Endotoxins chromogenic methods

Harada, T. Morita, T. Iwanaga, S. Nakamura, S. Niwa, M. A new chromogenic substrate method for the assay of bacterial endotoxins using Limulus hemocyte lysate. In Biomedical Applications of the Horseshoe Crab (Limulidae) Cohen, E., Ed. Alan R. Liss, Inc. New York, 1979 209-220. 

The samples of products are incubated with Limulus amoebocyte lysate at 37°C. If endotoxins are present a solid gel forms, indicating the presence of endotoxins. The British Pharmacopoeia (2002) describes six separate methodologies for the test for endotoxin. These are (A) gel-clot limit test (B) gel-clot semi quantitative (C) turbidimetric kinetic method (D) chromogenic kinetic method (E) chro-mogenic end-point method and (F) turbidimetric end-point method. There are checks for interfering factors. Any validated method may be used, but the gel-clot method is the referee test in the case of dispute. Coloured products cannot be tested by... 

A number of studies have investigated interferences with the LAL assay and have attempted to optimize the assay (Douwes et al., 1995 Hollander et al., 1993 Thorne et al., 1997). These studies have demonstrated that results may vary depending upon the sample matrix, the extraction method and the assay method. Other constituents present in the sample may interfere with the LAL assay and cause inhibition or enhancement of the test or aggregation and adsorption of endotoxins, resulting in under- or over-estimation of the concentration. Techniques such as spiking with known quantities of purified endotoxin and analysis of dilution series of the same sample have been described to deal with these interferences (Hollander et al., 1993 Milton et al., 1990 Milton et al., 1992 Whitakker, 1988). Studies in the laboratories of the authors of this chapter have demonstrated within-Iaboratory coefficients of variation between 15 % and 20 % in routine assay work. When extra care is taken to optimize precision this can be reduced to under 5 % in the endpoint chromogenic assay (Thorne, unpublished data). Several interlaboratory comparison studies have been performed and demonstrate much greater variability. One in-depth comparison of two laboratories experienced in the LAL assay. 

The limulus amoebocyte lysate (LAL) test with chromogenic substrate is faster than the gelation method, and it can be automated. The chromogenic substrate is attached to p-nitroaniline, that is released when reacted with the endotoxin-activated enzymes. The free p-aniline is read at 405 nm. 

Generic ELISA method for measuring immunoglobulins and other analytes 175 Analysis of endotoxin by chromogenic LAL 178... 

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