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why do my hybridomas seem to stop producing antibodies when screened 2 weeks lat


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#1 lizh

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Posted 21 March 2011 - 01:36 AM

Hi guys

I am growing hybridoma cells to obtain monoclonal antibodies. After doing limiting dilution and plating the cells out on 96 well plates, 1 cell per well, they were left to grow over 1 week in the incubator. After 1 week they were screened using ELISA, and positive clones were identified (value 0.4 Abs). I expanded the positive clones into more 96 wells and screened them after 4 days. This time the expanded positive clones did not give a signal when screened on ELISA. Why did the positive clones give a negative signal, indicating that no antibody is detected? What should I do next?

Regards
lizh

#2 Chelo

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Posted 21 March 2011 - 04:08 AM

Hi guys

I am growing hybridoma cells to obtain monoclonal antibodies. After doing limiting dilution and plating the cells out on 96 well plates, 1 cell per well, they were left to grow over 1 week in the incubator. After 1 week they were screened using ELISA, and positive clones were identified (value 0.4 Abs). I expanded the positive clones into more 96 wells and screened them after 4 days. This time the expanded positive clones did not give a signal when screened on ELISA. Why did the positive clones give a negative signal, indicating that no antibody is detected? What should I do next?

Regards
lizh


Hi Lizh,
One possibility is that your hibridomas are genetically unstable and that they lose the needed chromosomes to produce the antibodies while replicating. In theory, this happens around 10-15% of the time. If you are missing ALL your hibridomas, consider to have some contamination coming from the old lymphocytes (how many days after fusion do you clone the hybridomas?). In any case, a OD = 0.4 doesnt look promising to start with.

#3 lizh

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Posted 22 March 2011 - 05:30 PM


Hi guys

I am growing hybridoma cells to obtain monoclonal antibodies. After doing limiting dilution and plating the cells out on 96 well plates, 1 cell per well, they were left to grow over 1 week in the incubator. After 1 week they were screened using ELISA, and positive clones were identified (value 0.4 Abs). I expanded the positive clones into more 96 wells and screened them after 4 days. This time the expanded positive clones did not give a signal when screened on ELISA. Why did the positive clones give a negative signal, indicating that no antibody is detected? What should I do next?

Regards
lizh


Hi Lizh,
One possibility is that your hibridomas are genetically unstable and that they lose the needed chromosomes to produce the antibodies while replicating. In theory, this happens around 10-15% of the time. If you are missing ALL your hibridomas, consider to have some contamination coming from the old lymphocytes (how many days after fusion do you clone the hybridomas?). In any case, a OD = 0.4 doesnt look promising to start with.


Thanks chelo!
3 weeks after fusion the cells are subcloned. Screening is done 2 weeks after fusion, followed by subcloning a week later. How do I get rid of the contamination from the old lymphocytes? I have attempted subcloning thrice and each time the same thing happens: a positive clone always turn up negative eventually.

#4 HBImolbiol

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Posted 24 March 2011 - 09:23 AM



Hi guys

I am growing hybridoma cells to obtain monoclonal antibodies. After doing limiting dilution and plating the cells out on 96 well plates, 1 cell per well, they were left to grow over 1 week in the incubator. After 1 week they were screened using ELISA, and positive clones were identified (value 0.4 Abs). I expanded the positive clones into more 96 wells and screened them after 4 days. This time the expanded positive clones did not give a signal when screened on ELISA. Why did the positive clones give a negative signal, indicating that no antibody is detected? What should I do next?

Regards
lizh


Hi Lizh,
One possibility is that your hibridomas are genetically unstable and that they lose the needed chromosomes to produce the antibodies while replicating. In theory, this happens around 10-15% of the time. If you are missing ALL your hibridomas, consider to have some contamination coming from the old lymphocytes (how many days after fusion do you clone the hybridomas?). In any case, a OD = 0.4 doesnt look promising to start with.


Thanks chelo!
3 weeks after fusion the cells are subcloned. Screening is done 2 weeks after fusion, followed by subcloning a week later. How do I get rid of the contamination from the old lymphocytes? I have attempted subcloning thrice and each time the same thing happens: a positive clone always turn up negative eventually.



How are you performing your ELISA? What is your antigen, blocking solution, detection antibody? What controls are in place? How long do you develop to get an OD of 0.4? A truly positive clone should have a very strong absorbance over a short time period. I am guessing that your signal is a non-specific false positive due to adsorption of IgGs in the media to the wells.

#5 lizh

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Posted 24 March 2011 - 11:17 PM

the ag is a purified allergen protein, which I had used to inject the mice previously. Blocking solution was 3% BSA in PBS-tween. Detection Ab was anti-mouse IgG. Positive control was serum from mice which were injected with the allergen. The developing time was 10 mins.

Looking back my previous experiments I think it might be contamination from non-positives in 96-well plate. For transfering the supernatant from 96 well plate to ELISA plate, I used the same pipet tips; so a non-positive cell could have entered into the same well with the positive cell and eventually overgrown the positive clone.

Will repeat limiting dilution again using frozen stocks, this time with fresh pipet tips for each well.

#6 garzaa1010

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Posted 28 October 2011 - 12:04 PM

Hey guys I'm sorry I know this post is Kinda old now. But I had the same problem. I had a great antibody that was competing well but It just disappeared. Now I believe it is due to mycoplasma contamination or it is the lymphocyte problem you mentioned. I have been treating my cells with normicin since I first fused them but my mycoplasma test have still come up positive. However the cells still look nice and healthy with great colonies that clone out fine. Is there anything I can do to maybe recover those cells or get them reactivated pumping out antibody again?




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