10 replies to this topic

[vojera]

Hi!

I haven't done very many ELISAs and since we have no postdocs in our lab and my PI is strictly DNA/molecular-based, I'm pretty much trying to learn how to do them from mat/meths in papers (ha!).

My first problem is that I'm trying to detect (and quantify) one protein out of a mixture of proteins (plant soluble proteins). The protein is definitely there in high quantities (confirmed by many Westerns) but my ELISAs are not picking it up. I'm using alkaline phosphatase and I get some colour change after many hours' incubation but not much. My positive controls (purified protein of interest) always come up without any problems.

To further complicate things, I did some work on this protein a year or so ago and never had any problem picking it up. The difference now is that the tissue I'm working with has been freeze-dried and ground into a powder, as opposed to a TSP extraction from fresh tissue with carb/bicarb buffer.

Could it be that the freeze drying has altered my protein somehow and that it makes no difference for Western because my samples are denatured anyway before loading?



My second question is, sometimes my standard curve is a bit backwards, as in the lower concentrations actually give a higher OD than the higher concentrations. Could this mean I haven't properly optimised the Ab concentrations for my ELISA?


Thank you for any help you can give me - I'm stranded in an ELISA-knowledge-free world and I'll be grateful for any tips you can give me, however small.

Edited by vojera, 06 November 2009 - 07:38 AM.

[Prep!]

hi vojera.. these are the things i cud think of
1) yes the protein can be affected in terms of relatied impuriteis by freeze thaws which might alter the structure and ultimately the binding to your coated plate.
2) are u using a monoclonal antibody for coating?? this might be another one of the reasons why your ELISA is too specific and not your western (considering u using polyclonals for western detection)
3) in youe sds-wb, the proteins are gonna migrate based on charge so they are not jumbled together so may be t is giving good positive singals but while u doing an ELISA all proteins are tiogether.. may be affecting the binding again... try to partially purify it and do an ELISA moight just help...
4) as far as your standard curve is concerned.. ya may be the titer is not optimum.. but if dint use to happen earlier then the prob might not be just it!!
Hope this helps...
Best Luck!!!

[vojera]

Hi! thanks so much for your reply! I'm using the same polyclonal Ab for both the western and Elisa because I couldn't buy an antibody commercially and had to have a custom one made (polyclonal was all I could afford!)

I definitely think you're right in saying that my protein isn't binding to the plate properly so I'm going to look into that. I ended up making an appointment to talk to one of the professors in the dept whose lab does loads of stuff like this, to see if he can offer me any advice. Here's hoping it goes well!

[Prep!]

Best luck then.. do write in with the conclusions of the discussions....

[Gerard]

"Could it be that the freeze drying has altered my protein somehow and that it makes no difference for Western because my samples are denatured anyway before loading?"
I don't think so, I've used freeze dried plantextract and even freeze dried after a ether extraction to remove fats and had never a problem.

From your question I can't see if your coating is with the plant extract or has to picked up by the coating. If the plant extract is used for coating and  you think it's possible that the target protein doesn't getting coated enough you can try another buffer then carbonate at pH 9 perhaps PBS at pH 7.4 or a citrate buffer at pH 5.8.


"My second question is, sometimes my standard curve is a bit backwards, as in the lower concentrations actually give a higher OD than the higher concentrations. Could this mean I haven't properly optimised the Ab concentrations for my ELISA?"
Could be, but another reason can be that other proteins from your extract deactivate your substrate and maybe another substrate as HRP-coupled could help.

[sgt4boston]

Just curious..are you using the same polyclonal for capture and detection in your elisa?  

It sounds like you adsorb the protein to the plate and then detect with your polyclonal-conj.  In this case, you might have so much non specific protein in your prep that the specific protein is not adsorbed because the surface is coated with the contaminate proteins.  Changing the pH or coating conditions will not help.  You will need purified protein for coating or do a sandwhich assay.

Freeze drying can affect your proteins they can be denatured unless you have optimized your lyophilization matrix and lyophilization cycle.  Can you run samples with/without freeze drying; also test the matrix you are using for the lyophilization. Usually the matrix will not affect binding unless you have surfactant or drastically change the pH of the matrix.

The backwards curve...higher signals at low end and lower signals at high end...known as "hook effect". Your dose response cuve can only have a certain range...you may be going beyond the range of the curve.

[vojera]

Hi!

I'm coating my plate with my extracts (and in the case of my std curve, my purified protein of interest) overnight, then washing, incubating with my Ab (polyclonal, raised in chicken), washing again, incubating with my secondary Ab (anti-chicken-alk phos) and then detecting. It's a protocol I got from a paper and I never realised I could do it another way! I’ll have a look into that sandwich assay, it may work better for me. If I understand correctly, in that case I would coat the plate with my primary Ab, then incubate with the samples, then wash, incubate with the secondary and detect?

Also, I can’t change to HRP because our lab is broke and I can’t afford a new detection system and antibody. It may be possible to borrow some from other labs if they’re feeling generous!

As for the hook effect problem, what is the best way to combat this? Should I narrow my curve? Or do a series of primary and secondary Ab dilutions and see which works best? Sorry for all the questions, I have so little experience with this work.

Thank you all for your answers!

[sgt4boston]

Your calibrators are purified.
Your samples are impure.
The antigen is high MW ? (confirm?)
Your goal is to develop a quantifiable test elisa.
You have polyclonal chicken ab specific for antigen
you have anti-chicken ap
you have substrate.

the sanwich assays require ab coated plates'/wells.  You may be able to use your polyclonal for both.  BUT. you can NOT use the anti-chicken AP.  You will have to label some of your chicken ab with AP yourself.  


Another approach...
label latex particles with your chicken antibody (adsorption).  you will not need your anti-chicken ap.  You will run a turbidimetric test.  The particles will agglutinate in presence of antigen.  The agglutination is quantifiable.  All you need is a spectrophotometer and inexpensive latex particles.   Check seradyn, polysciences, spherotech, varian, for simple protocols.

[sgt4boston]

After much thought I think the latex particle route is best for your application and budget.

[vojera]

Just in case anyone is looking through old topics and had the same problem as me, I finally sorted things out, so here is what I did, and it might help you.

It turned out I was coating my plates with far too much protein. I had been using the equivalent of 0.1mg dried tissue per well and now I'm using 0.005mg/well, a big difference! I loaded a plate with lower and lower dilutions of my sample and ran the elisa and, lo and behold, my abs started to jump when I went to those really low dilutions before dropping off again.

I then set up a checkerboard titration of my primary Ab with a low and high positive control (and a low and high negative control) and used the dilution that gave me the best absorbance (and correlation between the two concentrations) and the lowest background abs in my negatives.

Since I've had to significantly drop my standard curve in order for my protein of interest not to be competed out of binding to the plate, I have also solved my problem of my backwards curve, indicating that it was indeed the hook effect that was causing me problems.

It took a while but I feel that having sorted out this problem I'm better prepared for such problems in the future. Thanks to everyone for your advice!

[Prep!]

that s great vojera.. thanx for sharing the solution!!!
best luck!!