A rapid method to produce a sensitive Limulus amoebocyte lysate (LAL). I. Evaluation of inter and intra batch differences in LAL and hemolymph from Limulus polyphemus.

Abstract

An improved method for preparing Limulus amoebocyte lysate (LAL) is described and compared with two other well-known procedures. In five Limuli, three different bleeding procedures and three different cell rupture methods were studied. Increased sensitivity of LAL was accomplished by avoidance of the anticoagulant N-ethylmaleimid (NEM) in the bleeding procedure, optimal methods for mechanical cell rupture and use of pyrogen free conditions. Reaction of LAL with as little as 10(-15) gram Lipopolysaccharide (LPS) per ml was generally obtained. To evaluate a possible explanation for the differences in reactivity of LAL between different Limuli, found in one experiment, six Limuli were bled, and the pH and the concentration of protein, Ca2+, Mg2+, Cu2+, Na+ and K+ were determined in LAL and cell-free hemolymph. Inter and intra batch variations were found in LAL, but there was no correlation between the sensitivity of LAL and the content of the above mentioned constituents in LAL and cell-free hemolymph. Experiments with use of NEM in the bleeding procedure and addition of NEM to homogenized cells in different concentrations, showed that NEM inhibits the reactivity of LAL to LPS. It is concluded that the modified method of producing LAL by bleeding without NEM and by using optimal methods for mechanical cell rupture is quick, simple and produces a very sensitive reagent for the Limulus test.