Pros and cons of different endotoxin detection readouts

Bacterial endotoxins are strong pyrogens that may cause fever, inflammation, and even septic shock if they gain access to the human bloodstream. Endotoxins, which are an integral component of the outer membrane of gram-negative bacteria, are ubiquitous in the environment. Therefore, medical devices and pharmaceutical products intended for parenteral use should routinely be subjected to endotoxin testing. The Limulus Amebocyte Lysate (LAL) assay is the standard test for endotoxin detection,1 which has been approved by regulatory institutions across the world and has been included in the US Pharmacopeia.

Limulus amebocyte lysate (LAL) assay methods

The LAL assay can be performed using three main methodologies –gel-clot, turbidimetric, and chromogenic.2,3 The gel-clot method is based on the incubation of protein extracted from horseshoe crab blood lysate with endotoxin, leading to a clotting reaction. The turbidimetric and chromogenic methods are variations of the gel-clot method. The turbidimetric method relies on the formation of turbidity (cloudiness) after endogenous substrate cleavage, whereas the chromogenic method is based on chromogen production after the cleavage of a chromogenic substrate.

LAL gel-clot method

The LAL gel-clot method is qualitative, sensitive, and easy-to-use. It is the simplest LAL method which relies on the formation of a gel clot and can be assessed visually. LAL is extracted from primitive amoebocytes in horseshoe crab blood. The endotoxin’s lipid A component interacts with pro-clotting enzymes in the LAL and activates a coagulation cascade (including factor B, factor C, and a proclotting enzyme), modifying the amebocyte coagulogen, and leading to gelation and clot formation.

The PYROSTAR™ ES-F single-test and multi-test kits contain high sensitivity, endotoxin-specific (ES-F) reagents that are not activated by (1→3)-β-D-glucan and thus reduce the rate of false-positive results. The same kits that are used to perform the gel-clot method can also be utilized to perform a kinetic turbidimetric assay (KTA).

LAL kinetic turbidimetric assay

The LAL turbidimetric assay is a quantitative, automated, highly sensitive LAL assay, which is also based on the gel-clot reaction. It relies on the formation of turbidity of the test solution after endogenous substrate cleavage and prior to gel formation. A faster increase in turbidity indicates higher endotoxin levels that can be quantified using a spectrophotometric instrument, such as the Toxinometer®, or a microplate reader.

FUJIFILM Wako offers three KTA kits: PYROSTAR™ ES-F single-test for the Toxinometer®, PYROSTAR™ ES-F multi-test for the Toxinometer®, and PYROSTAR™ ES-F/Plate for the Microplate Reader, all of which are endotoxin specific. The PYROSTAR™ ES-F single-test and multi-test kits, used on the Toxinometer® measurement system, have enhanced sensitivity with a range of 0.001–10 EU/mL. The PYROSTAR™ ES-F/Plate kit, which enables engagement in high-throughput LAL testing, has a quantitative range of 0.01–10 EU / mL.

LAL chromogenic assay

The LAL chromogenic assay is also a modification of the gel-clot reaction and allows for highly sensitive and automated LAL measurements. The LAL chromogenic assay relies on the addition of a specific chromogenic substrate, which mimics the cleavage site in coagulogen, to the LAL. The clotting reaction causes hydrolysis of the chromogenic substrate, releasing the yellow chromogen p-nitroanilide (pNA), and enabling reaction visualization.

FUJIFILM Wako’s Limulus Color KY series includes the Limulus Color KY single-test kit for use on the Toxinometer® measurement system and the Limulus Color KY Multi-test kit which can be utilized on either the Toxinometer® or a microplate reader. These quantitative, kinetic, chromogenic assays have an extremely high sensitivity with a quantitative range detection limit of 0.0002 EU/mL for the single-test format and 0.0005 EU/mL for the multiple-test format. They also include endotoxin-specific reagents and enable the ability to perform high-throughput LAL testing when utilizing the microplate platform.

What are the pros and cons of the different endotoxin detection readouts?

The LAL assay can be performed using three principal methodologies: gel-clot, turbidimetric, and chromogenic. All three methods are widely accepted and have found broad applications in the practice (Table). The gel-clot LAL method is the simplest one; however, it is still very sensitive and reliable. The turbidimetric and chromogenic assays enable endotoxin quantification and more high-throughput assessment. However, they require the use of additional automation, whereas the gel-clot method does not. FUJIFILM Wako’s chromogenic assay has the lowest detection limit across the three methodologies, rendering it especially useful for cases with extremely low endotoxin concentrations. The PYROSTAR™ gel-clot and turbidimetric kits include the same line of endotoxin-specific reagents and can be used during different stages of pharmaceutical product or medical device development.

Thus, even though all LAL assay methods are sensitive, reliable, and straightforward to use, the selection of an individual assay should be based on researchers’ needs and tailored to the specifics of the tested products.

 

References

  1. Mehmood Y. What is Limulus amebocyte lysate (LAL) and its applicability in endotoxin quantification of pharma products. In: Growing and Handling of Bacterial Cultures. 2019 Jan 8. IntechOpen.
  2. Joiner TJ, Kraus PF, Kupiec TC. Comparison of endotoxin testing methods for pharmaceutical products. International Journal of Pharmaceutical Compounding. 2002;6:408–409.
  3. Wheeler A. Comparing endotoxin detection methods. Pharmaceutical Technology. 2017;41(11):58–62.

 

 

Table. Characteristics of  FUJIFLM Wako assays for endotoxin detection

 

Technique

Quantitative/

Qualitative

Detection range

Test format

Key features

PYROSTAR™ ES-F Single gel clot technique

Gel-clot

Qualitative

0.015–0.25 EU/mL

Single-test

Easy-to-use

Sensitive and accurate

Does not require instrumentation

The reagent can be used conveniently and in small increments

PYROSTAR™  ES-F Multi gel clot technique

Gel-clot

Qualitative

0.015–0.25 EU/mL

Multiple-test

Easy-to-use

Sensitive and accurate

Does not require instrumentation

Cost advantage at higher test numbers

PYROSTAR™  ES-F Single KTA assay (Toxinometer®)

Turbidimetric

Quantitative

0.001–10 EU/mL

Single-test

Highly sensitive

Quantitative assessment

The reagent can be used conveniently and in small increments

PYROSTAR™ES-F Multi KTA assay (Toxinometer®)

Turbidimetric

Quantitative

0.001–10 EU/mL

Multiple-test

Highly sensitive

Quantitative assessment

Cost advantage at higher test numbers

PYROSTAR™  ES-F/Plate (Microplate Reader)

Turbidimetric

Quantitative

0.01–10 EU/mL

Microplate format

Sensitive

Quantitative assessment

High-throughput

Cost advantage at higher test numbers

Limulus Color KY Single  (Toxinometer®)

Chromogenic

Quantitative

0.0002–2 EU/mL

Single-test

Highest sensitivity

Quantitative assessment

The reagent can be used conveniently and in small increments

Limulus Color KY Multi (Toxinometer®)

Chromogenic

Quantitative

0.0005–5 EU/mL

Multiple-test

Highly sensitive

Quantitative assessment

Cost advantage at higher test numbers

Limulus Color KY Multi (Microplate Reader)

Chromogenic

Quantitative

0.0005–5 EU/mL

Microplate format

Highly sensitive

Quantitative assessment

High-throughput

Cost advantage at higher test numbers