We offer a three-phase reliable and affordable rat and mouse hybridoma development services for producing custom monoclonal antibodies.
ProMab has successfully generated approximately 3000 custom monoclonal antibodies over the past 11 years. Therefore, our experience allows us to provide our customers with realistic estimates of the chances of success for a particular project. In vivo systems are, by their very nature, somewhat unpredictable, and while the vast majority of our custom antibody projects are successful, it is of paramount importance to ProMab that our experience be used to provide our customers with accurate, and not just “Best Case” scenarios.
The aim of a ProMab Biotechnologies custom monoclonal antibody project is not only to generate an antibody that recognizes the immunizing antigen, but to raise an antibody that works for your specific application. ProMab’s highly technical staff, with decades of experience spanning immunology, microbiology, biochemistry, cell and molecular biology will be there to guide you through the process of designing an appropriate antigen, and the subsequent strategy required for the generation of a successful antibody.
One of the most important aspects of generating an antibody will be in the screening of polyclonal sera during the immunization stage, and supernatant from subsequent hybridoma clones. While ELISA and Western Blotting (if recombinant protein antigen is used) are included in the project, it may be required to examine other, additional screening methods. As with any biological validation process we will assist you in determining the appropriate controls to use, for example, whole cell lysate for Western Blotting, or tissue sections for immunohistochemistry.
The first step will be to work closely with you to determine the exact type of antigen, peptide or recombinant protein that will be most appropriate for your specific project. Not only can we provide technical assistance with the stage, but can also generate the antigen for you, whether that will be a peptide, or recombinant protein antigen. Once this has been determined, the process of generating a custom monoclonal antibody will be divided into three phases. Phase I will include the immunization of Balb/C mice with antigen, and subsequent screening of polyclonal sera ELISA. The sera exhibiting the highest titer by ELISA, and positive in other appropriate screens, will be used to determine which mouse to move forward to fusion.
While many companies will suggest rapid production of monoclonal antibodies, this will largely depend on the response raised by immunized mice to the respective antigen. In general the response against a recombinant protein antigen will be stronger, at an earlier date, than that seen against a peptide antigen, and while it is certainly possible to obtain a robust immune response, one that will allow us to move forward with fusion in Phase II, it may well require a 4th or 5th injection of antigen (and on rare occasions a 6th or 7th boost). These details are shown in the schematic below outlining our standard immunization procedure.
Fusion of splenocytes to mouse myeloma cells and subsequent screening, and isolation of antigen-positive hybridoma cells will constitute Phase II of the project. The procedure involves fusion of the two cell types and sequential screening of pools of hybridoma clones, from an initial count of approximately 3000 fusion clones, down to a smaller ELISA antigen-positive population. Conditioned media, “supernatant”, will then be removed from ten of the final 2-3 ELISA-positive clone population pools and sent on to you at your facility, for evaluation by your specific in-house assay system(s).
As soon as you have determined which two of the 10 clones are producing antibodies that best fit your needs, we will enter phase III, the final phase of the development procedure. At this point we will isolate the clones of interest by limited dilution, expand their number in culture, and isotype them. At this point cells will be ready for cryopreservation. In addition to the frozen vials of cells we will send to you (if shipped within the US, or depending on import restrictions, live hybridoma cultures in T25 flasks may be sent if so requested), we will normally retain 2 vials of each hybridoma for a 6 month period. This period can of course be extended, as can the number of vials being stored for a small additional fee.
To order, please fill out a Custom Monoclonal Antibody order form and e-mail at firstname.lastname@example.org. For further questions, please refer to our support pages. ProMab also offers a variety of other custom antibody services including human custom antibodies, hydridoma sequencing, and custom polyclonal antibodies. Additional services, and related prices, connected to the generation of a custom monoclonal antibody are listed below.
Additional Monoclonal Antibody Services:
|Peptide||Peptide synthesis and conjugation||$500|
|Peptide synthesis without conjugation||$350|
|Screening||Additional clones (>10) in Phase II||$50 per clone|
|Extra screening with IHC||$300|
|Additional clones (>2) in Phase III||$300 per clone|
|Cell-line adaptation to serum free medium||$500~1,000|
|Ascites||Ascites production (<11 mice )||$30/mouse+$200 setup fee|
|Ascites production (11-50 mice)||$26/mouse+$200 setup fee|
|Ascites production (51-99 mice )||$24/mouse+$200 setup fee|
|Ascites production (>99 mice )||$20/mouse+$200 setup fee|
|Antibody culture in bioreactor, per liter||$1,100|
|Antibody culture in flask, per liter||$800|
|Antibody purification by protein G from one liter of medium||$300|
|Antibody purification by protein G from ascites (20mg)||$300|
|Labeling||Antibody conjugation with FITC, per mg||$600|
|Antibody conjugation with HRP, per mg||$600|
|Antibody biotinylation, per mg||$600|
|Other||Cell banking and storage of 10 ampules after first two months||$30/month|
|Antibody epitope mapping by peptide library||$1,200|