PyroGene
Recombinant Factor C
Endotoxin Detection System

Catalog Number: 50-658U, 50-658NV

TABLE OF CONTENTS

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TITLE

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Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Safety Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Reagents Supplied and Kit Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Materials and Equipment Required but not Supplied . . . . . . . . . . . . . . . . . . . . .6
Specimen Collection and Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Sensitivity Setting for Fluorescent Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Preparation of Endotoxin Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Testing Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Types of PyroGene® Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Performance Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Product Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Patent Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Other Technical Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

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INTENDED USE
PyroGene is intended for use as an in vitro end-product endotoxin test for humanand animal parenteral drugs, biological products, and medical devices. This product is
not intended for use in the detection of endotoxin in clinical samples or the diagnosisof human disease.
The PyroGene test utilizes recombinant Factor C (rFC), an endotoxin- sensitive
protein. rFC is used in combination with a fluorogenic substrate, an incubating
fluorescence microplate reader and appropriate software to detect endotoxin. A
minimum detection limit of 0.01 EU/ml and a measurable endotoxin concentration
range of 0.01 to 10 EU/ml can be achieved.
WARNINGFor In Vitro Diagnostic Use Only. PyroGene is not intended to detect endotoxemia
in man or animals, or for use in clinical diagnosis, patient management, or for thequalification of blood or blood products.

activate a proclotting enzyme into a clotting enzyme. The chromogenic LAL assay(Lonza’s Kinetic-QCL®, KQCL) uses a synthetic chromogenic peptide substrate that can becleaved by the clotting enzyme, resulting in a product that exhibits a yellow color. The
turbidimetric assay (Lonza’s PYROGENT ®-5000, KTurb) uses the native substrate, coagulogen,which can be cleaved into coagulin. Coagulin then begins to self-associate, resulting
in an increase in turbidity. The densities of the yellow color (OD 405 nm) and turbidity(OD 340 nm) are correlated with endotoxin concentration.
Studies have demonstrated the ability of Factor C to selectively recognize endotoxin
and activate the protease cascade. To create an endotoxin-specific assay, Factor C has
been purified and cloned.5,6,7,9,10,11 When activated by endotoxin binding, recombinant
Factor C acts upon the fluorogenic substrate in the assay mixture to produce a
fluorescent signal in proportion to the endotoxin concentration in the sample.
Figure 1. Schematic drawing of the endotoxin detection mechanisms in the LAL
system and the rFC system.

Endotoxin Detection

SAFETY PRECAUTIONS
The toxicological properties of the reagents supplied have not been tested. Thesereagents are not considered hazardous according to the OSHA Hazard Communication
Standard 29 CFR 1910.1200. It is recommended that the NIH guidelines forrecombinant DNA experiments be followed along with the use of standard laboratory
precautions.

FC

The protease cascade and rationale of traditional LAL tests are illustrated in the LAL
pathway in Figure 1. Factor C, the first component in the cascade, is a protease
zymogen that is activated by endotoxin binding5, 6, 7. In this pathway, Factor B (FB) is
activated by Factor C. An alternative pathway, the Factor G (FG) pathway, can be
activated by glucan binding8. Downstream, Factor C and Factor G pathways individually

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Recombinant Factor C
GlucanFCFBBACKGROUND
A Gram-negative bacterial infection of Limulus polyphemus, the horseshoe crab,
may result in fatal intravascular coagulation.1 At the molecular level, it has been
demonstrated that endotoxin activates a serine protease catalytic coagulation cascadethat results in the gelation of Limulus blood. This cascade is used in the Limulus Amebocyte
Lysate (LAL) 1, 2, 3, 4 endotoxin detection method.

LAL

Endotoxin

FG

FB

Proclotting
EnzymeEndotoxin

FG

rFC

Clotting
EnzymeKQCL

Chromogenic
Substrate

KTurb

Coagulogen

Fluorogenic
Substrate

Product
Yellow Color
Coagulin (gel clot)
Turbidity

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rFC
Product
FluorescencePRINCIPLE

MATERIALS AND EQUIPMENT REQUIRED BUT NOT SUPPLIED

Recombinant Factor C is activated by endotoxin binding, and the active moiety
created then acts to cleave a synthetic substrate resulting in the generation of afluorogenic compound (Figure 1, rFC pathway). The assay is carried out in a 96-wellplate. Fluorescence is measured at time zero and after a one-hour incubation at 37°C + 1°C
in a fluorescence microplate reader using excitation/emission wavelengths of 380/440
nm. The difference between the one-hour reading and the time zero reading ($RFU) is
corrected for blank $RFU fluorescence. The log net fluorescence is proportional to thelog endotoxin concentration and is linear in the 0.01-10 EU/ml range. Endotoxin in asample is calculated relative to a standard curve.

1.

STORAGE CONDITIONS
1.

The PyroGene kit, catalog numbers 50-658U, 50-658NV, is stored at 2-8°C.

