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Kinetic-turbidimetric LAL method for Endotoxin Detection (PYROSTAR™)

28th February 2019

Kinetic-turbidimetric LAL method for Endotoxin Detection (PYROSTAR™)Bacterial endotoxin is the lipopolysaccharide (LPS) component of the cell membrane of Gram-negative bacteria. The presence of endotoxin can be detected by a specific and efficient method known as the Limulus Amebocyte Lysate (LAL) assay. The LAL assay was developed in the 1960s and is based on the clotting reaction of haemolymph from the Atlantic horseshoe crab with the antigens present on the microbial surface, such as endotoxins. Various reagents have been developed to optimize the detection efficiency of the LAL assay and to make the process faster and more convenient.

PYROSTAR™ ES-F, manufactured by FUJIFILM Wako, is one of the widely used reagents for the detection of bacterial endotoxins and contains Limulus Amebocyte Lysate, Carboxy-methylated Curdlan (CMC), buffers, and both monovalent and divalent cations.

PYROSTAR™ ES-F is highly sensitive and can detect endotoxin with a quantitative range of 0.001 to 10 EU/ml, depending on the sensitivity used. This assay, as well as, the manufacturing facilities associated with it are also fully licensed by the US Food and Drug Administration (FDA).

PYROSTAR™ ES-F contains lyophilized turbidimetric lysate reagent that allows for the simultaneous measurement of multiple samples. It is also a unique reagent as it contains Carboxymethylated Curdlan (CMC) that has been co-lyophilized with the LAL making the reagent completely endotoxin-specific. This proprietary formulation prevents the interference of β-glucans present in the bacterial cell membrane that can affect the outcome of the LAL assay. 

The method used for the detection of endotoxins using PYROSTAR™ ES-F reagent is kinetic- turbidimetric or KTA. The kinetic- turbidimetric assay can quantitatively assess the endotoxin level of a product with a greater degree of precision relative to any other methodology in use.

PYROSTAR™ ES-F can be purchased in multi-test and single-test configurations, as well as, with or without Control Standard Endotoxin (CSE). CSE is a lyophilized reagent that has been developed from E. coli and is used as the assay standard to confirm the sensitivity of the LAL agents, prepare test controls and can validate the product test methods.

The CSE vial contains 500 ng of endotoxin. Each vial is tested in comparison to the FDA Reference Endotoxin and the conversion factor (EU/ml) is determined. To reconstitute the CSE, gently pull and remove the rubber stopper from the vial to release the vacuum. Determine the volume of LRW that is to be added to the vial to yield a 1,000EU/mL solution.  Add the determined volume of LRW to the vial and replace the rubber stopper.  Invert and shake the vial vigorously several times for 2 minutes.

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Reconstituted CSE can be stored for one month at 2-10°C. CSE should not be stored at temperatures below freezing. When using solution that has been previously stored, shake vigorously for one minute prior to use.

Kinetic turbidimetric reagents require a multi-tube or microplate reader along with endotoxin-measuring software to obtain quantitative values for endotoxin. The detection of endotoxin with PYROSTAR™ ES-F is possible without it being affected by (1 → 3) - β-D - Glucan in the sample. A small amount of sample can also be used to carry out the assay as only a sample volume of 100 µL is needed for the tube reader and 50 µL for a microplate reader.  Also, due to the buffers contained in the reagent, there is only a negligible impact on the readings from sample pH values, thus making it possible to achieve stable endotoxin measurement.


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By: Lisa Komski In: Kit LAL