I am wondering if streptavidin or avidin can bind PCR tubes or a 96-well plate. I saw many products of 96-well plates coated streptavidin for ELISA. For my experiment using the ELISA sandwich method, I have a problem where it is detected with antigen and no antigen. I think streptavidin binds not only biotinlyated antibody, but also the 96-well plate directly. Could you let me know your experience of this problem or could you give your advice? Thank you
Are you coating the plate yourself? If so, make sure you block the plate wells after antibody coating as unproper blocking is the main cause of false positives. Blocking can be done with BSA, FBS or even skimmed milk. There are many protocol to do so.
Good luck
You would need to ensure your 96 well plates have an affinity for binding and moreso a particular affinity for binding avidin/strepavidin. I don't think they can bind to PCR tubes though. Yes, you can easily buy coated 96 well plates but coating them yourself may not be as simple/easy. If that's your problem, then yes, it's possible that your strepavidin may be binding your plate directly because the plate was initially used for that purpose, maybe you are reusing plates? Are you using a kit for this experiment? Look into these issues.
Are you coating the plate yourself? If so, make sure you block the plate wells after antibody coating as unproper blocking is the main cause of false positives. Blocking can be done with BSA, FBS or even skimmed milk. There are many protocol to do so.
Good luck
Yes, streptavidin perfectly binds to many different ELISA plates (I think not to PCR tubes). But if you coat your plates, block them with an inert protein and dilute all the subsequent reagents with the same blocking solution, non-specific background should dissappear. You also need to titrate your reagents to determine the optimal concentrations to produce a good signal/noise ratio.
If you give more details about your assay, perhaps you will receive more specific advice.
There have been some interesting discussions about ELISA backgrounds in researchgate before. Try to find them as well.
thank you for your advice. i tried to do elisa within from streptavidin and second antibody ,which has HRP without coating ag and capture antibody. i got result in no detection in this plate. so i concluded that streptavidin doesn't bind plate directly. but it still remains problem. it still detected in ag and no ag. i saw streptavidin has 4 binding site. so i think, it works something in ELISA.
do you have another suggestion?? thank you for your attention.:P
Are you blocking and diluting samples and conjugate in the same blocking solution?
Did you try different concentrations of the reagents?
Are you using BSA, milk or which blocking agent?
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