ELISA, BDNF, Biotinylated Conjugate - (Jan/07/2013 )

Hi,
I am working for BDNF measurement in human bloods.For this, I need to get my capture and detection Ab's working. My detection Ab is mouse monoclonal biotin conjugated. One-site ELISA worked for my biotin conjugated Mab. For last couple of weeks I have been optimizing the concentration of Mab's. My concentration for capture is 3ug/ml and for detection Ab is 2ug/ml. My problem is that, at lowest concentration of detection Ab, my blank is too high. I tried using different blocking buffers, washing buffer. My supervisor says that I dont have any problems with buffer. Does Acetic Acid interfere the biotin? Can you guys please help me out with the biotin conjugated detection Ab (mouse monoclonal) protocol?

Thank you.

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.

Thank you very much. My blocking incubation time was 2 hrs at RT in a shaker (100RPM). I tried it with O/N incubating. My capture Ab incubation time is O/N at 4^oC. For antigen(sample) its 90 mins, detection is 1 hr and HRP is 1 hr at RT in a shaker (100RPM). I didn't have any idea about NaCl. I will increase the NaCl concentration and perform it. And yeah the world of science is really confusing, once you are into it, you keep on loving...
Once again thank you very much for the information.

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!

Need to talk to my supervisor. I can't do it myself. Will let you know. Thanks a lot for sharing the information.

Have you been working with only one serum lot? Many humans have circulating antibodies specific for animal immunoglobulins, and can form a bridge between the capture and detection antibody especially if both capture and detection antibodies are from the same species. For such an assay format, it is standard practice to include either mouse serum in the sample diluent to saturate human anti-mouse antibodies within the sample or to use a heterophilic blocking reagent. Scantibodies has such products and has detailed information on their use.
Do you get a similar level of background among multiple lots of human matrix?

Not yet started working on human blood. I have been trying to figure out the combinations of Antibodies that works for my rest of the project on human blood. Presently, I have been doing experiments on rhBDNF protein to find the best Ab pair combination.

So what matrix are you using to dilute your recombinant human BDNF in?

Hello everybody.
I have a question regarding ELISA.
my sample is cell culture supernatant. usually, for long storage, at what degree I should store my sample?
Is that Ok if I store at -70C? instead of -20C?

-70C is probably better than -20C for long term stability of proteins that can bear freezing.