It is unusual not to get antibodies to KLH. Likely causes are ELISA failure ( do you have the polyclonal sera on each plate as a positive control?); supernatants were possibly tested too soon ( they should be clearly yellow i.e. slightly acidified by metabolism); or, finally, the cells that are growing are not hybridomas.

Yes, I have the sera positive controls on all ELISA plates and they show a positive result for KLH and the hapten target, thus showing the assay is valid. I am testing another set of colonies today. I have considered the possibility that they are not hybridomas. Thank you.

Okay, it's great that you have the positive controls, so the ELISA test is working well. If you haven't made hybridomas before, they are very distinctive under phase contrast, usually quite bright and often perfectly round, growing in quite dense colonies. By contrast, you will probably have a lot of dull elongated cells and very small splenocytes. Hopefully you just screened your cultures a little too soon. If you get a negative result for a yellow supernatant on an ELISA plate that has a positive control, then it truly is a negative culture. Good luck!

My fusion colonies tested today were also negative. In conclusion, I think that my fusion did not work. My PEG is about 5 years old, although I have the original ampules stored in the refrigerator. Also, perhaps my HAT media did not work either. Otherwise, the splenocytes (some very tiny cells were present in the fusion wells) would have all died off. Maybe the colonies I am seeing are the SP2/O's that should have died off in the HAT media. The HAT is stored frozen but is also about 5 years old. I am going to order new HAT and PEG and try again. Any thoughts on the HAT and PEG being too old? Thanks again!

I haven't stored reagents for that long and it makes sense to replace them.

However, how many days post fusion are you? if you had a failure of HAT medium, all wells would show growth. I’m just wondering if you are testing your cultures too soon? Are the supernatants distinctly yellow rather than orange?

The supernatants are more orange than yellow. I started screening colonies at Day 14, then Day 21 and yesterday's screening was Day 28 post fusion. I have enough cells in some wells that they are dying, maybe due to overcrowding. I have replaced some of the HAT media with HT media once I took out sup. Yes, you are right about the failure of HAT media. I didn't have growth in all of the wells. When I screened at Day 21 and 28, I included some of the previous wells tested also....all negative for KLH and hapten. Positives for KLH should have been most evident...but nothing. Thank you, Nick!

BTW, I am seeing some elongated stretchy cells in with the colonies. I'm not sure what they are but could they be causing a loss of antibody? Sometimes they appear to be engulfing the cells of the colonies but there usually enough cells that I think I would pick up the antibodies if they were there.

Yes, this is normal. Usually, the hybridoma colonies grow far more quickly and will re-seed new positions in the well before eventually dominating. I'm not sure what exactly is growing in your wells, but I'd expect the cultures to have been positive well before day 28. I think you probably have a total failure of the fusion step, or almost total failure and too few hybridomas to have any chance of getting ELISA positives. Good luck with the new reagents!

Thanks so much for your input, Nick. You have been most helpful. I'll give it another try!

Best regards, Mary Ann

Great thread. I'd add that yes, HAT can get too old (happened to me once), although if the Sp2/o cells didn't die, they would have quickly overtaken the cultures in all the wells. I don't know about PEG because we use electrofusion. But it does seem that the fusion itself failed. Good luck for your next run!

Thank you for your comments and mentioning your own experience. I will order new reagents next week and try again. Best regards!