Monoclonal Antibody Development Service

Alpha Lifetech Inc. can provide epitope prediction, peptide synthesis, protein expression, polyclonal, or monoclonal antibody production as well as antibody purification from cell culture, ascites fluid or whole serum.

Alpha Lifetech Inc. is experienced in providing customers with various stable cell line services including hybridoma cells, genome editing cell lines, knockdown cell lines, and overexpression cell lines. We try to offer advanced custom cell line services to every client and our services are proven and trusted.

Antibody Development Strategy

1.Antigen design and preparation
These antigens can be proteins, peptides, or other molecules. Designing immunogens requires a comprehensive approach that integrates knowledge of antigen structure, immunology, and delivery systems. Alpha Lifetech has antigen design technology that can design immunogens for customers in need.
Alpha Lifetech provides recombinant protein products for customers to choose from. We have a variety of protein expression systems, such as E. coli expression system, yeast expression system, baculovirus-insect expression system, mammalian expression system, etc., to produce the recombinant proteins you need for research.

2.Animal immunization
Alpha Lifetech can provide immunogens to customers, and immune animals such as mice, rabbits, sheep, alpacas, guinea pigs, hamsters, etc. are available for you to choose from.

3.Monoclonal antibody production methods

● Mabs Production by Hybridoma technology
Hybridoma is a hybrid cell produced by the fusion of two kinds of cells, namely mouse myeloma cell line and plasma cells (lymphocyte B), in which the heterozygous cell can produce continuous antibodies against the antigen of interest in vitro. The production of a hybridoma cell line consists of three parts: antigen design (haptens, small molecules, and peptides), animal immunization, cell fusion, and positive clone screening. Utilizing unique immunization methodologies, we obtain exceptionally high cell-fusion efficiencies (1-10 hybridomas per thousand B cells) and high antibody titers, maximizing our success rate in subsequent analytical tests that include biochemical screening, in-vitro cell-based assays, and animal studies. 

● Mabs Production by phage display technology
Phage display offers another method for generating monoclonal antibodies. B-lymphocytes are isolated from animal blood and their mRNA is extracted. Through PCR, this mRNA is converted into cDNA, which amplifies all the VH and VL segments. These segments are then cloned into a vector, typically as single-chain variable fragments (scFv), alongside the PIII protein of a bacteriophage. Subsequently, this construct is used to infect E. coli, resulting in the creation of a library containing approximately 10^10 cells through inoculation with a helper phage. E. coli is then able to secrete the bacteriophage harboring the VH and VL segments as part of its coat. Following this, specific VH and VL segments targeting the antigen of interest can be selected and used to reinoculate E. coli with the bacteriophage. Cells containing the plasmid are then isolated and sequenced.

4.Fusion screening

● Cell Fusion And Selection
The harvested B cells are then fused with myeloma cells, which are cancerous cells that can proliferate indefinitely in culture. The fusion is typically achieved using a technique such as polyethylene glycol (PEG) fusion.
After fusion, the hybridoma cells are cultured in a selective medium that allows only the hybridomas to survive. The medium usually contains aminopterin, which prevents the growth of unfused myeloma cells.

● Screening and Clonging
The resulting hybridoma cells are screened for the production of antibodies specific to the target antigen. This is typically done using techniques such as enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and flow cytometry.

Once hybridoma cells producing the antibody are identified, they are cloned to generate a population of identical cells. This ensures that the antibody produced by each hybridoma cell is identical.

5.Antibody Functional Verification
Alpha Lifetech provides antibody functional validation, such as Western blotting, immunohistochemistry, or functional assays, to characterize monoclonal antibodies and confirm antibody specificity, affinity, and functionality.

6.Antibody optimization
Purification of antibodies from culture supernatant using techniques such as protein A or protein G affinity chromatography, and Alpha Lifetech can also provide antibody modification methods to improve antibody affinity.

7.Antibody production
Alpha Lifetech can evaluate candidate antibodies for high-throughput screening applications and large-scale production.

FAQS

1. How to choose the right fusion hybridoma?
During the fusion process, 10-15 96-well microtiter plates are seeded with the fusion hybrid mixture. Each plate is fed HAT-IMDM selection media and maintained in a carbon dioxide incubator at 37 degrees Celsius. 9 - 14 days after the fusion, hybrid supernatants are tested for the presence of the specific antibody of interest.
Fusion of plasma cells with their myeloma counterparts is not 100% effective. Even under optimal conditions and the most effective stimulation, cell fusion still produces a mixture of unconfluent and confluent cells that need to be separated. To improve the efficiency of the selection process, myeloma cells used for fusion lack HGPRT, a key enzyme in the nucleotide salvage pathway. The mixture was then cultured in an HAT medium, where only cells with the HGPRT enzyme, hybridomas that inherited HGPRT from plasma cells, were viable.

2. How to screen hybridoma cell lines for antibody activity?
Evaluation of hybrids is a critical step. Generally, many companies provide hybridoma, clone, and subclone supernate screening by enzyme-linked immunosorbent assay (ELISA), Western blot assay, and fluorescence-activated cell sorting (FACS). To reduce costs, scientists may choose to perform all screening of supernates in their own lab.
Hybridoma supernates to be tested may range from 500 to 1,440 samples and will be ready to test by days 9- 14. They could arise over a three- to five-day period or may all be ready on the same day, so it's important for the client lab to be prepared weeks in advance with working specific secondary screening assays of choice.

3. Why do positive hybridomas need to be subcloned?
After positive Wells were identified during the initial ELISA screening procedure, hybridomas were transferred to a larger volume, i.e., 24-well plates, in preparation for the subcloning procedure. Subcloning of hybridomas is usually performed by a limited dilution method to ensure the isolation of stable monoclones. The method involves diluting hybridoma cultures and dispersing them into 96-well plates to achieve monoclonality (one cell per well). To reduce the risk of developing mixed hybridoma populations, at least two limited dilutions should be performed.

4. What are the benefits and limitations of using hybridoma technology for antibody discovery?
Benefits: Hybrid cell lines have been praised for their ability to produce antibodies with high affinity, stability, and specificity in the most cost-effective manner.
Limitations: In contrast to in vitro antibody generation techniques, the development of hybridomas and their corresponding myeloma fusion partners requires significant time.