Quality Control of Food and Drinking Water

Phil Black - PII Editor 07/04/2022

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Tracking Down Pyrogens: The Unique ES Buffer enables Specific Detection of Endotoxin with All common LAL Reagents – Independent of the Manufacturer and Without Additional Test Systems

Coagulation Factor Sourced Sustainably From the Blood of the Primeval Horseshoe Crab

pfeilschwanzkrebs — The Atlantic horseshoe crab has a special immune system. If the blood of this ancient species comes into contact with endotoxin, it forms a clot around the pathogen which prevents further infection of the wounded area.

LAL test — Limit value tests (BET/LAL) are used to preclude contamination during manufacture. They are based on a special factor harvested from the blood of the horseshoe crab.

Solid food, egg or meat/fish products: Before market release, foodstuffs and their raw materials must be checked for sterility during production. In particular, bacterial endotoxins must not enter the human organism. If these gram-negative bacteria cell membrane components get into the bloodstream of humans or animals, they can cause high fever, which can be fatal to weakened patients. Special limit value tests (BET/LAL) are used to preclude contamination during manufacture. They are based on a specific factor derived from the blood of horseshoe crabs. In the presence of endotoxin, this factor reacts with the pathogen, triggering a biochemical coagulation reaction.

The contamination can be detected on the basis of the gel formation. However, interferences such as fluctuating pH values or fungal components, which may be contained in plant-derived sample supplements, can cause a similar reaction, resulting in false positives. In order to eliminate this interference, FUJIFILM Wako Chemicals Europe GmbH provides a unique and exclusive endotoxin-specific (ES) buffer for untreated LAL reagents. It is already formulated for all common detection methods and is easy to use so that it can be integrated into the quality control without much effort. While all LAL reagents manufactured by Fujifilm Wako are already endotoxin specific, other commercially available reagents are not. With the ES buffer as an additive, these now become endotoxin specific, and allow the determination of the actual endotoxin concentration in the sample. A change to other test systems is not necessary.

Endotoxins – components of the cell membranes of gram-negative bacteria – are extremely heat-stable, resist many standard sterilisation methods, and need no viable bacteria in order to be active. If they get into the bloodstream of humans or animals via food during digestion, they quickly react with immune cells and cause a severe inflammation reaction and fever. However, the Atlantic horseshoe crab (Limulus polyphemus) has a very special immune system. If the blue blood of this ancient species comes into contact with even the smallest quantities of endotoxin, as an immune response it forms a clot around the pathogen which prevents further infection of the wounded area. This is done by a special blood factor which forms the basis of the bacterial endotoxin test (BET) or limulus amoebocyte lysate (LAL) test. This is often used to test egg products intended for further processing, for example in pasta. In addition, the rapid and highly sensitive test can be used to detect dead and live Gram-negative bacteria in liquid and solid foods, meat products and other raw materials used in food production. The coagulation factor is harvested from horseshoe crab blood in a sustainable way without harming them. To protect the animals, Fujifilm Wako has also joined several organisations, including the Horseshoe Crab Conversation Fund and the US Fish and Wildlife Service.

ES Puffer — Interferences can be prevented with the endotoxin-specific buffer supplied by FUJIFILM Wako Chemicals Europe GmbH. When this is added to the sample, it blocks undesired cascades and neutralises pH fluctuations.

“Numerous foodstuffs as well as their raw materials can be contaminated during the preparation, production or packaging phases, for example through insufficient cleaning of the process equipment or already contaminated raw materials such as the delivered milk,” reports Yannic Werner, Sales Manager for Laboratory Chemicals at FUJIFILM Wako Chemicals Europe GmbH. “The test is intended to prevent contamination before sale and consumption.” However, untreated LAL reagents also react to fungus cell wall components – more precisely, to (1→3) β-D-glucan (beta-glucan) – likewise causing the coagulation cascade. This leads to a false positive BET result. Interference like fluctuating pH of the sample, for example caused by addition of an acidic medium during formulation, can impair the accuracy of endotoxin determination. That's why FUJIFILM Wako Chemicals Europe GmbH, a sister company of the FUJIFILM Wako Chemicals U.S.A. Corporation, offers an endotoxin-specific buffer that blocks the activation cascade of fungus and permits only that of gram-negative bacteria. This ensures highly precise and quantitative determination without false positives. In this regard, the buffer is suitable for use as an additive in all current and untreated LAL procedures from various manufacturers. The ES buffer is available individually or as an integral part of FUJIFILM Wako’s product line of endotoxin-specific reagents.

