I need to do isotyping on some purified rat monoclonal antibodies. The isotyping is done using in-house light/heavy chain coated on ELISA plates. Plates were blocked with 10% CBS overnight. The clones have concentration ranging from 0.2mg/ml to 0.7mg/ml and I already diluted the antibodies 1:50 with same blocking buffer. I have 10 clones and the signal is light up for all isotypes for 8 out of 10 clones. Please help with suggestions on how to reduce non specific signal. I did not have problem before
There are couple of things you can check.
1. It is possible your culture is not clonal. They make different isoforms. Also newly generated hybridomas tend to do some isotype switching. Doing more subcloning of your culture will help.
2. Make sure your protein used for coating light/heavy chain is highly pure.
If it is an experimental error with your procedure, you can try using some commercially available isotyping kits (we also sell 5 minute lateral flow rat isotyping kit too). Thanks.
Hi Das,
Thank you for the reply. I don't think it is due to different isoforms. The whole plate has signal for all possible isoforms, which are impossible. If it's just 1 or 2 different isoforms then maybe it's true that it's not clonal. The proteins I used to coat plate is in-house, and they have been working fine for other clones. I'm trying to see if anybody had experienced this problem and how to solve it. Since we are hybridoma development team, we generate A LOT of clones. It gona become very costly if we purchase commercial isotyping kits. =(
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