PYROSTAR™ Neo+

Recombinant Endotoxin Detection

A wildlife-conscious recombinant alternative to the conventional LAL assay. Designed for bacterial endotoxin testing for research, pharmaceutical and medical equipment development. 

Key Features

  • Non-animal derived

  • Recombinant cascade reagent (rCR) assay

  • Colorimetric (KCA), use with absorbance plate reader
    or Toxinometer®

  • High sensitivity testing with less lot-to-lot variation

  • Stable storage after dissolution

  • New optimised formulation for difficult sample matrices

  • For optimal performance, the reagent is lot matched to
    a specific Control Standard Endotoxin (CSE) lot stated
    in the CoA.

Wildlife Conservation in Mind

Not reliant on Atlantic horseshoe crabs for raw materials as it uses recombinant DNA technology.

3Rs of Animal Testing Compliance

3Rs principles of replacement, reduction, and refinement of animal testing. Replacing traditional animal derived reagents and reduces the impact on wildlife.

Without Compromising Performance

3-Factor system which mimics the same cascade as traditional LAL. Same high sensitivity as the PYROSTAR™ ES-F.

Increased Reproducibility

More consistent results as there are less lot-to-lot variations due to use of recombinant technology as opposed to animal derived materials.

Endotoxin Specific

Endotoxin specific assay, which eliminates the risk of false positives from β-D-glucan

Good Scalability

Assay has the capacity to grow to meet increasing demands in the future.

Storage Stability

Stable storage after dissolution (4 hrs at 2-8°C and 2 weeks at -30°C).

High Sensitivity

Has a detection range of 0.001 to 50 EU/mL, depending on requirements.

Simple to Switch to

Uses the same instrumentation and method as the LAL test.

Background

Lipopolysaccharide (LPS), also known as endotoxin, is a component of the outer membrane of Gram-negative bacteria. Endotoxins causes severe symptoms such as fever and shock in the human body, testing of bacterial endotoxins in research, quality control, development of pharmaceuticals, medical devices and healthcare laboratory consumables is essential.

Bacterial endotoxin testing (BET) is performed using LAL reagents that applies the clotting cascade of Atlantic horseshoe crab (Limulus polyphemus) blood. The introduction of synthetic alternatives replaces traditional LAL with recombinant reagents that are non-animal derived and no longer reliant on the Atlantic Horseshoe Crab*(1,2,3). In compliance with the 3Rs principles of replacement, reduction, and refinement of animal testing.

First introduced was the recombinant Factor C (rFC) reagent which uses recombinant Factor C from the clotting cascade to detect endotoxins using fluorescence. The next generation includes the recombinant Cascade Reagent (rCR), which uses all three factors of the clotting cascade to detect endotoxins by absorbance similar to conventional LAL reagents*(4).

PYROSTAR™ Neo+ is a new rCR using a recombinant 3 factor system, utilises the Kinetic Chromogenic Assay (KCA), offering the same high sensitivity as PYROSTAR™ ES-F to detect endotoxins.

The reagent is composed of Factor C, Factor B, and a proclotting enzyme prepared by recombinant technology to replicate the equivalent LAL clotting cascade. It contains a pNA chromogenic group, enabling detection of the assay results on an absorbance plate reader using a colorimetric method. The recombinant clotting factors, chromogenic substrate, and a buffer component are co-lyophilised.

It has improved specificity with the absence of Factor G and eliminates interference due to reaction with (1 → 3)-β-D-glucan. Additionally, it has an optimised formulation to support handling of difficult sample matrices which cause interference.

Welfare of the Atlantic Horseshoe Crab

Atlantic Horseshoe Crabs (Limulus polyphemus) are found along the North Atlantic coast from Maine down into the Gulf of Mexico*(4). Harvested directly from beaches, their blood is used within the biomedical industry and within the commercial fishing industry as bait. This species is estimated to be around 445 million years old, predating dinosaurs, and is classed as a ‘Vulnerable’ species by the IUCN*(5).

Ecological benefits - The Atlantic Horseshoe crab is considered a keystone species within its environment. The species plays a vital role maintaining the marine food web, being an essential prey resource for; Turtles, Alligators, Horse Conchs, and Leopard Sharks*(6). The Red Knot, a well-known migratory bird, consumes crab eggs as a food source, adapting their migration pattern along the coastline to match that of peak spawning season.

Horseshoe Crabs are also considered an indicator species, helping determine the quality of the marine and intertidal ecosystems*(5), more crabs equate to an overall healthier environment. As an integral species to these ecosystems, the population success of the species helps ensure a positive influence on the wider ecosystem.

Risks - Prolonged harvesting direct from coastal habitats disrupt the natural flow of nutrients within the food web, with the potential to cause ecosystem collapse. Ecosystem collapse and disruptions in the food web can lead to negative impacts on economies reliant on these systems.

When harvested for blood extraction, improper treatment and handling including the storage and transportation can lead to a mortality rate as high as 30%*(5,7). Further stress can be incurred due to the extended periods of time away from the aquatic environment (inducing hypoxia), triggering the coagulation of the blood and damaging the LAL agent*(5,7,8). Up to 40% of the individuals’ blood is extracted before release back into the marine environment. Mortality and stress factors extend into the release and can have negative impacts on the success of the individual’s survival and future spawning*(8). It is immeasurable to determine mortality rates based on resulting trauma once returned to the ocean and thus further spawning impacts, irrelated to the disruption in spawning cycles which are caused with the original removal of breeding individuals from the beaches.

Control Standard Endotoxin

The Control Standard Endotoxin (CSE) from FUJIFILM Wako is a lyophilised product composed of 6 x 500ng vials of endotoxin. Through serial dilution it can be used to create positive control samples and sample curves for GCA, KTA and KCA methods. Purified from E. coli UKT-B strain that serves as an accurate and dependable standard for endotoxin testing.

CSE can be reconstituted with LAL Reagent Water (LRW) or equivalent. A Certificate of Analysis (CoA) can be provided for lot matching the Neo+ reagent with CSE for optimal performance.

  • Endotoxin derived from E. coli UKT-B

  • Control and standard curve preparation

  • Potency of CSE supplied to match each lot of LAL

  • Reconstituted CSE can be stored at 2–10°C for 1 month

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