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PyroSmart NextGen® Recombinant Cascade Reagent (rCR)
No Animal Content – Horseshoe Crab Blood Free
Same Cascade, Instrument & Preparation Steps
No Cross Reactivity With 1,3-β-D-Glucan
To Review Analytical Performance & Comparability Testing Data,
Request Technical Brochure | Download Product Brochure
With the proposed new standard, Chapter <86> from The USP Expert Committee to include rCR recombinant reagents, there’s never been a better time to take the PyroSmart NextGen® challenge!
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Recombinant Cascade Reagent (rCR)
PyroSmart NextGen® recombinant Cascade Reagent (rCR) is a sustainable recombinant LAL reagent technology for Bacterial Endotoxin Testing (BET). Utilizing the same LAL cascade as traditional LAL reagents, while eliminating the potential for 1,3-β-D-glucans cross reactivity, PyroSmart NextGen® delivers all of the quality and consistency of results you have come to expect from ACC LAL reagents.
PyroSmart NextGen® can be used for a wide variety of endotoxin tests, ranging from standard water testing to samples requiring high sensitivity, such as intrathecal products and those requiring high dilutions to overcome interference.
PyroSmart NextGen® Package Insert
PyroSmart NextGen® Product Brochure
- BPB Reports Downloads
- Vol. 6 No. 2 p.68-75 2023 Title: A Demonstration of the Validation Process for Alternative Endotoxin Testing Methods Using PyroSmart NextGen® Recombinant Cascade Reagent - Corresponding author: Madeline Kelley
- Vol. 6 No. 1 p.11-15 2023 Title: Evaluation of Recombinant Cascade Reagent PyroSmart NextGen® and Limulus Amebocyte Lysate Equivalency in a Plate and Tube Reader for Bacterial Endotoxins Testing - Corresponding author: Madeline Kelley
- Vol. 5 No. 5 p.105-114 2022 Title: Advanced Recombinant Cascade Reagent PyroSmart NextGen® for Bacterial Endotoxins Test as Described in the Pharmacopeias - Corresponding author: Ingrid Stevens, Norihiko Ogura
The sensitivity for recombinant chromogenic assays is determined by the lowest standard concentration on the standard curve used for the assay. The maximum sensitivity of PyroSmart NextGen® is 0.005 EU/mL when run in an incubating microplate reader (or 0.001 EU/mL when run in Pyros Kinetix® Flex tube reader).
Sample to Lysate Ratio
PyroSmart NextGen® is used at economical volumes:
- Pyros Kinetix® Flex tube reader: 4:1 using 200 µL of test sample : 50 µL of reagent
- Microplate reader: 1:1 ratio using 50 µL of test sample : 50 µL of reagent
The PyroSmart NextGen® reaction mixture is incubated at 37±1°C and read in a microplate reader or tube reader equipped with a 405nm filter. The time of incubation is dependent on the lowest standard concentration in the standard curve, with 0.005 EU/mL achievable in 2500 seconds in a microplate reader and 0.001 EU/mL achievable in 3000 seconds in a tube reader, respectively. Software is used to construct the standard curve and calculate the endotoxin concentrations.
PyroSmart NextGen® is a lyophilized product with an excellent shelf life of 3 years from the date of manufacture.
PyroSmart NextGen® is provided as co-lyophilized with the chromogenic substrate and as such it is ready-to-use following a simple reconstitution (using 2.8mL of the supplied reconstitution buffer).
PyroSmart NextGen® reagent is provided as a pack of 2 vials of reagent and 2 vials of reconstitution buffer. This is sufficient for a total of 96 wells (approx. 50 tests/vial).
2.8 mL/vial (approx. 50 tests/vial)
Each kit contains
2 x PyroSmart NextGen® Reagent
2 x Reconstitution Buffer
Interested in participating in our comparability study of LAL vs. rLAL?
- RSE as a standard (std. curve, PPC)
- Using validated systems only
- Coordinated testing allowing for concurrent analysis of the samples by LAL vs. rLAL
- Using endotoxin specific LALs
- Results expressed as Relative Recovery %
- Relative recovery % is within 50 to 200%
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Frequently Asked Questions
1. Will PSNG perform the same as naturally sourced reagents?
Yes, PSNG is a kinetic chromogenic reagent. It contains recombinant Factor C, Factor B and pro-clotting enzyme, thus it continues to rely on the cascade mechanism in order to amplify the signal. It is co-lyophilized with the same chromogenic substrate as other chromogenic reagents. Therefore it is homogeneous with Pyrochrome® and Chromo-LAL. This presents a huge advantage to the end user: they keep the method, only switch out the reagent.
2. What’s the difference between Recombinant Factor C (rFC) and Recombinant Cascade Reagent (rCR)?
Recombinant Factor C reagent contains only recombinant Factor C. This enzyme acts as a biosensor for endotoxin (specifically binds to it). But since the cascade amplifying the signal is missing (no Factor B and pre-clotting enzyme), Factor C reagents are used in combination with fluorescence read out in order to achieve sensitivity levels comparable to the cascade reaction. This means that a fluorescent plate reader has to be used (along with a specific microplate). So this means different method, different readers, different SOPs for rFC reagents. Recombinant cascade reagent (e.g. recombinant TAL (= PyroSmart) or recombinant LAL (= PyroSmart NextGen™) are homogenous to current LAL reagents (Pyrochrome® or Chromo-LAL) in that they contain the full cascade (3-enzyme cascade).
3. Does PSNG use any raw material from the horseshoe crab?
No. PSNG is completely animal free.
4. Will new instrumentation be required to use PSNG?
No. Due to the cascade based feature, you can utilize the same instrument and preparation procedures.
5. How are recombinant reagents produced?
It is a biopharmaceutical production based on the use of expression system as cell line clones, that contain (thanks to genetic engeering) DNA coding Factor C, Factor B and pro-clotting enzymes from Limulus Polyphemus. The cell lines express the final glycoproteins Factor C, Factor B and pro-clotting enzyme which are then collected, purified and formulated into PyroSmart NextGen™.