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Biotinylated BDNF - (Jan/06/2013 )

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?



Tell us exactly what your capture antibody is, what you biotinylated detection antibody is, the diluents that you are using, and how you are applying your streptavidin conjugate, the blockers you are using etc. Without this information, it is difficult to provide constructive advice

-Ben Lomond-

Capture is msMab#9 or (BDNF#9) at 3ug/ml, detection is Biotinylated msMab#1 or (BDNF#1) at 2ug/ml. I am following kit provided by Biosensis Pty but not the antibodies provided in the kit. My blocking buffer is Superblock Blocking Buffer in PBS (ThermoScientific).I dilute the Streptavidin-HRP in a sample diluent buffer at 1:100 dilutions and add 100ul per wells. My incubation time is O/N for capture, blocking - 1 hr, Antigen(sample) - 90 mins at RT in a shaker(100RPM), detection - 30 mins at RT, 100 RPM and HRP - 1 hr at RT, 100RPM. I hope this could help you. Thank you.