2 replies to this topic

[LoverOfWorms]

Hello fellow pioneers of the natural world!

I am currently making some MAb's to a protein mixture I've concocted (and affectionately termed wormspit), and I'm going through a great deal of antigen coating all of these ELISA plates to screen for antibody-producing hybridomas (which sucks - worms don't spit a lot).  As I've done some work with Westerns before, I was thinking about the possibility of stripping ELISA plates like stripping western blots.  Has anybody done this before?  If so, give me a shout and let me know how it turned out.  

If I don't hear anything from my esteemed fellow forum users, I will perform the following experiment to test whether I can get it to work:
1. Perform standard ELISA (the ones I've been doing), making sure some wells "light up"
2. Add stripping solution (the 2-mercaptoethanol kind) to each used well, incubate at 50 °C for 1 hour
3. Add detection reagent, see if anything "lights up"
4. Reprobe using same methodology as step 1, see if anything "lights up"

Hopefully this works - I'll post the results once I get them unless you guys dissuade me in the meantime

Cheers,

LoverOfWorms

[bob1]

I think the problem will be that the stripping will remove the antibody from the plate (weakish interaction) rather than the protein from the antibody (strong interaction) as the stripping of westerns is basically destroying the antibody and phosphatase/peroxidase as far as I can tell.  It also removes some of the protein on the blot.  

You could give it a go with non-critical samples and see if it works.

[klinmed]

LoverOfWorms, on Jul 3 2010, 12:16 AM, said:

Hello fellow pioneers of the natural world!

I am currently making some MAb's to a protein mixture I've concocted (and affectionately termed wormspit), and I'm going through a great deal of antigen coating all of these ELISA plates to screen for antibody-producing hybridomas (which sucks - worms don't spit a lot).  As I've done some work with Westerns before, I was thinking about the possibility of stripping ELISA plates like stripping western blots.  Has anybody done this before?  If so, give me a shout and let me know how it turned out.  

If I don't hear anything from my esteemed fellow forum users, I will perform the following experiment to test whether I can get it to work:
1. Perform standard ELISA (the ones I've been doing), making sure some wells "light up"
2. Add stripping solution (the 2-mercaptoethanol kind) to each used well, incubate at 50 °C for 1 hour
3. Add detection reagent, see if anything "lights up"
4. Reprobe using same methodology as step 1, see if anything "lights up"

Hopefully this works - I'll post the results once I get them unless you guys dissuade me in the meantime

Cheers,

LoverOfWorms

I´m pretty sure your method will not work and you will just remove your Ag from the plate. Even if you do succeed in stripping some of the Abs, I think you will experience significant background problems.

Why not reverse your assay.

1) Label your "worm spit" with biotin. You need very little protein to do this. Should work with crude preps.

2) Coat plates with goat anti-mouse immunoglobulin (0.3 ug/well) , incubate, wash.

3) Add Mabs, incubate, wash.

4) Add biotinylated "worm spit" (need to titrate to find optimal amount, but will be very small), incubate, wash.

5) Detect with a streptavidin-HRP tracer.

Hope this helps.