The production and maintenance of a hybridoma cell begins with the fusion of a specific antibody producing B cell, to a cancer B cell called a myeloma, which does not produce an antibody by itself. Fusion results in an immortalized line called a hybridoma that will faithfully produce a specific antibody against a single epitope called a monoclonal antibody. Once produced, proper maintenance and culturing is required to maximize the performance and continued production of the antibody in question. Have you ever wondered how this is done? Are you fusing cells to produce your own monoclonal antibodies or wondering about your culturing options. This is your opportunity to ask questions about monoclonal antibody production and the culturing of mouse hybridoma cells.
I am a manager of a monoclonal Mab core lab for the university of Colorado, we have a history of successful hybridoma production but recently we have had problems with getting hybridomas from a mouse that shows a strong signal. On the first screening we will get strong positives that would traditional result in good hybridomas, but recently I have seen the positives stop producing the Mab after the initial screen. Few make it to cloning but don’t produce Mab after that. Do you have any idea why this is happening? We’re using mice that have had at least 3-4 injections with the protein of interest and are screened by Elisa titration assay and by a western. A traditional fusion is done with PEG to SP2/0 cells and plated into 96well plates. They are normally screened in 7-10 days. Media is IMDM+15% FBS +HAT
Thanks for your question and providing all the details. I think you are writing that this is happening with one animal and not all the animals you are recently harvesting cells from. Assuming this is the case, I think you got a bad mouse . You also mentioned that spleen cells from the animal initially … Continued
I am looking into protocols for making hybridomas and notice that some people grow their myeloma cells in 8-azaguanine and other don’t. What is the purpose?
Some people grow their myeloma cells (Sp2/0, Sp2/0-ag14, NS-0 and NS-1 cells for example) in 8-azaguanine or 6-thioguanineThe reason for this is to ensure that the cell line is 8-azaguanine or 6-thioguanine and HGPRT negative. Azaguinine and 6-thioguanine are competitors of guanine. This phenotype is necessary for the myeloma cells to be sensitive to the … Continued
or cloning hybridomas via the limiting dilution method (1-2 cells per well), what are your thoughts on the following: (1) serum conc=20% (hybridoma qualified FBS) (2) PEC feeder cells-approx. 2000-5000 per well (3) conditioned medium I make conditioned medium from the myeloma fusion partner (P3X or SP2/0). Is there a better cond. medium to use? What experience, if any, do you have with rat-mouse hybridomas? Anything special I need to know about cloning, growing, maintaining these hybrid cells?
This is a good set of questions that are all related around how to isolate a single cell into the well of a 96 well dish. The goal of course is to get that single cell to grow into many. The problem is that initial single cell in that big well gets lonely and doesn’t … Continued
I am looking for the most inexpensive way to culture hybridoma cells in serum free conditions. I am starting with classic media, is there anything that is serum free that I can add to classic media to allow for serum free culture or do I need to use a pre-made serum free medium for hybridoma cells.
Moving cells from serum-containing to serum-free media is not so easy since adaptation is required. To my knowledge there are no additives such as a non-animal derived substitute that can be added to a basel (classic) medium that will make it perform like a serum containing medium. For your information there are media such as … Continued
Do i need to inject a 100% pure antigen in order to get a desired results or can I use a 80% purity antigen for injection, as the antigen I am dealing with will only grow along with the bacteria. I’m also using non spf female balb/c mices for the injection. I’m wondering if all these factors may affect to get the desired results.
About your antigen, if you were making polyclonal antibodies the purity would be a problem. Technically, by making monoclonal antibodies you can still get the one you want. But I do envision some problems. Obviously your search for the MAb you want will be made more difficult due to the 20% non-antigen directed Abs. The … Continued
Hi, I would like to know about the mouse myeloma cell line P3X63Ag8.653 azaguanine resistancy period upon subculture. My cells have undergone 9 passages in total and then I decided to cryopreserve it. Upon revival last month they show 70% viability. My question is,even though the cells has been sensitized with 8 azaguanineprior to buying , do i still need to sensitized it because the cells has undergone few passages? I’m also wondering if myeloma cells can undergo infinite passages and will the cells loose the sensitization to azaguanine along the way? What would be the maximum passage for the myeloma cells because i will need to revive the cells again for fusion and it will undergo few passages again.
P3X63Ag8.653 is a subclone P3X63.Ag8 which was originally selected based with 8-aza-guanine sensitivity. Periodic growth in 8-asa-guanine ensures cells are HGPRT negative and thymidine kinase negative. Growth on 6-Thio-Guanine is useful for periodic selection for HAT sensitivity. Also remember that cells for fusion should not be passaged for a long times so keep frozen stocks … Continued