Test Procedure

Movie of Test Procedure (Flash Movie; link to file on homepage biocheck.de)

Common advice:

• Bring all reagents to room temperature.
• Proceed without interruption.
• Avoid drying of the membranes.
• Pipette reagents onto the gap not covered by the membranes.

Dogs / cats:

1. Strip shortly moisten with wash buffer and pat out on paper.
2. Coat membrane first with 250 µl starter solution and incubate 5 minutes. Pat out carefully on paper.
3. Add directly after moistening 200 µl serum in the gap beside the membrane. Incubate 60 minutes at room temperature under constant shaking at 30 rpm. Tip the serum and wash the membranes three times with 1 ml wash solution (about 1 ml Wash buffer is a filled strip-gap); pat out on paper.
4. Add 250 µl wash buffer in the gap beside the membrane. Incubate 5 minutes at room temperature under constant shaking at 30 rpm. Wash membrane as described in step 2.
5. Add 250 µl detection antibody in the gap beside the membrane. Incubate 90 minutes at room temperature under constant shaking at 30 rpm. Wash membrane as described in step 2.
6. Add 250 µl Streptavidin conjugate in the gap beside the membrane. Incubate 20 minutes at room temperature under constant shaking at 30 rpm. Wash membrane as described in step 2.
7. Add 250 µl substrate solution (BCIP/NBT) in the gap beside the membranes. Incubate in the dark (i.e. cover with aluminium foil or opaque cover) 20 minutes at room temperature, under constant shaking 30 rpm. Wash membrane as described in step 2.
   (Time over all 195 minutes)
8. Let membrane dry sufficiently and analyse strip.

Horses:

1. Strip shortly moisten with wash buffer and pat out on paper.
2. Coat membrane first with 250 µl starter solution and incubate 5 minutes. Pat out carefully on paper.
3. Add directly after moistening 250 µl serum in the gap beside the membrane. Incubate 60 minutes at room temperature under constant shaking at 30 rpm (tilting shaker). Tip the serum and wash the membranes three times with 1 ml wash solution (about 1 ml Wash buffer is a filled strip-gap) ; pat out on paper.
4. Add 250 µl wash buffer in the gap beside the membrane. Incubate 5 minutes at room temperature under constant shaking at 30 rpm. Wash membrane as described in step 2.
5. Add 250 µl 1 st detection antibody in the gap beside the membrane. Incubate 90 minutes at room temperature under constant shaking at 30 rpm. Wash membrane as described in step 2.
6. Add 250 µl 2 nd detection antibody in the gap beside the membrane. Incubate 60 minutes at room temperature under constant shaking at 30 rpm. Wash membrane as described in step 2.
7. Add 250 µl Streptavidin conjugate in the gap beside the membrane. Incubate 20 minutes at room temperature under constant shaking at 30 rpm. Wash membrane as described in step 2.
8. Add 250 µl substrate solution (BCIP/NBT) in the gap beside the membrane. Incubate in the dark (i.e. cover with aluminium foil or opaque cover) 20 minutes at room temperature, under constant shaking 30 rpm. Wash membrane as described in step 2.
   (Time over all 290 minutes)
9. Let membrane dry sufficiently and analyse strip.