Antibody Position on ELISA plate - (Jun/14/2006 )

hello,

i am would like to ask a question on the elisa plates used for detection, these plates which we get are pre coated with abtibodies and they give us a number of antibodies on the plate. i would like to know how do they know that these many antibodies are in the right position, that is the light chain are facing upwards and by mistake are not facing downwards.
is there any method by which i can detect and estimate how many of the antibodies on the plate are having the light chain facing upwards and how many are down.



thekid

hello,
not many people care about position of the Ab on the plate.
most of them cares and counts only the Ab which is been coated in right direction (Y) coz this is the one which will bind to Ag of interest. Abs which are coated in wrong direction are never considered in the experiment unless it has cross reactivity.

the propotionality between right direction and wrong direction of antibodies depends on probability. if we assume that there are 3 directions where Ab can attach to the plate
up direction (Fab upside)
down direction (Fc upside)
horizontal direction (neither Fab nor Fc up side)

then we can say 33% of the Ab which have been coated are in up direction.
this can be increased by increasing the concentration of coating Ab concentration.

does it make sense
gud luk
payeli.

how can you increase the percentage of antibodies in the right position by increasing the concentration, all possible binding positions will increase linearly.

thus 33% will always be the amount of corretly bound antibody, all that an increase in concentration will do is bind more antibodies to the well, this is not neccesary under normal conditions there are genrally more than enough antibodies in the correct position.

also i dont think it is as simple to say there are only 3 ways that the antibody can bind to the plate, as there are numerous ways, all slight variation of one another, some will still allow analyte binding while other not.

to the kid, antibodies bind the plastic at random, but following manufatures specification, the amount of antibody used to coat the plates far outnumbers the analyte being being bound that you dont have to worry about the position of the antibodies being bound

you can force the issue by precoating the plate with protein A or G. then all of the bound antibody will be in the proper orientation.

hi,
in regard three positions of the Ab, i was trying to explain the situation with example, u r right that it has got lot of different orientation how Ab can bind to the plate.

At the end the concentration of Ab whihc we use for coating is important and it must be constant to compare with other results.


bye
payeli

don't forget that there is an upper limit of coating concentration (about 10ug/ml usually) above which you start to lose out due to steric effects. Precoating with Protein A or G is the best way I've heard to optimise coating in th eright orientation.

i have used protein a coated plates to capture the antibodies.. it works.. but u should see the suitability of the method for ur needs.. the protein A or G will help to orientate the antibodies so tht the variable region would be accessible to the antigens.... but for most assays.. sometimes this is not a big issue .. in the sense tht the amount of antibodies coated on ur plates.. would show if the amount of antibodies are sufficient for detection... i think in terms of costs... u should try workin without the protein A and if the results are not satisfactory u can try protein A...