3 replies to this topic

[vojera]

Hi!

I'm setting up an ELISA to look at antibody titres in mouse serum and I've never done this before (nor has anyone in my lab as we're a plant lab).

This is the background:
I have tobacco plants expressing a bacterial antigen. I want to see if that antigen can elicit an immune response. So I have five groups of mice as follows:

1. Mice gavaged with soluble fraction of tobacco extract (containing 100ug of the antigen) and CTB as an adjuvant
2. Mice gavaged with soluble fraction of wild-type tobacco extract and CTB (this is my negative control)
3. As group 1, with a subsequent sc boost of purified antigen (with alum)
4. Mice injected with purified antigen (with alum)
5. Mice injected with purified antigen (with alum) (with a few weeks' gap).

I have collected serum and BAL from these mice, but for now I just want to see the levels of antigen-specific IgG1 and IgG2a in the serum. (Later I will check the IgA in the BAL).


I know roughly how to do an ELISA (I've used it to determine how much of my antigen I have in my tobacco extracts), but I'm not sure on a few things.

1. Do I still need to leave a blank on the plate with just PBS? Or do I count my negative (i.e. wild-type sample) as my blank?
2. Do people find it better to leave the serum on the plate for 2 hours at rt or overnight at 4C?
3. Is PBST with 5% milk powder an adequate block?
4. When I get my results, how do I interpret them? How do I go from a page of absorbance values to a graph?

Sorry if I haven't been very clear, this is a bit confusing to me.

Thanks for any advice you can give me.

[K.B.]

1. It costs you nothing to include blank with PBS and you get information how high really is the background from the negative control.
2. When I was working with rabbit serum or ammonium sulphate purified antibodies and plant proteins (MW ~40-70kDa) I tried both and haven't noticed any difference so I used one that was much more convenient for me.
3. Yes.
4. The most simple way is to assume that positive = 100%, negative = 0%, and use linear regression. Although when you make a dilution series they quite often follow sigmoidal curve and that requires more specialized software to manipulate.

[vojera]

K.B., on 14 September 2010 - 05:34 AM, said:

1. It costs you nothing to include blank with PBS and you get information how high really is the background from the negative control.
2. When I was working with rabbit serum or ammonium sulphate purified antibodies and plant proteins (MW ~40-70kDa) I tried both and haven't noticed any difference so I used one that was much more convenient for me.
3. Yes.
4. The most simple way is to assume that positive = 100%, negative = 0%, and use linear regression. Although when you make a dilution series they quite often follow sigmoidal curve and that requires more specialized software to manipulate.

Thanks for your feedback. I think I'll have to check out one of the immunology labs in the department to get help with software if that is the case.

[sgt4boston]

Table Curve (from Systat.com) is an inexpensive product that will suite your requirements. For Data analysis you can use Med Calc for a limited number of times without purchase.