ELISA for casein - need help setup ELISA for casein (Sep/29/2005 )

Hi there,

Hopefully someone can help me with the following
I am trying to set up a sandwich ELISA for casein.
I use affinity purified sheep anti-bovine casein for the capture AB (diluted in carbonate coating buffer, pH 9.6), then wash (PBS-tween pH 7.3) and block with 0.1% gelatine or 1% BSA in PBS buffer, wash, add casein in different dilutions (in blockbuffer), wash and add HRP conjugated sheep anti-bovine casein.
My problem is that the background is way too high and the signal/noise ratio is therefore too low.
I have done chessboard titrations for both antibodies and just a very little improvement is obtained by this optimization. I also see often that the negative control (just buffer as a sample) gives a higher OD than the lowest calibrationpoint.

I getting depressed so can please someone give me tips or tell me what I am doing completely wrong because I am not that specialized in ELISA's.
These 2 antibodies are the only ones I could find commercially and this limitates me so hopefully someone can help me while I can still use these antibodies.

Thanks very much
Marjolein

i have a little improvement by longer incubation times with the blockbuffer and samples and also increasing the volume of the wash and blockbuffer, but only a little improvement is obtained by this (at least now i get a normal curve, no strange points and negative control is not higher than the lowest standard), but still i only get a s/n ratio of about 5-6. so the main question is, how can i get a lower background??

i also tried increasing BSA concentration in the blockbuffer to 2% but this seems to increase my background and i get a lower s/n ratio

should i use a specific ELISA plate for casein antibodies? i use 'standard' ELISA multiwells that give good results with IgG and Lactoferrin but perhaps I need different plates for casein?

Ashish, as you see i increased the washing but the result was not much better, which protein do you mean for optimizing? he capture AB? i already did that, i don't have to do that after better (bigger volume) washing do I?

do you think a commercial blockbuffer should give better results?

Hi
Just a couple of thoughts. How pure is your BSA (bovine serum albumin) might it have contaminating casein? I would avoid using it. Increase the gelatine or try goat or sheep serum at 5%. Also try no block at all. Do you know whether your capture ab is actually capturing or is the casein attaching directly onto the plate. Try coating alternate columns with and without the capture ab. As for plates, certainly some plates are better than others for proteins depending on hydrophilic/hydrophobic properties. Check with the rep, alternatively email me as i dont want to advertise plates on forum.
Regs
parky

Parky,
I did not know that BSA could contain casein? but the bsa block gave better results than the gelatine block so i think the bsa is ok but i could try goat of sheep serum for sure.
when i don't coat the capture ab but directly the casein itself then the background is a lot lower, just for pure casein samples this is no problem but my samples contain other proteins that attach to the wells aswell and the signal for casein drops when i coat the standard in the sample background so this direct elisa is no option for my samples
i suppose the capture ab is actually capturing (that is what the supplier say!), i get a curve with pure casein just not a big n/s ratio. i will email you tomorrow with the information about the plates i use.
thanks for your help sofar!

What is your detection antibody diluted in?

I find that when I changed the dilution buffer for just my secondary antibody, it dropped my background in ELISA a lot. Regardless of the blocking buffer - 3 to 5% BSA or 3 to 5% casein.
In the end, I think I used 1% whole calf serum in PBS to dilute my secondary antibody in. My antibody was of goat origin.

The other possibility is that you could increase your tween concentration in your washes to make the washes more stringent.

It is definitely true that most BSA (large bulk kind) have other contaminants, such as casein.

Are you getting decent OD's for your standard curves? (0.1 thru 2+)
Lastly, though I'm sure you aren't doing this, make sure that your PBS (and any dilution buffer after the detection antibody) does NOT contain Na-azide. This destroys the HRP enzyme.

Good luck

I dilute my detection AB in the blockbuffer (tried both with and without extra 0.05% tween-20)

when i try a direct elisa (coat casein on the plate) i get a good background (below 0.1) with a good n/s ratio but this i cannot use because of my matrix sample, because of either other proteins in the matrix that will also coat on the plate of other components in the matrix but casein in the matrix drops dramatically in OD compared to the same concentration casein in buffer, therefore i have to return to the sandwich but the background is still too high, i do get a good max. OD (2+) that is no problem but with a background of 0.8 that is a problem.
no i did not use na-azide in the buffers so that is not it


i will try to use a different serum for blocking but most serums are bovine right? so perhaps that will be difficult or very expensive to use?
i will also try to increase the tween-20 for washing


a few questions for you:
what is the difference for the buffer you use for the secondary ab and the samples??

the assay you talk about is that a casein assay or a elisa in general?

thanks!!!

when do you use a coatingbuffer pH 9.6 and when pH 7.2?

i am back at the starting point of the setting up of the elisa again , my results cannot be reproduced day to day and optimization is therefore impossible.
also i see in the standardcurve a strange thing, half way it dips down and then starts to increase again. i suspect that the capture ab is not well attached and perhaps pulled off the plate by washing and the antigen, what is the best way to make this attachment stronger?

You need to optimize the coating buffer / pH. Passive adsorption ab should not come off. Plenty documents on line from corning etc on elisa methods.

If you are worried about bsa you can obtain blocking buffer from kem en tec or call east coast biologics for a fish based material. Be careful with commercial bsa blockers (kpl) many are not clean (ie not fraction v only).

When washing with surfactant follow with dh20 or use dh2o and let sit for 15 sec prior to decant and blot.

good luck