Monoclonal Antibodies - Hybridoma Screen (Jul/22/2009 )
tonix37 on Nov 11 2009, 12:45 AM said:
hi all,
wirly thanks for your help.
You say the results are similar, but are they exactly the same?
In the cases I have tested with both ELISA the result are double-positive. Not all samples are tested by both immunoassays. We have an established cutoff at which the mean +2 sd of the negative control is positive, and high positive sd +4, and is consistent in both ELISA, the high positive clones are found in both ELISA, and negative clones are negative in both ELISA
How many colonies do you get per well?
The problem occurs in different steps of the cell fusion: in the primary wells fo cell fusion, and in the wells of cell clonnings at 1 cell/well, with 1 clone microscopically visible.
Do you perform any media changed between initial 96-well plating and initial screening with anti-gamma? Ho many?
In the case of primary cell fusion wells (postfusion) we did HAT media change (day 7), HT (day 11), 10% FCS (day 18), hybridoma clones are very apparent and we do the ELISA from primary wella between the day 20-25 . In the case of cloning, once we limit dilution add 200 microliters / well and we ELISA 7-10 days.
Is every positive well with both secondaries?
Yes. Not always we do the double ELISA, the ELISA with FTR (time resolved fluorescence) is a screening test to perform before colorimetric test in order to select and expand positive hybridomas more quickly. With qualification + = mean +2 sd negative control, + + = mean +4 sd the negative control wells are positive with both ELISA. The scale is different because the colorimetric ELISA signal are between 0-3, while the signal we FTR are between 0-90000.
Does every end up being IgG antibody in the end?
Although serum hiperimmune have IgG, the resulting hybridomas are all IgM and IgA and IgG3 (postive primary well cloned- positive clon (1cell/well) recloned)
If not, what is a rough percentage for your postive wells double / single positive wells / Resulting IgG / IgM Resulting
I haven't result of all isotypes, but over 40 positive, 37 IgM: 2 IgA: 1 IgG3
I do not know what happens... What do you think about:
- Batch of fetal calf serum can influence the hybridoma growth rate, and IgM-secreting hybridomas grow faster than IgG-secreting hybridomas ?
- We use a layer of macrophages as feeder layer in the early days postfusion, but we found the same problem while to generate other monoclonal and most clones were IgM hybridoma using the enhanced supplement supplement from Sigma. What kind of supplement could be better to grow the hybridomas on the first days postfusion, or after cell cloning??
thanks in advance for your ideas, comments, suggestions and time
wirly thanks for your help.
You say the results are similar, but are they exactly the same?
In the cases I have tested with both ELISA the result are double-positive. Not all samples are tested by both immunoassays. We have an established cutoff at which the mean +2 sd of the negative control is positive, and high positive sd +4, and is consistent in both ELISA, the high positive clones are found in both ELISA, and negative clones are negative in both ELISA
How many colonies do you get per well?
The problem occurs in different steps of the cell fusion: in the primary wells fo cell fusion, and in the wells of cell clonnings at 1 cell/well, with 1 clone microscopically visible.
Do you perform any media changed between initial 96-well plating and initial screening with anti-gamma? Ho many?
In the case of primary cell fusion wells (postfusion) we did HAT media change (day 7), HT (day 11), 10% FCS (day 18), hybridoma clones are very apparent and we do the ELISA from primary wella between the day 20-25 . In the case of cloning, once we limit dilution add 200 microliters / well and we ELISA 7-10 days.
Is every positive well with both secondaries?
Yes. Not always we do the double ELISA, the ELISA with FTR (time resolved fluorescence) is a screening test to perform before colorimetric test in order to select and expand positive hybridomas more quickly. With qualification + = mean +2 sd negative control, + + = mean +4 sd the negative control wells are positive with both ELISA. The scale is different because the colorimetric ELISA signal are between 0-3, while the signal we FTR are between 0-90000.
Does every end up being IgG antibody in the end?
Although serum hiperimmune have IgG, the resulting hybridomas are all IgM and IgA and IgG3 (postive primary well cloned- positive clon (1cell/well) recloned)
If not, what is a rough percentage for your postive wells double / single positive wells / Resulting IgG / IgM Resulting
I haven't result of all isotypes, but over 40 positive, 37 IgM: 2 IgA: 1 IgG3
I do not know what happens... What do you think about:
- Batch of fetal calf serum can influence the hybridoma growth rate, and IgM-secreting hybridomas grow faster than IgG-secreting hybridomas ?
- We use a layer of macrophages as feeder layer in the early days postfusion, but we found the same problem while to generate other monoclonal and most clones were IgM hybridoma using the enhanced supplement supplement from Sigma. What kind of supplement could be better to grow the hybridomas on the first days postfusion, or after cell cloning??
thanks in advance for your ideas, comments, suggestions and time
OK, I still can't see a definitive answer so I'll just throw out a few more questions and observations:
It's stupid, but I assume your mouse sera titering is antigen specific-ELISA with the Sigma anti-moIgG(Fc) secondary?
Your antigen is protein, or is it carbohydrate/glycoprotein/other?
We don't use feeders so I don't know how they may contribute to the situation. Instead, we use either Hybridoma Cloning Factor (HCF) from Bioveris (expensive) but really all you need is to add IL-6 to HAT media with 10% FBS (less than 10 ng/mL is more than enough) which is what we have moved to.
We screen wells from the fusion on day 10. Not sure if feeders induce slower growth that the factors we add, but we get good signal by day 8-10 with a standard antigen-specific ELISA with IgG-Fc secondary.
If you have a well that comes up IgG specific, you can immediately isotype it with a strip and see if it that confirms the isotype, or if it is double positive. Or you could get a mu-specific secondary and screen all of your plates with anti-IgG and anti-IgM ins separate ELISAs at the same time (we do this regularly). Maybe that Sigma antibody is crap (not likely but you never know) - we use a pretty good polyclonal from Jackson. (don't use the Sigma Aldrich antibody, it's not helping you find gammas)
Can't say anything about the FBS lot theory, but screening at an earlier time-point and expanding the next day should eliminate any growth differential issues.
-wirly-
Hi,
Does anyone know of specific journal articles that I could use as references for the fact that the isotype of antibodies can be affected by the type of antigen used?
-MouseMab-