conformation specific monoclonal antibody - how to confirm? (Jul/31/2007 )

Hi all,
i am working on monoclonal antibody production
recently i have done a screen of all the monoclonals by indirect elisa in which there will be a change in the O.D in the presence of oligo when detecting with a particular mab.
surprisingly i have got only one out of approximately 90 clones which shows a difference
that means either the mab is preventing the binding of oligo to the protein by exclusion method or it is freezing the enzyme in a specific conformation which doesn't allow the enzyme to bind to the oligo.
so how can i confirm the difference in these two possibilities
any suggestions?
thanks in advance
Leelaram

hi
can u explain me your indirect ELISA?
how does it work?
what is this oligo ?
is your protein of interest is an enzyme?

in my experience you get atleast 4-5 clones for confirmational epitiopes (this very generalized statement).

rgds
sravan.

[quote name='payeli' date='Aug 7 2007, 03:11 AM' post='107461']
hi
can u explain me your indirect ELISA?
how does it work?
what is this oligo ?
is your protein of interest is an enzyme?

in my experience you get atleast 4-5 clones for confirmational epitiopes (this very generalized statement).

rgds
sravan.

Hi sravan,
i use a 32mer oligo which contains the specific sequence recognized by the antigen
yes, the antigen is an enzyme which catalyzes cleavage of the oligo
i have done the indirect elisa by first coating the elisa plates with antibody and then capturing the antigen
then either oligo or antibody is incubated with it and then vice versa to check for the difference in the O.D
and in case of only one mab out of nearly 80 clones i could see a difference in the O.D indicating that the antibody is not binding in the presence of oligo or not allowing the oligo to bind. but what i want to confirm is that is the antibody freezing the antigen in particular conformation so that oligo is not able to bind or is it simply competing the same binding site like oligo?
any suggestions are highly welcomed
thanking you in advance
Leelaram

hi,
u do not incubate both oligo and detector Ab at the same time? in this case competion betweem oligo and Ab is not there, but they may bind to same site!
when u say 80 clones, all these are recognizing the enzyme right? (are u sure that those 80 clones are from different clones but not from same single or few clones? ).
what is the origin of capture antibody ? detector antibody is ofcourse from mice and this is wat you want to test?
all other clones are binding to your antigen in the absence of necleotide?

after looking at your question,
i would proceed with an experiment as followed

conditon- keep capture Ab and enzyme concentration as constant
add increasing conc of oligo ( then incubate with capture Ab (conc is constant but maximum)).

if you have any dose response, then you have the answer!!

Result:
if you have decreasing signal intensity upon increasing oligo conc- oligo and Ab competes for the site, this also explains that antibody is not freezing the enzyme but may be the availability of nucleotide is limited by what ever reason.

to find out whether your Ab is freezing the enzyme or not, experiment is bit complicated.

let me know what do u think about above experiment.

open for your comments
gud luk



[ but what i want to confirm is that is the antibody freezing the antigen in particular conformation so that oligo is not able to bind or is it simply competing the same binding site like oligo?
any suggestions are highly welcomed
thanking you in advance
Leelaram
[/quote]

hi,
that's a nice idea
i will try it out
thank you
Leelaram