REAGENTS SUPPLIED AND KIT CONFIGURATIONS
1.
2.
3.
4.

5.

1.
2.
3.
4.

Disposable endotoxin-free glass dilution tubes (13 x 100 mm, #N207 orequivalent).
2.
Individually wrapped sterile measuring pipettes.
3.
Automatic hand-held pipettes with sterile, individually wrapped or racked tips.
4.
Disposable sterile microplates. Note: Prior to routine use, microplates should be
pre-qualified (#25-340 or equivalent).
5.
Reagent reservoirs (#25-364 or equivalent).
6.
Eight channel pipettor.
7.
Fluorescence microplate reader (#25-344, FLx800 TBIE reader
with Ex 380/20 and Em 440/30).
8.
Software (WinKQCL® V 3.0.1, or higher).
9.
Timer.
10. Vortex Mixer.
11. For 50-658NV, LAL Reagent Water (#W50-640 or equivalent).
TM

Catalog No. 50-658U contains:Two vials of rFC Enzyme Solution, R50-658, 1.2 ml/vial
Two vials of the Fluorogenic Substrate, S50-658, 6.0 ml/vial
Two vials of the rFC Assay Buffer, B50-658, 5.0 ml/vial
Two vials of the E. coli Endotoxin, O55:B5, E50-643, lyophilized. The reconstitution
volume of the vial is stated on the Certificate of Analysis included with each kit
and is calculated to yield a solution containing 20 EU/ml. Reconstituted endotoxin
is stable for 4 weeks at 2-8°C.
Two vials of LAL Reagent Water, W50-640, 30 ml/vial. This water is used to rehydrate E. coli endotoxin and to prepare endotoxin standard and sample dilutions.
Catalog No. 50-658NV contains:
Thirty vials of rFC Enzyme Solution, R50-658, 1.2 ml/vial
Thirty vials of the Fluorogenic Substrate, S50-658, 6.0 ml/vial
Thirty vials of the rFC Assay Buffer, B50-658, 5.0 ml/vial
Ten vials of the E. coli Endotoxin, O55:B5, E50-643, lyophilized. The reconstitution
volume of the vial is stated on the Certificate of Analysis included with each kit
and is calculated to yield a solution containing 20 EU/ml. Reconstituted endotoxin
is stable for 4 weeks at 2-8°C.

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SPECIMEN COLLECTION AND PREPARATION

Determination of appropriate sensitivity setting

Careful technique must be used to avoid microbiological or endotoxin
contamination. All materials coming in contact with the specimen or test reagents must
be endotoxin-free. Clean glassware and materials may be rendered endotoxin-free byheating at 250°C for 30 minutes. Appropriate precautions should be taken to protect
depyrogenated materials from subsequent environmental contamination.

NOTE: This procedure should be conducted as part of the performance qualification forany new lot of reagents, after major equipment maintenance (such as lamp
replacement) or when reader performance is questioned. A periodic re-verification isrecommended as part of any on-going equipment maintenance program.

From experience, most sterile, individually wrapped, plastic pipettes and pipette tips
are endotoxin-free. However, these materials should be tested before regular use.
It may be necessary to adjust the pH of the sample to within the range 6.0-8.0 using
endotoxin-free sodium hydroxide or hydrochloric acid. Always measure the pH of analiquot of the bulk sample to avoid contamination by the pH electrode. Do not adjust
unbuffered solutions.

1.

Prepare a 1 EU/ml endotoxin solution (see Preparation of Endotoxin Standards).
Allow reagents to equilibrate to ambient temperature before mixing.

2.

Using WinKQCL Software, select Validation/FLx800 Reader/SensitivityTest thenclick the New button.

3.

Enter appropriate information in blank fields and confirm settings for the test.
Endotoxin Concentration Unitage
EU
Time Between Reads (hh:mm:ss)
01:00:00
Excitation Filter (nm)380:20Emission Filter (nm)440:30
Reads per Well
4
Delay before reading (millisec.)
150
Optics Position
bottom
Wells to Use: Pre-defined(D6,7; E6,7; F6,7)
Sensitivity (select by checking)30, 35, 40, 45, 50, 55

4.

Add 100 Ml of the 1 EU/ml solution to wells D6,7; E6,7; F6,7.

5.

Pre-incubate covered plate with standards at 37°C + 1°C for 10 minutes.

6.

Prepare working reagent by mixing rFC enzyme solution, rFC assay buffer andfluorogenic substrate at 1:4:5 ratios, respectively. 100 Ml + 400 Ml + 500 Ml is asufficient quantity for the sensitivity test. Refrigerate any unused reagents after
opening. Uncontaminated previously opened vials remain effective throughout the
lifetime of the reconstituted endotoxin if returned to refrigeration. NOTE: The orderof mixing should remain consistent. Add enzyme last to the combined buffered
substrate. Mix thoroughly but gently. Avoid vortexing of the working reagent mix.
NOTE: Once prepared, the working reagent cannot be stored.

