Dear Flow-ers I have a question about in vivo use of a monoclonal and the use of Flow to study its attachment to the target cells. Several months ago we used an anti-CD18 monoclonal antibody (mouse IgG1 raised from clone BAQ30A) to study reperfusion injury in a pig model of ischemia/reperfusion. At that time the monoclonal was raised in ascites. To monitor its activity we took blood samples at different intervals after administration (1.5mg/Kg) and found that we could detect, by FACS, mouse IgG1 (we assume the monoclonal) on the surface of circulating neutrophils. The signal was apparent within 2 minutes of administration and remained bright for 24-48hours. A few weeks ago we tried a similar experiment (dosing at 5mg/Kg), this time the monoclonal was raised in a bioreactor. We found that it was contaminated with about 100EU of endotoxin/ml, so the experiments were stopped until we cleaned up the material. After several tries we got the concentration of endotoxin to 20-30EU/ml, still high but we though that it could be manageable if we also lowered the dose of material to 3mg/Kg. We just finished doing 3 additional animals and found that: 1- The monoclonal was not protective (it had been when we first did it) 2- The neutrophils did not seem to light up until well pass 2 hrs post dosing (in previous experiments we saw a signal within 2 minutes) Has anyone in this conference had any experience with endotoxin Does it change the in vivo activity of a monoclonal?(in vitro the monoclonal inhibited the adhesion of pig PMNs to endothelial cells). Does the nature of the monoclonal change with its source (ascites vs bioreactor)? Any suggestions will be very much appreciated. Ed Cabrera Procter and Gamble Pharmaceuticals Mason, OH
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