Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States
- Prepare endotoxin standard curve. Take 100 µl of 12 EU/ml endotoxin up to 1000 for 1.2 EU/ml. Place in wells of an Immulon 2 (Dynatech; ELISA plate) the following:
- Use endotoxin-free (‘LAL Reagent’) water for all dilutions. Charles River provides endotoxin at 12 EU/ml (after reconstitution with 1.5 ml ‘LAL Reagent’ water. Vortex to reconstitute and vortex immediately before use. Store at 4ºC for up to 2 wks after reconstitution (should test to see if we can freeze aliquots).*
- Prepare test samples (µl of 1 nM lacritin [= 12.3 ng/ml of pRB2] or 1 nM lacritin fragment, or of current working concentration):
- Incubate in 37°C incubator for 5 min.
- Add 50 µl of ‘LAL Solution’ and mix on rotating platform in 37°C incubator for 18 minutes.
- Add 100 µl of ‘Substrate-Buffer Solution’ mix on rotating platform in 37°C incubator for 8 minutes.
- Read on plate reader at 405nm. Look at analyzed graph to get EU values for test samples and see standard curve. Divide by volume applied.
This method is based, with permission, on an original protocol available here.