Protective antigen (PA) analysis
toxemia
SEPARATION, TOXEMIA
Following separation, 80 μL of serum was aliquoted into one vial for toxin analysis by electrochemiluminescence (ECL) immunoassay. Vials were pre-loaded with 10 μL of Protease Inhibitor Cocktail Set III from Calbiochem (Catalogue #539134) and stored at-20°C. Just prior to use, the vials were thawed and 80 μL of serum and 10 μL of 0.2M EDTA (Fisher Catalogue #S311-100) were added sequentially to the vial. Vials were then closed and specimens vortexed for 5 seconds. Specimens were tested using a real-time ECL assay (up to treatment) or were stored at ≤-60°C until assays were performed (post-treatment/ infusion). All frozen, stored specimens were batch-tested.For both rabbits and NHPs serum specimens were analyzed in real-time according to the collection schedule to identify the onset of toxemia for initiation of treatment. Specimens from the untreated control animals were also tested in real-time if corresponding specimens from the treatment groups were available at that time point. Prior to infusion (PrTI) and post-infusion (PoI) specimens from the treatment groups and specimens from the untreated control group that did not coincide with specimens being analyzed in real-time were preserved frozen and batch-tested at a later time.