Hi everyone,
I am growing the hybridoma cells to get the antibody. My problem is when I tested the antibody by running the SDS gel. I have no expected band at all. I have checked both original supernatant and after purified by protein G column (millipore). Therefore, I suspect that the cell line didn't secret the antibody from the beginning.
I am using the 11B11 clone which produce anti mouse IL4 monoclonal antibody. This cell line was purchased from ATCC. I suppose it should be a genetically stable cell line? Cells grow very well in RPMI supplemented with 10% FBS, L-glutamin, pen/strep . I split the cell line roughly every 4-5 days and collect the supernatant for antibody purification. I get about 1mg for 100ml of hybridoma supernatant which might be just contaminated from FBS.
I wonder if anyone has suggestions on how could I improve the production?
Thanks,
Yiju
Hi, in my opinion you should subclone your cells as so as possible because the hybridomas are very unstable. I do this to recover the Mab producing cells: add 100 ul of cells ( from a 25cm2 flask) to 10 to 15 ml of medium (5% FBS) and add 100 to 150 ul medim to a 96 well plate, let the cells multiply to confluence them do an DAS-ELISA for IgG producing cells( mouse IL4 is an expensive antigen) , expand those cells ( about 10 positive clones) in a 24 plate and the finally do an INDIRECT-ELISA to your antigen mouse IL4. Expand only cells from wells that give a higher values in ELISA ( ABOUT 3 TO 5 wells , from 24 well plate). then select one to expand to 500 to 1 liter. Purified your Mab from this volume. Use PROTEIN-A-SEPHAROSE TO remove the bovine from your Rat Mab them PROTEIN-G-Sepharose to purified your Mab. http://www.kpl.com/docs/techdocs/PURIFIGG.PDF
I looked up the specs on this hybridoma at ATCC and I think there's two major possibilities. 1: you're right, and the hybridoma isn't producing antibody (at least not a lot), or 2: it's producing it but you're not seeing it.
1. If you're right, and the hybridoma isn't producing, then I don't know how to help you. You might start with a call to ATCC and ask them how to confirm that you've got the right clone, and what you should be looking for.
2. If the hybridoma is secreting antibody, but you're not seeing it, there could be a couple reasons for this, so here are some questions for you:
a. What are you using to determine your 'expected band'? Does it not even show up in a coomassie stain? If it does show up in the gel, maybe it's just not transferring properly? Or maybe it even makes it to the blot, but you're not seeing it once it's there? Any anti-rat polyclonal secondary on a Western blot should show you where your rat primary ended up. If it's truly not on the blot, but you do see a band in a lane for a positive control (a different rat antibody at high concentration, say a cheap unconjugated rat-anti-mouse secondary).
b. is protein G really the best thing for pulling down Rat IgG 1 isoforms? Protein G binds bovine igGs stronger than it binds rat (see http://www.piercenet.com/files/TR0034-Ab-binding-proteins.pdf). You could be choking the column with residual BOVINE igg from your serum, and not having any beads left over to grab your antibody of interest. This is why lots of folks like to grow hybridomas serum-free.
Let me know if these suggestions help.
Hi Yiju
Do not grow the hybridoma to extensively. Prepare a big production and keep going back to the original cells. Hybridomas are not really stable as they contain different numbers of chromosomes due to the fusion process they tend to loose them as well. In case you find a good old batch do a small expansion and freeze back. Some hybridomas need to be subcloned from time to time (google subcloning for help). Considering the FBS contamination, you can either use low IgG FBS or serum-/protein-free medium for production (after an adaptation).
Best, Marco
Ises Abrahamsohn, Univ. Sao Paulo Brazil
Many hybridomas produce relative low Ab concentrations that mixed with the protein of FCS are not enough t oshow a band in SDS gel PAGE.
A clear band in a gel you will only obtain after concentrating your sups 10 X or more.
You should test your supernatants by ELISA. If you have the Ag, coat the 96 w test plates with it , then add your ? supernatant , then an Ab directed to the Ig of the species your myeloma is produced (anti-rat Ig if the hybridoma is secreting rat Ab or anti-mouse-Ig in case of a mouse hybridoma). This second Ab is labelled and then the other indicated developing reagents.
By ELISA you can test the sups of cells grown in 24 wells plates. Seed the plates with live cels at 5 X 10 4 cels per mL in complete medium with . keep looking under the scope t ocheck that they are multiplying. When media start to yellow wait 24 hs and havest the sup; Centrifuge harvest the sup (sterile) and test immediately or within 24h . Do not freeze-thaw at this point because you may loose part of the Ab bound to the plastic Eppendorf tube. The test will give you qualitative information.A common cause for a hybridoma t ostop or reduce Ab output is infection by Mycoplasma. The cells wit h low infection still look "normal" but fail to produce the Ab. You should test the culture s and your FCS for Mycoplasma sinc e the cells may have come already infected to your lab when for manother lab. FCS of poor quality is often contaminated check with your supplier. Finally if you checked these aspects it can happen tha twith time cels lose the insertion and so it is necessary to reclone them looking for the high producing wells. If it is an easily obtainable hybridoma it is not worth , better buy or get another sample from a colleague or related lab.
Hi, in my opinion you should subclone your cells as so as possible because the hybridomas are very unstable. I do this to recover the Mab producing cells: add 100 ul of cells ( from a 25cm2 flask) to 10 to 15 ml of medium (5% FBS) and add 100 to 150 ul medim to a 96 well plate, let the cells multiply to confluence them do an DAS-ELISA for IgG producing cells( mouse IL4 is an expensive antigen) , expand those cells ( about 10 positive clones) in a 24 plate and the finally do an INDIRECT-ELISA to your antigen mouse IL4. Expand only cells from wells that give a higher values in ELISA ( ABOUT 3 TO 5 wells , from 24 well plate). then select one to expand to 500 to 1 liter. Purified your Mab from this volume. Use PROTEIN-A-SEPHAROSE TO remove the bovine from your Rat Mab them PROTEIN-G-Sepharose to purified your Mab. http://www.kpl.com/docs/techdocs/PURIFIGG.PDF
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