Colorimetric and turbidimetric analysis of endotoxins using the absorbance detection mode

Introduction

Bacterial endotoxins, in particular lipopolysaccharides (LPS), are structural components of the outer membrane of Gram-negative bacteria that can activate the innate immune system in a way that leads to elevated temperature and fever.^1,2 Such reactions are potentially life threatening and can be triggered even by trace amounts of endotoxins which is why their detection is crucial for quality assurance in the pharmaceutical and biotechnological in-dustries. These tests are therefore an integral part of the regula-tory requirements for the safety of drugs, medical products and biological therapeutics.

Limulus Amebocyte Lysate (LAL)-based tests are an established and sensitive method for the detection of endotoxins.^3,4 These tests are based on the coagulation cascade of amebocyte cells derived from horseshoe crabs (typically Limulus polyphemus but Tachypleus spp. are also used in Asia). This cascade is specifically activated by the presence of LPS triggering an enzymatic reaction that leads to clotting of LAL. Endotoxin assays, which quantify the strength of the coagulation reaction, are used to determine the amount of endotoxin present in a test sample. Over the years, several absorbance-based assays have been developed for this purpose that are still widely used today. In this application note, we highlight two absorbance-based endotoxin quantification assays that rely on distinct measurement principles to monitor a LAL clotting reaction. 

Assay principle

Endotoxin quantification assays with an absorbance-based read-out are typically based on two measurement principles. Both utilize the clotting reaction of LAL in the presence of endotoxin. In each case, different concentrations of endotoxin are prepared and their signals measured. These measurements are used to construct a standard curve which serves as reference for the calculation of endotoxin concentrations of unknown samples. Adding LAL starts the clotting reaction that can be quantified on a microplate reader. One way to do this is to measure the turbidity of the test sample by monitoring the light transmission through a sample. The more clotted LAL is present in the sample, the less light reaches the detector mainly due to light scattering (Figure 1). The PYROSTAR™ ES-F/Plate LAL assay kit can be used to perform a turbidimetric LAL clotting assay.

Other assays, such as the Limulus Color KY Test employ a colorimetric readout for the quantification of endotoxin in a sample. Here, a chromogenic substrate is added to the assay that is cleaved by the activated enzymes of the LAL clotting cascade in the presence of endotoxin. Higher concentrations of endotoxin lead to increased enzymatic activity and higher amounts of converted substrate. In this case, more of the yellow chromogen para-nitroaniline (pNA) is released from the substrate. The increase in light absorption due to the increase in pNA is then quantified by the microplate reader.   

Results & Discussion

LAL clotting could be quantified with both the colorimetric (Figure 2A) and the turbidimetric assay kits (Figure 2B). The speed of the clotting reaction depended on the endotoxin concentration in the sample, with higher endotoxin concentrations leading to a faster increase in the absorbance units (OD) quantified by the microplate reader. In comparison, the signals derived from the colorimetric assay were significantly higher than those from the turbidimetric assay, but ultimately the lower assay threshold of 0.015 OD was achieved by the same number of standards in both assays.