Conventional limulus amebocyte lysate tests involved procedures to prevent contamination by atmospheric endotoxins. To address this problem, the authors have proposed a technique in which the sampling, reagent mixing, and reaction steps are carried out consecutively in a single tube. Since reagents do not come in contact with the atmosphere, the new technique promises stable determination of the concentration of endotoxins in dialysate fluid. An aqueous solution of endotoxin simulating dialysate fluid was sampled in a silicone rubber tube from a sterile infusion bag, then mixed with an indicator in the same tube. After reaction at 310 K, measurements were made of light absorbance at 405 nm and its linearity with endotoxin concentration was determined. Results showed a high degree of linearity (correlation coefficient of not less than 0.99) at endotoxin concentrations of 0-15 pg/ml. The time for the reaction was shortened to 12 min, in which case the response time was 15 min. It is suggested that this new test for determining endotoxin concentration using limulus amebocyte lysate reagent, in which all three steps - sampling, mixing, and reaction - proceed continuously in a single tube, offers higher reliability, greater ease of operation, and shorter response time than conventional tests.
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