Monoclonal antibody production - no cells show high O.D. - (Aug/17/2006 )
Hi all,
I have previously posted the same question but couldn't get further replies
sorry for writing again but I am interested in the observation we have got while doing the monoclonal antibody production. It goes as follows:
I am trying to raise monoclonal antibodies against a mycobacterial protein.
After fusing the immune spleen cells and fusing them with sp2/o cells and then aliquoting into 96 well plates, in the screening of supernatants by ELISA we have observed that few wells show very high O.D. but to our surprise, no cells at all.
Is it routine thing in mab work or is it peculiar to few types of antigens.
I am eagerly waiting for a possible explanation but couldn't find any until now.
The supernatants of such wells i have pooled and then added to sp2/o myeloma cells and observed that the sp2/o cells are dying and the death looks like apoptosis under phase contrast microscopy.
I would be very grateful for a reasonable explanation which would save lot of other experiments i am planning with the supernatants.
Leelaram
May be the antibodies produced by hybridoma are killing the cells.
Hi Minni Mouse,
thank you very much for the reply
Even we are thinking that the antibodies might be killing the cells
To check whether it is caused by the antibodies against the antigen used for immunization or by some other antibodies we are trying to purify antibodies by protein A column and also by antigen coupled to sepharose beads.
Hope we will get some interesting results
Thanks a bunch
Leelaram
I have previously posted the same question but couldn't get further replies
sorry for writing again but I am interested in the observation we have got while doing the monoclonal antibody production. It goes as follows:
I am trying to raise monoclonal antibodies against a mycobacterial protein.
After fusing the immune spleen cells and fusing them with sp2/o cells and then aliquoting into 96 well plates, in the screening of supernatants by ELISA we have observed that few wells show very high O.D. but to our surprise, no cells at all.
Is it routine thing in mab work or is it peculiar to few types of antigens.
I am eagerly waiting for a possible explanation but couldn't find any until now.
The supernatants of such wells i have pooled and then added to sp2/o myeloma cells and observed that the sp2/o cells are dying and the death looks like apoptosis under phase contrast microscopy.
I would be very grateful for a reasonable explanation which would save lot of other experiments i am planning with the supernatants.
Leelaram
Hi Leelaram,
If the supernatants you’re screening really contain antibodies, then you should have seen the hybridomas producing them. After all, your main goal is to isolate the hybridomas producing the antibodies against your epitope. Once you have the right hybridoma, it’s easy enough to produce and collect the antibodies later on.
I’ve never done any fusion but during my undergrad, I was following around a PhD student when he was recloning one hybridoma line using feeder cells. I had to look at the plates every day (how exciting was that!). Starting from literally one cell you could see them multiply and eventually form colonies right before your eyes (of course a lot of them were non-producers).
I don’t think that a high-titered supernatant was produced by only a few cells and if the supernatant is indeed toxic and killing off your hybridoma then the cells should not have proliferated enough to secrete a good concentration of antibodies. You should probably try another screening assay.
Casey
hi casey,
thank you for your suggestion
the supernatants when used for western were showing specific band of the antigen but no band at BSA loaded. So, i think the high O.D. we were seeing could be because of specific antibodies only. but the puzzle is are these antibodies secreted by fused cells only? if yes where are the cells gone. If not, then what cells are secreting them.
awaiting for your valuable suggestion
Leelaram
I think the fusion between the immune spleen cells and the sp2/o myeloma cells may have failed. The high OD is due the antibody produced by the spleen cells. Unfortunately, spleen cells died after a few days if they are not fused with myeloma cells (immortal cell).
Hope this may help.
thank you for your suggestion
the supernatants when used for western were showing specific band of the antigen but no band at BSA loaded. So, i think the high O.D. we were seeing could be because of specific antibodies only. but the puzzle is are these antibodies secreted by fused cells only? if yes where are the cells gone. If not, then what cells are secreting them.
awaiting for your valuable suggestion
Leelaram
Hi Leelaram,
I agree with Minnie, I think that you should fix your fusion first. Your goal is to isolate the hybridomas, right? BTW, do you see the same specific band when you use the sera of your immunized mice?
KC
Dear researcher
I had the same results during my previous work for hybridoma production,
after deep analysis i get the answer
it is that during transport the supernatant from culture wells to screening ELISA plate alittle drops may be scattered to the adjacent wells so you will find high OD in 2 adjacent wells one of them contain cells and the other not.
happy to hear from you
m_youssef_mabs@yahoo.com
hi leelaram ! i completely agree with minie. infact the high titres you are getting in the initial screening is because of the specific antibody which might be produced by the spleen cell . it is quite common to get false positive results in the initial screening. some unfused spleen cells continue to produce antibodies in the plate and eventually die off when their life span is comleted. that is why you are getting results in the screening but no cells in the plates. that is why they say that you should screeen your plates atleast twice if not thrice to make sure that your clone is perfect.hope
all the best
