ELISA for antibody titre using Insoluble recombinant protein - Newbie question (Oct/26/2008 )

Hi all,

I am totally novice to ELISAs.

I am trying to figure out how to coat my plates with an insoluble protein purified from bacteria, and acquire antibody titre data from this.

It appears that it is possible to solubilize the protein in 2%SDS and then coat, but I also read somewhere that Triton might be better for such purpose?..

I would really appreciate other's expert input on this as well as example protocols..

Also, may I ask how much protein should I be coating?

Thank you so much in advance

Jerry

Protein should be in solution for coating purposes. Coat at pH slightly higher than isoelectric point of the protein. 100 - 200 ul per well. Coatings I have done are usually without surfactant...maybe someone out there can comment on this. If your protein concentration is high enough your surfactant may be diluted out and not play a role in the coating. 100 ug of protein per well would be a good starting point. You can titer your coating protein. You will need to block exposed well surfaces with BSA or equivalent. Basic steps:

1. Coat with protein 100 - 150 ul/well, Titer protein coating. Make control with different protein coating (ie BSA or other bacteria protein)
2. Incubate RT (30 min - 2 hr) or 37C 15 min +
3. Decant
4. Wash (BSA/pbs etc)
5. Block (fill well, 300 ul) You can add sucrose in the blocking buffer to make your plates stable after drying.
6. Incubate RT or 37C
7. Decant
8. Dry inverted
9. Store sealed 4C

Next experiment
Test for ab reaction with coating protein v. non-specific control
1. Add ab/buffer or sera to wells (50 ul)
2. Incubate 30 - 2 hr
3. Wash 3-4X BSA/PBS
4. Add conjugate anti-ab; will have to titer concentration of the conjugate
5. Incubate
6. Wash
7. Substrate
8. Incubate
9. Stop solution
10. Read