Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

PrPc quantification by ELISA

We want to study the influence of dimethoate on total PrP protein content in infected and non infected cells. We choose a simple sandwich ELISA test which can be easily performed in a P3 laboratory. Only the 96well plate is read on a spectrophotometer, and this plate can be sealed and disinfected before going out of the P3 laboratory.

Technical characteristics:
Our ELISA is realised with the following antibodies, given by J Grassi (CEA Saclay, France): Monoclonal mouse anti PrP SAF61, and biotinylated 3F4. Revelation is done with streptavidin-HRP conjugate (Amersham) and TMB substrate. Standard curve is done using recombinant PrP obtained by RTS system (Roche). Detection threshold is at 1ng/ml of PrP with a background signal of 0,4 OD(450nm), plateau is around 3,5 OD (450nm) with 50ng/ml.

Detailed protocol
- Maxisorb 96-wells plate are coated 30min at 37°C with SAF61 at 10µg/ml in PBS, saturated with PBS-1%BSA one hour at 37°C and washed 5 times with PBS-0.1%Tween20.

- Samples (recombinant PrP standard, 50µg of protein from PrP knocked out mouse brain as negative control, or 50µg of protein sample from cell culture extracted with 1%DOC, 1%Triton X-100 buffer) are diluted in 50µl of PBS-1%BSA-0.1%Tween20-0.1%Triton X-100 and incubated one hour at 37°C.

- After 5 washes as above, 50µl of biotinylated 4F2 at 2.5µg/ml in PBS-1%BSA-0.1%Tween20 is incubated 2 hours at 37°C and washed again.

- HRP-stretavidin (Amersham ref RPN1051) is diluted 1/1600 and 50µl is incubated in each well one hour at 37°C.

- Three washes in PBS-0.1%Tween20 and two with PBS are followed by 100µl of TMB substrate and incubation of 25minutes at 37°C

- Reaction is stopped by 100µl H2SO4 0.5M and absorbance is read at 450nm.

Informazioni correlate

Reported by

Université Joseph Fourrier
Faculté Médecine & Pharmacie, Domaine de la Merci
38700 La Tronche
France