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#180587 Testing recombinant proteins through indirect ELISA for aspergilloma diagnosis

Posted by Yamafx on 31 July 2018 - 03:26 AM in ELISA and Immunoassay



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,





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