Signal disappearance in ELISA - (Oct/22/2007 )

Hi all,
i am doing ELISA for the detection of human serum antibodies. basic procedure is coating the antigens with PBS (maxisorp plates), blocking with skim milk, adding diluted serum (in blocking buffer) and detection using HRP-TMB. washing with PBS-tween 0.05%, accept after the serum - PBS tween 0.1%.
i used to have signals ranging of 0.2-2 ODs. lately i've changed my DDW (for the PBS) from RO to sterile tissue culture grade and all of my signals dissapeared (now ranging from 0.05-0.2).
can anybody suggest me a possible cause for the steep decrease? i'm not sure the water is the problem, any other suggestions would be mostly wellcomed.
thanks,
yahalom

hi,
add your diluted secondary antibody solution (20ul) to the substrate solution.
u should see color, that means the buffer conditons are fine for HRP activity. but this can not rule out the intactness of Ab-HRP. to avoid this porblem u need to use fresh aliquote of HRP conjugated sec antibody.

if above conditios does not work, then use saline for coating your antigen and observe how the experiment goes!!!

gud luk
sravan

[quote name='donot lie for ever' date='Oct 26 2007, 05:03 AM' post='115271']
hi,
add your diluted secondary antibody solution (20ul) to the substrate solution.
u should see color, that means the buffer conditons are fine for HRP activity. but this can not rule out the intactness of Ab-HRP. to avoid this porblem u need to use fresh aliquote of HRP conjugated sec antibody.

if above conditios does not work, then use saline for coating your antigen and observe how the experiment goes!!!

gud luk
sravan


Thanks sravan
the HRP is fine - and fresh aliquotes are being used each time, i don't want to change coating conditions relative to my old experiments (i.e. replacing PBS with saline).

I can't think of anything obvious, apart from something in the sterile water, but that is totalyl counter intuitive. Can you tell us why you want to change from RO to the top-shelf water? My personal opinion is that ELISA is such a blunt instrument that there is nothing to be gained by going overboard with the water, as long as it is better than tap water! Ro should be fine!

If nothing else has been changed, try water from a couple of different sources, including a repeat run with RO water.

This might seem to be an insult to your ability, so I'll apologise right now, but are you sure you added the salts to the water, and didn't accidently use straight water for the washing? Similar things have been done by almost every person running a gel, so you won't be the first. Or the last.

may be your skim milk powder goes off or forms lumps

You may wish to use a new skim milk powder.


Hope this may help.

you are absulutly right about the RO - but this is purely a desparation move...
regarding the salts - i doubled and tripled checked it,
regarding th milk - also checked.
so you think it is possible that something is wrong with the antigens (loss of activity due to freeze thaw?, proteases?)
again
thanks for all replies,
yahalom

hey,
when you have problem with your ELISA, you can not stick to the standardized protocol. Here your goal is to try and find the problem. once you know the problem then u can come back to the original protocol.

If you use RO water can reproduce your results?

sravan


Thanks sravan
the HRP is fine - and fresh aliquotes are being used each time, i don't want to change coating conditions relative to my old experiments (i.e. replacing PBS with saline).
[/quote]

hi all
i think i know the problem - too little incubation at 37 with the Abs. - i guess Abs don't bind well when they don't get the proper conditions....
when i increase incubation at 37 i get my signals back.
can you suggest the best time for 37 incubation with the Abs in order to increase the signal, yet not to increase the non- specific signal?
do you think 1 h is enough? would you suggest adding ON incubation at 4 in order to strengthen the Ab-Ag interactions?
thanks for all suggestions,

yahalom