titer
For protein antigens or viral antigens, the serum titer of immune ELISA is>10^5, and for peptide antigens (carrier protein conjugation), the serum titer of immune ELISA is>10^4.
Mature Immune Program
The immunization frequency should not be less than 5 times (unless specifically requested by the customer, a 5 time immunization program should be set).
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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