Nithiananthan, Ahilan; (1992) New techniques for the detection of the limulus amoebocyte lysate endpoint. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The circulating blood cells of the marine horseshoe crab, Limulus polyphemus, lyse and the contents clot in the presence of endotoxins. The discovery of this phenomenon led to the commercial extraction and preparation of Limulus amoebocyte Lysate (LAL) for the in vitro testing of endotoxin contamination in parenteral products and sterile medical devices. The most commonly used test of the LAL's reaction with endotoxin is the qualitative Gel-clot technique. This involves inverting the test solution through 180° after incubation at 37°C for one hour and observing for a firm gel; indicating a minimum level of endotoxin contamination equivalent to the sensitivity of the LAL. To date the techniques for quantitative determination of endotoxin activity have relied upon the optical changes taking place within the LAL. Preliminary theological studies of the solution surface during the reaction of LAL with endotoxin, using an Oscillating Ring Surface Shear Rheometer (ORSSR), demonstrated measurable changes and characteristic trends. Although the measurements were poorly reproducible this technique offered several advantages over the other techniques for LAL-endotoxin reaction endpoint determination. Based on the important advantage that it allowed application of the LAL to coloured, opaque and/or viscous products, a project aimed at developing the accuracy and easier application of the ORSSR for this test was initiated. To this end mechanical, electrical and computer program developments to the ORSSR were made and validated using the standard surface film formers gum acacia and protein collagen. In order to propose a LAL-Surface Rheological Assay (LAL-SRA) for endotoxins it was necessary to investigate the effect on and the reproducibility of the surface rheological properties of the film formed by: (1) endotoxin concentration, (2) LAL concentration, (3) bacterial origin of the endotoxin, (4) formulation excipients and (5) metal ions. During the course of these investigations parallel studies were conducted into the effect of these factors on LAL sensitivity using the LAL-5000 Kinetic Turbidimetric technique and where applicable the Gel-clot technique.

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