Detection of bacterial endotoxins using the PYROSTAR™ ES-F/Plate LAL assay

Introduction

Bacterial endotoxins, also known as lipopolysaccharides (LPS), are potent pyrogens found in the outer membrane of Gram-negative bacteria.^1,2 Their presence in pharmaceuticals, medical devices, and biological products can trigger severe inflammatory reactions in our immune system. These inflammatory reactions may lead to serious adverse events and compromise health. Therefore, the detection and quantification of endotoxins are crucial for ensuring product safety and regulatory compliance for food, medical, and other products.

The Limulus Amebocyte Lysate (LAL) assay is a widely used method for detecting bacterial endotoxins.^3-5 Two types of horseshoe crab have been used as sources of LAL: Limulus polyphemus from the North Atlantic and Tachypleus spp. from Asia. The LAL assay exploits the natural clotting response of blood cells (amebocytes) isolated from these animals to endotoxins. When exposed to endotoxins, LAL undergoes a series of enzymatic reactions that result in gel formation, which can be measured to determine the presence and concentration of endotoxins.

The PYROSTAR ES-F/Plate LAL test is sensitive, robust, suitable for small sample volumes, and is not activated by β-glucans which can lead to false positive results. It therefore ensures that products are free from harmful levels of endotoxins, safeguarding patient health and meeting stringent regulatory standards. 

Assay principle

The PYROSTAR ES-F/Plate kit is based on the classic LAL assay principle. In the presence of endotoxins, the LAL responds with a clotting reaction. Clotted LAL can be detected on a microplate reader in two ways which both depend on light scattering but use different types of readouts. The LAL assay can either be performed with a light-scattering readout on a nephelometry-based microplate reader or with an turbidimetric readout on a spectrometer-based absorbance microplate reader (Figure 1). In a nephelometer, scattered light is measured due to detection taking place at an angle to the incident light. If an absorbance-based microplate reader is used, light that passes through the sample is measured - so all light that isn’t scattered. A reference measurement is used to determine the total light produced by the spectrometer, which allows an indirect statement to be made about the amount of light scattered by the sample.  

Here you will find the protocol files for BMG LABTECH’s Smart Control Software (CLARIOstar Plus and VANTAstar) and the Omega Software (NEPHELOstar Plus), the used plate layout and the corresponding templates for automatic evaluation of generated data in MARS. You can download these files from the overview page, import them into your BMG LABTECH software and use them to evaluate your analysis.
 

Results & Discussion

Figure 2 shows kinetic data of the PYROSTAR ES-F/Plate kit. Samples containing endotoxin displayed an increase in assay signal over time. Higher endotoxin concentrations led to a faster increase in measurement signal until the signals reached a maximum compared to samples with lower endotoxin concentrations. The nephelometric readout (figure 2A) and turbidimetric readout (figure 2B) gave comparable results regarding to the separation of endotoxin standards.