Strange results using sandwich Elisa - All antigen concentrations show same result - same optical density! (Dec/12/2008 )
Just remembered, not sure it is of any importance, however I wash my plates by applying 300ul of washing buffer (0.1% Tween in PBS and DI water) to each well, I leave it stand for about 1min, empty contents into sink, tap the plate (very sharply and hard) against clean tissue sheets until no more droplets appear on tissue, and then I repear 4x. Is this the right thing to do?
Also for how long can the antibodies usually last if stored as aliquotes in the freezer (-20 degree celcius)? My trainer suggested that the antibodies he got with him must have degraded on the way. But I also tried expt again with antibodies that I found stored in freezer and still got same results!
Sounds OK to me, but you only have to be really careful about removing droplets of wash solution the final time you wash. In between, I usually just empty the plate and flick it a couple of times.
I presume the Ab is meant to be stored at -20C, right? Silly question, I know, and potentially insulting, but it has to be asked. Next question: did the Ab go through a few rounds of freeze-thaw?
How long do you block the plate after coating with your mAb?
Hi Swanny...not insulting at all...the more questions the better.
Abs are stored at -20 degree celcius yes. As far as i know the Ab was not exposed to freeze and thaw cycles since I found them all divided into tiny eppendorf tubes already aliquoted.
????!!!! Except for the 3rd Ab. All 5x that I ran the ELISA I varied the Abs (3x I used the Abs given to me by the trainer and 2x I used the ones left in the freezer by the previous cell biologist EXCEPT for the 3rd Ab which belonged to the trainer all of the 5x). So if the Abs given to me by the trainer degraded on his way to Malta then could be the last 2x i ran the ELISA it didnt work due to the degraded 3rd Ab.
Will send you the last set of results tomorrow morning.
Have you tried the next (it worked for me)?
Antigen-Block-AB2-detectAB-TMB where you coat overnight with different conc of antigen. Doing this you might get an idea if AB2/AB3 are doing their jobs right. In my case this certainly gave a nice result with increasing OD's. I also did this for AB1 and its respective detection-AB, and this also worked fine. I just don't understand why it does not work in the sandwich but it does in the indirect-ELISA. Maybe in your case this experiment could be worthwile. It just gives some extra information about AB2 and AB3.
Happy holidays to you all, Bertrand
A few comments: using protein free blockers to avoid cross reactivity or diluting blocking agent for same purpose. The immunogen would have to be contaminated with "BSA" or other protein and, thus, the ab you have would be no-specific to your antigen.
the term cross reactive is used for antibodies reacting to structurally similar antigens such as antibodies to hCG reacting with FSH, LH or antibodies to amphetamine reacting to structurally similar drug compounds.
Hopefully, your antibodies are affinity purified or monoclonal? The blocking agent will have no bearing on the reaction between the antibodies. It will have a bearing on the reaction of the antibodies to the protien blocker (if they are non-specific).
From all the information you have provided it seems the blocking and washing steps are correct. The coating of the plate is correct. I think you have reaction between the antibodies in absence of antigen OR the titer of the conjugate is extremely high.
I belive you said your 3rd (conj ?) ab reacts with coating ab in absence of ag. So either the coating ab is binding the 3rd or the 3rd is binding to the coater. Can you purchase another species of anti-2nd ab conjugate? or mix some purified non-specific ab same class/species as the coater with your 3rd and allow to react for 1 hour then adjust dilution of conju and use in the test. This would block the non-specific binding.