From Western To ELISA - HELP!? - Are my parameters acceptable for use in ELISA? (Oct/11/2008 )

Dear Reader,

I have a C-terminal polyclonal antibody raised in rabbit which is highly specific to my ~100kDa protein of interest. The antibody works on the denatured protein, I assume it also works on natively conformed protein but I have only done denaturing PAGE and Westerns with it.

Is this a potential problem?

I also have access to an N-terminal polyclonal antibody AND purified protein for a standard.

I do protein extraction from leaf-tissue using an SDS-based buffer. Someone told me recently this would be a problem for ELISA. They also told me I would have to use a Triton-based (non-ionic) buffer for extracting protein for use in ELISA. Is this true? I am told (by our collaborator) that our protein is not very soluble in Triton.

Can I use my current cellular extract in Sandwich-ELISA?

Also, which antibodies should I use of my C-terminal AND N-terminal polyclonal's? Does it matter?

Also, my collaborator said she was worried about "possible cross-reacting proteins giving a false signal"? Is this a problem if I have 2-different antibodies? How do I determine if this will be a problem? When I perform PAGE and Western Blot I occasionally get non-target sized bands but only at very high expression levels and I always assumed it was degredation/assembly products.

Sorry for the many, many questions, but the closer I get to deciding to try ELISA the more questions it seems I have.

Cheers,

M

The terminal portion of the proteins are usually random coiled, so abs anti C or N terminals have good probability to recognise both the denatured protein or that naturally conformed. On the other hand, sometimes the aminoacid composition of the antigen and the surrounding aminoacid environment could affect the interaction ag-ab because of either steric obstruction or strong influence on the terminal part conformation.
SDS-based buffer can really be a problem in ELISA, since SDS destabilises or completely denature (according to the concentration) proteins, thus it can affect abs conformation, which is essential for antigen recognition. The use of triton should create less troubles, but too high concentrations might influence ag-abs interaction as well.
For your sandwich ELISA you could try both abs (anti C and N terminal) as capturing abs and the other as primary abs. Of course one of the two possibilities might work better, but you cannot know until you try. The problem is that if your 2 abs were both raised in rabbit you have to conjugate one of them to a revealing enzime (usually AP or HRP), while if your abs anti-N terminal were not raised in rabbit, you can buy secondary abs without being compelled to perform conjugation. For the false signal, the fact you have two different abs is very good, since, with the use of them together in a sandwich, you can strongly increase the specificity of the assay. Try and test also the abs anti N terminal in WB. To be quite sure you do not have any false signal in ELISA you should use as negative control an extract from a leaf that does not not express your target protein.
good luck

Thank you so much for all the very useful advice. I'm not sure (yet) what my C-terminal was raised on. The N-terminal comes from Rabbit, so it is probably rabbit. Let's say it is not... I already have Rabbit Anti-body, I would then hope to capture with the C-terminal and sandwich with the N-terminal which I would probe with the anti-Rabbit secondary?

This raises three additional questions for me:

1. If the anti-bodies are both from Rabbit, is conjugation to an enzyme a simple process?
2. Is it possible and will it affect my signal if I were to do something like TCA-prep to concentrate my protein down and then put it into Triton? Is there another way?
3. If I am to take fresh plant samples for use in ELISA, what would be an alternative to SDS and DTT for extracting protein?

Thanks again for the good words so far!

Peace,

M

the process of conjugation is not difficult, but it is highly recommended to have solutions of abs that are as pure as possible. Some companies, such as pierce, sell activated HRP, AP, biotin which directly react with other molecules, abs included (in this case one of the most used conjugation method is that mediated by NHS, which reacts with aminogroups on the target protein). If you choose biotin (I recommend it, even though in the last post I forgot to mention), you will have to buy also extravidin HRP or AP conjugated, which is relatively cheap.
About the extraction methods, what I can tell you is that any molecule that influences protein folding is bad for this kind of test. I'm not experienced in the plant field, so I cannot propose any particular extraction methods. I think those using non-ionic detergent are to be preferred. Is your protein a membrane protein or a quite hydrophobic one? If not you can use very mild condition for its extraction, which may be compatible with ELISA. What about the cell wall? Do you need to use particular method to get rid of it or to disrupt it? An alternative to TCA-prep to concentrate your protein might be the use of a cut-off system (tube with molecular filter that allows to concentrate a solution of protein above a certain MW, after centrifugation), otherwise a dialysis in a solution with triton would be recommended, but...you risk to lose part of the protein. Is your protein so low concentrated? IF you can detect it by WB I think you can easily see it by ELISA, without preconcentration steps!

Both SDS and DTT may well wreck your protein (SDS denatures proteins and DTT breaks disulfide bonds). What exactly is the prupose of the ELISA? Are you trying to quantify the protein or to check whether it is present or not? And if you are quantifying, is the absolute concentration necessary, or just a relative concentration - i.e. is there more or less of protein X in this plant than the others? If you are just trying to prove tre presence of the protein or the relative concentration, you might just take the easy way out and extract it by physical means - i.e. mortar and pestle or homogenizer. Then you can use less harsh solvents. Obtaining the absolute concentration this way might be a bit tricky though...