Dry up method as a revised Limulus test with a new technique for gelatin inhibitor removing.

To detect endotoxins, Limulus test, especially its tube method, is recently used most widely. But this method has shortcomings considerably, for example, the lack of the objectivity on the judgement, the necessity of long handling time and the requirement of relatively large amount of Limulus lysate. To revise these shortcomings we established a new method. In our method, sample and Limulus lysate are mixed on a silicone coated slide glass and incubated at 37 degrees C for 30 min, then heated to dry up for the judgement. In samples which contain protein, the pretreatments with (NH4)2SO4, dilution and boiling are performed to remove gelation inhibitor. It was proved that this method could be applied to such samples as physiological saline, plasma, urine, transudate, exudate, cerebrospinal fluid (CSF) and suspension of Escherichia coli (E. coli). This method has advantages in its (1) objectivity of the judgement because of the clear difference of the dry up patterns between positive reaction and negative, (2) shortness of the handling time (results can be obtained within 2 hr from sampling), (3) requirement of little amount of sample and of Limulus lysate (a fifth volume of sample and a tenth volume of lysate are needed compared with the conventional method) and (4) sensitivity (0.1 or 0.5 ng/ml of lipopolysaccharide (LPS)).