Coating antigen, denaturated upon well bottom binding? - (Jan/23/2008 )

I am using as coating antigen a protein which contains a phospholipid inside. For some results that we have obtained, we strongly suspect that mAbs which are able to recognize the protein when this is in solution, are not able to recognize it anymore when the protein is attached to the bottom of the well. Does this means that the protein is denaturated upon binding? Could it be due to strong hydrophobic attraction between the phospholipid and the hydrophobic-binding sites of the well? The phospholipid is located inside the molecule, within a pocket, and its tendency to bind to the hydrophobic sites of the well could alter the structure of the molecule?? Anybody of you is experienced in attaching this kind of antigens to ELISA plates? Thank you very much.

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Hiya,
How experienced are you with ELISA's as there are many many different variables in attaching proteins to plates? I don't want to patronise you by suggesting all the alternatives if you have lots of experience!

I appeared to have difficulty with attaching my protein at first, i also have a highly sensitive conformational monoclonal, which means it will not recognise my protein at all if it is reduced. My protein also has stronly hydrophobic areas, but it seems to bind very well to the plate, still in a recognisable form. It is possible that your protein becomes unrecognisable - there are many variables to check first however.

Which;
plates are you using?
coating buffers?
how have you chosen Ab concentrations?
what are your incubation conditions?
what detection substrate are you using?

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hi,

raise polyclonal antibodies to ur protein, then u have higher chance of detecting in ELISA.. given situation like urs, dealing with monoclonal antibodies in ELISA is difficult.

did u perform WB, ur antibodiy recognized the protein of interest (i guess not)??

gud luk

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Hi Flour Power thank you for the answer and sorry for the delay:
Plates- I have tested "only hydrophobic binding"; "hydrophobic + hydrophilic (usually called "high binding" plates)
Coating buffers: sodium carbonate, pH 9.6 or PBS, pH 7.4
Secondary Ab concentrations: the recommended by commercial suppliers (other chosen, no better results)
Incubation conditions: RT everything except blocking step (which is at 37 oC 1 h, tested several ones, BSA 3%; Pierce Superblock; Caseine 1%).
Detection substrates: tested OPD-H2O2, and TMB-H2O2.
Probably you are more experienced than us, any advice will help!

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Hi House,

thanks for your answer. We did perform Western blot, the point is that my mAbs recognize the Ag in Western, ELISA, but NOT THE NATIVE FORM (on membrane, or in solution). This is why I am assuming that I have Abs against the denaturated protein (this raises the question if I denaturated the Ag when sonicating it prior to immunizing mice (in Freund´s).

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hey,
from the hints u have given, i wud conclude that, the antibody recognizing site on ur protein is not a hidden epitope as u can detect in ELISA in solution (unless ur protein is not denatured) and when u use the protein for coating, the epitope may be facing towards the bottom. hence, stearic hindrance could explain y ur antibody is not binding anymore.

i wud not assume that denatured ur protein during sonication least likely (if u think this is the situation use lysozyme n see).

to come across this problem i wud suggest u to go for a capture ELISA, have a different mAb to detect different epitope, coat them on the plate, add ur protein of interest, then come up with the current Ab which u have already.


wat do u say....

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