Denny

Thank you for the additional information.
One quick suggestion: I tested incubation times and dilution rates of the HRP, which has a high affinity for the biotin, and found that by decreasing incubation time to 30 minutes, my background was lower and sensitivity was unchanged.

For my difficult ELISA's I incubate with blocking buffer a minimum of 3 hours at RT and do not use a shaker, again aspirating the blocking buffer instead of washing it out has lowered background on most of my ELISA's.

Good Luck, you'll get it working, hang in there!

I'm not familiar with BDNF, and do not have any details about your protocol (incubation times, temp, etc...) but I might offer a few suggestions to lower non-specific binding.
1)Increase incubation time with blocking buffer, aspirate blocking buffer immediately prior to loading samples (a few wells at a time, not the entire plate). Some people wash the blocking buffer instead of aspirating. This doesn't work for all ELISA's but for me, I use this method in 90% of mine and it has made the biggest difference.

2) Perform a salt curve in your sample buffer and conjugate buffer. Increasing the amount of NaCl in the sample buffer and/or conjugate buffer may decrease non-specific binding, but may interfer with the binding of antigen to antibody. So test a range of increasing NaCl with the same amount of sample and controls (blanks) for each condition. Change one thing at a time!! Do not increase NaCl in the buffer for the N-HRP or S-HRP step, it interferes, but NaCl in previous steps is fine.

3) Increase the number of washes - I have some ELISA's that require 9-18 washes after sample, conjugate, N-HRP! Again, change one thing at a time.

Optimizing is a delicate dance between lowering non-specific binding while maintaining capture and detection of target antigen. To save precious sample, optimize with the standard curve antigen if you have ample supply.

Good Luck, don't give up, it's a process, but when you find that perfect combination it's well worth it! Hope this helps or gives you more ideas, you know much more about your mAB's and antigen than I do.

There is a wealth of good information on the web about adapting CHO adherent cells to suspension culture and serum free medium, search for "adapting CHO adherent cells to suspension". You can find information from Invitrogen/Gibco,Thermo Scientific Hyclone, and other suppliers of
SFM... about adaption methods as well.

We often use total protein with ELISA to normalize data between samples taken from different sources. If some samples have more total protein, they may contain more of the protein of interest which can lead to incorrect conclusions. It really depends on what the experiement is and what your source of sample is. If you are following a classical method for analysis that is accepted in the literature for the work you are doing, then that sounds like what you should do. I'm not familiar with your experiment but can offer this explanation for why I use the two measurements.

I'm sorry for the confusion, I use 100 - 200 x g to pellet cells with high recovery and viability. For difficult to pellet cells, increasing time works well.


Thanks for the catch Bob I re-read the original post and see that Engi was spinning cells at 750xg not RPM, my very very bad