Plant Viruses in ELISA

[Babybel56]

I have an ELISA plot of an infected woody plant (apple) and an infected herbaceous one (tobacco - sorry no more specific than that!). Infected sap from both has been mixed with a PBS-T buffer put on an ELISA tray to let the alkaline phosphatase cause a colour change.
The graph has absorbency at 405nm plotted against the dilution of the sap between arbitrary factors 1-4, and both lines follow a downward curve as you'd expect (as the dilution of the virus-containing sap gets greater its ability to activate the enzyme and cause colour change is diminished).

HOWEVER between dilution factor 1 and 2 for the apple there is an increase in enzyme activity (and so viral presence) before the normal curve is resumed. Is this something to do with it being a woody plant?

[JackBean]

I would rather suspect some inhibition of the phosphatase from the sap. How was it treated before the phosphatase was added?
(did you add anything, dilute into buffer, purify through some column?)

[Babybel56]

Leaf material was simply ground using calcite and coarse carborundum and the supernatant liquid removed, mixed with the buffer and put in the ELISA wells. Woody plants contain a greater concentrations of inhibitors don't they? I just can't figure out why that would mean a greater virus activity :s

[JackBean]

not really higher virus activity. You mix that extract with some antibody and measure the alkaline phosphatase activity, rigth?

[Babybel56]

Broadly speaking. A greater concentration of colour suggests higher concentration of viral antigen's doesn't it?

[JackBean]

yes, higher concentration of the phosphatase product.

However, the extract is coloured, rigth? It contains lots of balast compounds, which probably interfere with your assay, thus if you apply high amount of it, you inhibit the phosphatase reaction and you get actually lower activity. I've had something similar in my work. When I measure activity of my enzyme in crude extract, the more I add, the lower activity I actually obtain. Nevertheless, when I purify it, it is better and the activity grows with enzyme amount added.

[Babybel56]

I understand where you're going: so inhibitors (whether by design or just getting in the way) are dropping antigen/antibody complex formation for the woody plant.

But this doesn't quite explain why the colour gets higher at first and then drops (with increasing dilution, not over time). Is there an optimum concentration for the inhibitors to work at or something?

Btw I can't tell you how grateful I am for this! Hours of my life have been whiled away trying to figure this out.

[JackBean]

wait a second, I thought, that the problem is, that the most concentrated sample gave lower response than more diluted one...?

[JackBean]

that it was like ^\ (up and then down:)

[Babybel56]

Okay sorry my bad for being unclear! I have infected sap samples from two plants: an apple tree and a tobacco plant. The tobacco colour decreases with dilution as you'd expect, but the apple has an initial increase in colour before it starts to decrease in sync with the tobacco.

[JackBean]

yes, I think I understood it that way. It agrees with what I said.

[Babybel56]

Oh god yes sorry! No I see what's going on - cheers!

*starts rushing to finish the report*