new to ELISA Immunoassay - Flat standard curve suggestions to improve my assay please (Oct/12/2010 )

Hi there,
I am new to the ELISA assays and am trying to set it up for the past few days and have so far not been very succesful. I am basically getting a flat standard curve and the the negative control is as high a reading as the highest concentration of sample!

I am using maxisorp plates from nunclon.
Applying the coating antibody (Beta-tubulin (anti-rabbit) in 5% BSA for 2 hours at RT (0.4ng/ml)
Wash in PBST 3X
Coating with the protein IN 5% BSA (whole cell extracts) overnight at 4degrees titrated 32ug to 160ng
Wash 3X n PBST
Blocking in 5% BSA RT for 1hr
Applying the 2nd primary antibbody (in 5%BSA) for post translationally modified tubulin (anti-mouse) (1ug/ml)1hr RT
wash 3x PBST
2o mouse 1:10,000 (5%BSA)1hr RT
3 wash PBST
ABTS reagent for 20 minutes
The stopping reagent I have is 1% SDS if required
Read at 405nm

I have been told that this assay is very straight forward but so far I have not found it so!

When performing this assay i dont seem to get much of a colour change (only slightly so I know my reaction doesnt seem to be working properly) The colour change appears to be the same no matter how much potein present in the sample even in 0.

I have also tried this assay with the capture assay and get a similar looking standard curve. Is it that I need to block after adding the initial capture antibody?
Is it that I need to coat the wells. I am using a M9410-1CS plates fom nunclon and are supposed to be maxi sorp so......

If anyone could help me I would really appreciate it,

Cheers,
a really lost PhD student

I think you should try different concentrations of your coating antibody, 0.4ng/ml sounds too low for me, I used to do 1ug/ml. Also, I used to coat overnight in the fridge.

More important, you should block after coating before adding your sample (protein) so that non-bound sites in the plate are blocked, otherwise your protein could be sticking to the plate directly specially as they are maxisorp plates.
There's no need to block between the protein and the secondary antibody.

Hope this helps.

hello
I read this:
Applying the coating antibody (Beta-tubulin (anti-rabbit) in 5% BSA for 2 hours at RT (0.4ng/ml)
Wash in PBST 3X
Coating with the protein IN 5% BSA (whole cell extracts) overnight at 4degrees titrated 32ug to 160ng
Wash 3X n PBST

Your antibody is in 5%BSA and you are coating with 5%BSA. Your antibody should NOT be in a bsa solution. It should be purified in aqueous buffer. Your coating protein is then in 5% BSA as well. this also should NOT contain BSA but just be buffer such as PBS, TRIS, Carbonate, etc.

Basically you have been coating BSA instead of your antibody. BSA is only used to:
1. block the plates after ab is adsorbed
2. as carrier protein for conjugates
3. as carrier protein for calibrators
4. in wash solutions

Hope this helps!

Well spotte sgt

Definitely DO NOT use BSA for coating. As sgt said you've been coating your plates with BSA.