Carcinoscorpius rotunda cauda amoebocyte lysate for detection of endotoxins--its preparation, stability, sensitivity...

Carcinoscorpius amoebocyte lysate (CAL) was prepared from C. rotunda cauda by a modification of the method described by Mahalanabis et al. [Indian J Med Res, 70 (1979) 35]. Seasonal variation as well as batch variation was observed in the yield of haemolymph and the total lysate protein. In the presence of E. coli lipopolysaccharide (pure, free endotoxin) and E. coli and Salmonella cell suspensions (bound endotoxin), the CAL formed a gel after incubation at 37 degrees C. The gelling time varied from 10-90 min depending on the concentration of endotoxin used; higher concentrations formed gel more rapidly. The endotoxin detection capacity (sensitivity) of the lysate preparations was influenced by the season in which prepared, but not by the total protein content. Ten fold increase in the sensitivity was achieved by a purification step using chloroform. Although subsequent frozen storage with or without lyophilization did not alter the initial sensitivity, it was either decreased considerably or lost totally when the lysate was stored for 4 months at 4 degrees C or for 2 months at 30 degrees C. Under the same conditions, Limulus lysate was more stable. The lost sensitivity could not be regained by the incorporation of divalent cations (Ca2+ and Mg2+). The CAL preparations in general were able to detect as little as 10-100 pg of endotoxin or as few as 10(3) cells of E. coli or 10(4) cells of Salmonella and were comparable to LAL. CAL could be used successfully in lieu of Limulus amoebocyte lysate in the detection and assay of endotoxins.