immunkaskade — In the endotoxin interaction the first serin protease precursor (Factor C) is initiated from its inactive form; this in turn activates the second serin protease precursor (Factor B). The activated Factor B stimulates the proclotting enzyme and transforms it into a clotting enzyme. The coagulation enzyme splits peptide bonds within the coagulogen to get coagulin, the insoluble gel-forming protein produced in the gel clot test. Source: FUJIFILM Wako Chemicals Europe GmbH

ES Buffer Prevents False Positive Results

The buffer is a solution that resists pH change when an acidic or alkaline substance is added. It can neutralise small amounts of added acids or bases, thus keeping the pH of the solution stable. “In order to block the effect of (1→3) β-D-glucan on detection in untreated LAL reagents, our buffer also contains a special factor that saturates the cascade for beta-glucan, making the test insensitive to beta-glucan,” says Werner. If endotoxin is present, only its activity is permitted, causing a coagulation reaction. The mechanism is based on a cascade of two serin proteases, called Factor B and Factor C. When the proclotting enzyme is activated, it is converted to its active form, which splits the coagulogen into an insoluble clot gel, thereby increasing the turbidity of the solution. This action makes it possible to observe qualitative or quantitative effects, proving the presence of endotoxin as well as the amount of it present.

ES Reagenzien — FUJIFILM Wako offers the buffer as a separate solution for adding to untreated LAL reagents. In addition, the buffer is already included in a number of endotoxin-specific reagents. These include ready-to-use test kits.

The test itself can be done by one of three methods: gel clot, kinetic turbidimetric (KTA), or kinetic chromogenic (KCA). In gel clot, the determination is qualitative. The result shows only the presence or absence of endotoxin. The kinetic methods allow quantitative determination of the endotoxin content. To do so, either a microplate reader or a tube reader, such as FUJIFILM Wako's Toxinometer® Analysis System, can be utilised to automatically monitor a change in absorbance that occurs over a specific period of time. The ES buffer from FUJIFILM Wako is formulated for all three methods. Each vial contains solution for the reconstitution of 6 mL of reagent. The vials are pre-prepared, so that the LAL reagent can be easily pipetted into them, which also results in fewer potential sources of contamination. The simple handling enables the determination to be carried out in a short time.

Reagenz — The ES products are very easy to use. It is necessary merely to observe the maximum valid dilution (MVD); the more interferences there are, the higher this needs to be.

ES Buffer Already Included in BET Reagents

FUJIFILM Wako offers the buffer exclusively as a separate solution for adding to untreated LAL reagents. In addition, the buffer is already included in their ES reagents. “Each reagent contains buffer components that contribute to bringing most test mixtures to the pH range needed for precise, reliable results,” explains Werner. “They can simply be used with endotoxin-free water without the need for further additives or a buffer for the BET tests.” It is necessary merely to observe the maximum valid dilution (MVD): The more interferences there are, the higher this must be. Thanks to this effective formulation with buffer components included, double and triple determinations can be done reliably at consistently high quality. “For routine tests in a quality control lab, it is critical to establish a reproducible test method in order to trace any reaction unambiguously to endotoxin. This is ensured with our reliable ES reagents as well as the buffer. With little effort, these tests can be made endotoxin-specific without the need for a complete change to new systems,” Werner concludes.

For further information: www.labchem-wako.fujifilm.com and www.wakopyrostar.com