EndoLISA® Frequently Asked Questions (FAQ)

Is EndoLISA® a valid alternative to the LAL assay?                             
Yes, recombinant Factor C (rFC) based tests such as EndoLISA 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 EndoLISA) are also listed as valid alternative methods in the FDA "Guidance for Industry - Pyrogen and Endotoxins Testing: Questions and Answers".

TR33 released by the Parenteral Drug Association lists the EndoLISA technology in section 4.3.6 - Endotoxin Detection.

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. EndoLISA has been validated by an external GLP certified company. The validation certificate of EndoLISA 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 EndoLISA® licensed by the FDA?
No animal source is used in the production of EndoLISA®. As EndoLISA® contains no biologic 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 EndoLISA® equally sensitive to inhibition as the LAL assay?
The unique solid phase technology of EndoLISA® allows the removal of matrix factors from your sample, thereby significantly lowering disturbing effects. An initial inhibition/enhancement testing for validation is recommended.

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 EndoLISA® 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 EndoLISA® 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 EndoLISA®?
EndoLISA® 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?
EndoLISA® contains two 96-well microtiter plates to run 192 tests. The EndoLISA® plates are divided into 6 individual stripes, each containing 16 wells, so that you do not have to use the whole plate when you only have a few samples.

How many samples can be tested in one run?
EndoLISA® 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 EndoLISA® kit not contain β-glucan inhibitors?
EndoLISA® 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 EndoLISA®.

What is the detection limit of EndoLISA®?
EndoLISA® has a detection range of 0.05 to 500 EU/ml. A benefit of EndoLISA® regarding sensitivity is the possibility of detecting endotoxin in undiluted samples, whereas LAL often requires dilution up to 1000 fold.

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 EndoLISA®?
EndoLISA® copes very well with a wide variety of different product types and ingredients. See the list of tested components below. 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. As there are numerous products, individual testing by spike recovery is recommended.

Table 1: Highest tolerated concentrations of substances for valid LPS spike recovery, a comparison between EndoLISA and LAL assay.

 
Substance
Solvent
EndoLISA®
LAL assay
Buffer/pH
Acetate (pH 4.0)
100 mM NaCl
50 mM
12.5 mM
Acetate (pH 5.0)
100 mM NaCl
100 mM1)
6.25 mM
MES (pH 6.0)
100 mM NaCl
100 mM1)
5 mM
Potassium phosphate (pH 7.2)
100 mM NaCl
100 mM1)
50 mM
Imidazole (pH 7.4)
Water
500 mM
40 mM
HEPES (pH 7.5)
100 mM NaCl
100 mM1)
100 mM1)
Sodium borate (pH 9.0)
100 mM NaCl
100 mM1)
50 mM
Salt
NaCl
Water
1M
0.5 M
KCl
Water
1M
0.25 M
Chaotropic agent
Urea
Water
6M
0.5 M
Guanidinium chloride
Water
1M
0.05 M
Organic solvent
Methanol
Water
20 %1)
5 %
Ethanol
Water
30 %
0.5 %
2-Propanol
Water
20 %
0.2 %
DMSO
Water
10 %
2%
Detergent
SDS
Water
0.05 %
0.001 %
CTAB
Water
0.004 %
0.0001 %
Zwittergent 3-14
Water
0.02 %
0.005 %
Tween 20®
Water
2 %
0.1 %
Triton X-100
Water
0.02 %
0.005 %
Chelator
EDTA (pH 8.0)
Water
0.4 mM
0.4 mM
Citrate (pH 7.5)
Water
10 mM
10 mM
Protease inhibitor
Benzamidine
Water
100 mM1)
0.1 mM
PMSF
2-Propanol
5 mM
< 0.05 mM
Antibiotic
Rifampicin
Methanol
3.5 mg/ml
0.04 mg/ml
Chloramphenicol
Ethanol
3.5 mg/ml
0.1 mg/ml

 1) Highest concentration tested

The tested substances were dissolved in the respective solvent and dilution series of the samples were prepared in water or in 0.1M NaCl and subsequently spiked with 5 EU/ml LPS (E. coli O55:B5). EU/ml values and spike recovery (%) were calculated. The validity criterion of spike recovery was 50-200%.

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

Can I get an MSDS for EndoLISA®?
The kit reagents are not considered to be hazardous. However, if you need a Material Safety Data Sheet, it can be downloaded here.