EndoZyme® Frequently Asked Questions (FAQ)

Is EndoZyme® a valid alternative to the LAL assay?
Yes, recombinant Factor C (rFC) based tests such as EndoZyme are approved alternative methods to the LAL assay according to chapter 5.1.10 of the European Pharmacopoeia since 1 July 2016.

Recombinant horseshoe crab Factor C methods (including EndoZyme) are listed as valid alternative methods in the FDA "Guidance for Industry - Pyrogen and Endotoxins Testing: Questions and Answers".

The United States Pharmacopeia (USP 33, NF 28, 6.30) state that you are allowed to use an alternative method if the alternative is equivalent to or better than a compendial method and if it has been validated accordingly. EndoZyme has been validated by an external GLP certified company. The validation certificate of EndoZyme is available here.

Note: The requirements for validation of an alternative method are given in USP chapter 1225 (or in ICH Q2B Validation of Analytical Procedures: Methodology).

Is EndoZyme® licensed by the FDA?
No animal source is used in the production of EndoZyme®. As EndoZyme® contains no biologic products and is not intended for diagnostic use, no FDA license is required.The FDA "Guidance for Industry" from June 2012 states: If a manufacturer chooses to use a recombinant factor C-based assay, then method validation should be in accordance with the requirements of USP Chapter <85>, Bacterial Endotoxins Test, as described in the section for Photometric Quantitative Techniques, and USP Chapter <1225>, Validation of Compendial Procedures.

Is EndoZyme® equally sensitive to inhibition as the LAL assay?
EndoZyme® is a homogeneous test method (as LAL) best suited for less complex matrices such as environmental and water samples.

For complex sample matrices we suggest the heterogeneous test EndoLISA®. The unique solid phase technology of EndoLISA® allows the removal of matrix factors from your sample, thereby significantly lowering disturbing effects.

What kind of equipment do I need to run the test?
You’ll need a vortex mixer, standard laboratory pipettors, a multi-channel pipette, an incubator, a plate shaker, and a fluorescence microtiter plate reader (FLx800™ from Biotek with Ex 380/20 and Em 440/40 or equivalent). For calculation of the standard curve and the endotoxin concentration of your samples we recommend the Gen5™ software, for which we can supply a software template for the EndoZyme® assay.

Should I use glassware or plastic?
Endotoxin adheres to plastic surfaces more strongly than to glass surfaces. Therefore, we recommend using glass tubes to prepare your samples and your standard dilutions. Appropriate certified endotoxin-free glass test tubes can be obtained from Hyglos.

Can I use the same microtiter plate reader as I use for the kinetic LAL?
The kinetic LAL method uses absorbance readers. Since EndoZyme® uses a fluorescence substrate you’ll need a fluorescence plate reader. We recommend FLx800™ from Biotek Instruments.

Which filter does my fluorescence reader need for measuring the EndoZyme®?
EndoZyme® has been developed and validated with following filters:

Excitation filter (nm/band)         380/20
Emission filter (nm/band)           440/40

Hyglos has also tested the following alternative filter combinations:

How many tests can be performed with one kit?
EndoZyme® contains two 96-well microtiter plates to run 192 tests.

How many samples can be tested in one run?
EndoZyme® is run on a 96 well microtiter plate, 2x6 wells are needed for the standard curve in duplicate, so 84 wells are left for the samples. We recommend running each sample plus spike control (positive product control) in duplicate. Thus 21 samples can be measured per plate, including all the controls required by regulation as well as the standard curve.

How much sample do I need to run a test?
You need 100 µl of your sample per well. As we recommend performing a spike control, as well as setting up duplicates, you would need 400 µl of your sample in the desired dilution.

Why does the EndoZyme® kit not contain β-glucan inhibitors?
EndoZyme® specifically detects endotoxin, and does not react with β-glucans. Therefore there are no false positive results due to β-glucans, when performing endotoxin detection with EndoZyme®.

What is the detection limit of EndoZyme®?
EndoZyme® has a detection range of 0.005 to 50 EU/ml.

How is the spike recovery calculated?
[(amount of endotoxin found in spiked sample)-(amount of endotoxin found in sample)] / (amount of added endotoxin) * 100%

Example:

Spiking with 5 EU, 10 EU found in the sample and 14.5 EU found in the spike sample

(14.5 EU – 10 EU) / 5 EU *100 = 90 %

How can Endotoxin Units (EU) be calculated in mass units and vice versa?
It is taken as a rule of thumb that 1 EU corresponds to 100 pg of endotoxin. Please note that it strongly depends on the source of the endotoxin, because endotoxin activity is highly variable, depending on the bacterial strain.

Do you know of any products which cannot be tested with EndoZyme®?
EndoZyme® is a very sensitive detection method which is very good suited for non-complex samples. To measure endotoxins in complex samples we advise EndoLISA® which copes very well with a wide variety of different product types and integredients. But for example products with natural fluorescence may interfere with the measurement method itself. We do not recommend EndoLISA® for blood and plasma samples, as the endotoxin is masked in blood

Can I use EndoZyme® to detect endotoxin (LPS) in blood/plasma/sera?
We do not recommend EndoZyme® for blood samples. Endotoxin is masked in blood and therefore detection is not reliable, neither with EndoZyme®, nor with LAL assay.

Can I get an MSDS for EndoZyme®?
The Assay buffer contains Imidazol pH 7.0. Imidazol is considered hazardous. Avoid ingestion or contact with skin and eyes. All other components are not considererd hazardous, but have not been tested considering their toxicolobical properties. The Material Safety Data Sheet can be downloaded here.