ABC (Avidin Biotin Complex) in ELISA - (Jun/11/2014 )

Hi All,

has anyone had any success using ABC (avidin-biotin complex) to improve the sensitivity of their ELISA? I am trying the Vector labs  VECTASTAIN Elite ABC Kit and I'm getting significantly worse results than just using standard biotin followed by strep-HRP. Does anyone have any successful experience with this? There are publications reporting its efficacy but not much in the ways of specific protocols, and using the one that came with the product is not working for me. Thanks

Ben

i used one a long time ago. i followed the instructions as printed and was rewarded with good results.

If anyone comes across this, I would highly, highly recommend just get poly-HRP versus using the ABC kit. There were virtually no advantages to using the ABC kit versus pre-polymerized streptavidin-HRP. Fitzgerald and thermoscientific both sell them. I prefer Fitz because they sell polymers of different specified lengths (40, 80, 100) whereas thermo just sells it as poly streptavidin HRP - unclear how long the polymers are.

Either way, the ABC kit is basically rebuilding the wheel. I would recommend skipping it and getting poly-HRP, way easier and actually had better results for my assays

No such experience. Reporter intensity is a function of the localized enzyme activity, and improved sensitivity can be achieved by increasing the number of enzyme molecules bound to the target antigen. The multiple biotin binding sites in each tetravalent avidin molecule are ideal for achieving this amplification.  I only know that avidin-biotin complex (ABC)is one of the most common methods for amplifying the target antigen signal in IHC detection.