What you would like to detect? The specific antibodies in a serum sample?

1. forget the Maxisorp plates, all other plates are working as well and in most cases the background is lower.

2. Coating: 100µl is fine, but incubate overnight at 4 °C in your carbonate buffer. Cover the stack of plates with an empty plate. but: use 2 µg/mL only, not 2 mg!!!!!!!!

3. Washing is fine but not before using. If you would like to store the coated plates, remove as much from the coating solution and store the plates bottom up frozen.

4. Blocking: forget this, blocking is required in commercial assays only. Your plate has a binding capacity of about 100 ng/cm², so you are coating with an excess of protein anyhow. If you need really a blocking, make sure that there are no other proteins in the blocking buffer reacting with your coating protein. Beware only: fetuin is a very sticky protein.

5. Sample (serum/plasma containing the specific antibodies????) incubation buffer: use your washing buffer add NaCl to a concentration of 0.3 mol/L (if necessary you can use up to 0.5 mol/L) This doubled NaCl-concentration will l reduce unspecific binding. Add an inert protein like BSA at a concentration of 1 - 3%. Add phenol red (a pitch) only to get a slightly colored buffer. So will see much better where you are pipetting. Incubate at room temperature. Anything else will lead to edge-effects due to temperature / himidity grafdients over the plate.

Use an empty plate to prepare your samples and standards & controls. Don't do the dilution of those in the coated plates. You will get scratches at your coated surface and you will have different incubation times for the different wells. If I would like to run a 100 µL-assay, I used 150 µL - 250 µL as volume for the sample preparation.

6. Washing is fine.

and now I've a issue: just now the anti-pertussis-antibody?

7. conjugate incubation (anti-species-IgG-Antibody-HRP labelled (anti-human-IgG-HRP if you would like to detect human antibodies, Protein-A/G-HRP if you like to detect antibodies from rodents....) Your polyclonal anti NIBSC Bordetella pertussis PT serum (sheep) can be used as standard, but due to the fact that it is from sheep, you need to have an anti-Sheep-IgG-HRP. This requires also that all your samples are from sheeps?

8. Washing

9. substrate reaction: TMB, H2O2 (use an ready to use solution: http://www.seramun.com/products/substrates/horseradish-peroxidase.html

12. Color development: Incubate for 20 min at room temperature.

13. Stop: sure you can kill the peroxidase by adding that much H2O2, but a 1 mol/L H2SO4 is much easier and cheaper.

14. Read at 450 nm is fine.

Have a short lock for the ELISA schemes in the attached paper ... and send me an information via RG.

Article Enzyme Immunoassay Techniques. An Overview

Thank you very much for your help Stephan Kiessig, what we are doing is trying to quantify fetuin, but we are having problems with background. Regards

Sorry Stephan Kiessig what we want to detect is pertussis toxin, no fetuin, fetuin is used only to covering.

Than you still can stick with a couple of points:

5. Sample incubation buffer: as describes above

7. anti-pertussis-IgG incubation: a normal concentration for such an antibody is about 1 µg/ml if purified or monoclonal. Here you have to check for an optimal dilution by checkerboard titration.

8. Washing

9. substrate reaction: see above

12. Color development: see above

13. Stop: 1 mol/L H2SO4.

14. Read at 450 nm is fine.

some alternatives:

optimize the coating concentration. you are about 1000 time to high! checkerboard titration.

use for coating a monoclonal antibody vs. pertussis toxin line in a classical two site binding assay.