Hello,
I am testing several recombinant proteins (resuspended in PBS or His 50 mM) to diagnose Aspergillus with human serum with indirect ELISA.
I use PLATELIA commercial kit as a golden standard. I use one strong positive serum (250 UA/ml) and one negative serum (-4 UA/ml) from two patient as positive and negative control respectively.
The in-house ELISA protocol is classical (in duplicate and with and without protein for noise):
- Coating in carbonate buffer overnight (I test 50 ng/well and 250 ng of protein) +4°C, then 1 hour 37°C
- 3 washing PBS 0.05% Tween 20
- Blocking PBS 0.05% Tween 20 + 1% BSA
- 3 washing PBS 0.05% Tween 20
- Serum (I only used the positive and negative control for set up) diluted in PBS 0.05% Tween 20 + 1% BSA (1/100, 1/500, 1/1000)
- 5 washing PBS 0.05% Tween 20
- Conjugate (Specific Fc IgG Jackson) diluted in PBS 1% BSA (1/1000, 1/3000)
- 5 washing PBS 0.05% Tween 20
- Citrate buffer + OPD + H2O2; 10 minutes
- Stop with H2SO4 4%
- Spectro 490 nm
I tested 3 proteins for the moment and each time the OD of the positive and negative control are very close (around 1,5). Often the C+ OD < C- OD.
The noise is low (OD 0.060). I tested without serum and there is no non-specific reaction (OD 0.060).
The likely conclusion is that the tested prot are not specific to Aspergillus, but since I am not very confident I would like to have a confirmation from an expert.
I join one file for the prot Gel1.
Thank you for your help,
Yannick
Attached Files
-
ELISA_Gel1.xlsx 17.37KB
88 downloads