7.

At the appropriate software prompt add 100 Ml of working reagent to all wells that
contain the endotoxin sample. Click OK to initiate test. Note: leave the plateuncovered for the duration of the test.

Samples to be tested must be stored in such a way that all bacteriological activity is
stopped or the endotoxin level may increase with time. For example, store samples at
2-8°C for less than 24 hours; samples stored longer than 24 hours should be frozen.
SENSITIVITY SETTING FOR FLUORESCENT READER
Fluorescence signals are normally recorded as Relative Fluorescence Units (RFU).
Since the real fluorescence signal is converted to an electronic signal that can be“tuned” using the gain setting or sensitivity setting, RFU is an arbitrary unit. Dependingon the strength of the detected signal, the instrument can be adjusted to a higher
gain/sensitivity setting to boost a weak signal or tuned down to a lower gain/sensitivity
setting when the signal is too strong. It is useful to consult the FLx800 Reader
Operator’s Manual, Operation Section for the manufacturer ’s explanation.
TM

In the PyroGene assay, the 3-log endotoxin concentration range corresponds to the
3-log range in RFU. If the sensitivity is tuned too low, fluorescence at the lowest
standard will be difficult to detect; if the sensitivity is tuned too high, fluorescence at thehighest standard will be off-scale. Prior to conducting any assays, it is important todetermine the appropriate sensitivity for the PyroGene assay. The FLx800 has afluorescence range from 0 to 99999. In order to establish the 3-log fluorescence range
for the standard curve, an RFU range from 1000 to 10000 was chosen for the 1
EU/ml standard. The RFU range from 1000 to 10000 corresponds to a log range from
3 to 4, with a log mid point of 3.5. Thus, the target absolute net RFU for the 1 EU/ml isapproximately 3000 RFU. To maintain adequate separation between the blanks and thelowest standard in a routine assay, this value should be considered the minimum of adesirable range.
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8.

At the completion of the test a summary of the results with the calculatedsensitivity will be displayed. Using linear regression, the sensitivity value
correlated to log $RFU = 3.5 is rounded to the nearest integer. Successful assays
should result when using this setting as a minimum. Both the Analyst andReviewer should e-sign then print the report.
Sensitivity Setting Determined:_______________________
for Kit Lot :_______________________________________

PREPARATION OF ENDOTOXIN STANDARDS

Volume of Endotoxin Standard
added toLAL Reagent Water

10
1
0.1
0.01

0.5 ml0.9 ml0.9 ml0.9 ml

0.5 ml of 20 EU/ml solution
0.1 ml of 10 EU/ml solution
0.1 ml of 1 EU/ml solution
0.1 ml of 0.1 EU/ml solution

Prepare a solution containing 10 EU/ml endotoxin by adding 0.5 ml of the 20
EU/ml endotoxin stock solution into 0.5 ml of LAL Reagent Water in a suitablecontainer. This solution should be vigorously vortexed for at least 1 minute before
proceeding.

2.

Transfer 0.1 ml of the 10 EU/ml endotoxin solution into 0.9 ml of LAL Reagent
Water in a suitable container and label 1 EU/ml. The solution should be vigorously
vortexed for at least 1 minute before proceeding.

3.

Transfer 0.1 ml of the 1 EU/ml endotoxin solution into 0.9 ml of LAL Reagent
Water in a suitable container and label 0.1 EU/ml. The solution should bevigorously vortexed for at least 1 minute before proceeding.

4.

Transfer 0.1 ml of the 0.1 EU/ml endotoxin solution into 0.9 ml of LAL Reagent
Water in a suitable container and label 0.01 EU/ml. The solution should bevigorously vortexed for at least 1 minute before proceeding.

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The PyroGene assay has been optimized to be linear from 0.01 EU/ml to 10 EU/ml.
However, the individual user may choose to truncate the standard curve depending on
specific product testing requirements.
®

The E. coli endotoxin, O55:B5, is provided for user convenience. Other endotoxin
preparations may be used to prepare the standards; however, their performance in the
PyroGene assay relative to the current USP Reference Standard Endotoxin (RSE) must
be determined.

Volume of
LAL Reagent
Water

1.

In order to calculate the endotoxin concentration in unknown samples, each
PyroGene test must be referenced to a valid standard curve.
Because of the large concentration range over which endotoxin values can be
determined, it is possible to adjust the quantitative range of any given test by adjusting
the concentration of endotoxin standards used to generate the standard curve. A
minimum of three standards is required.

Endotoxin
Concentration(EU/ml)®

The supplied lyophilized E. coli O55:B5 endotoxin is reconstituted with LAL Reagent
Water to yield a 20 EU/ml stock solution. The reconstitution volume of the vial is statedon the Certificate of Analysis, included with each kit, and is calculated to yield a solution
containing 20 EU/ml. Reconstitute with the specified volume of LAL Reagent Water.
Shake vigorously for at least 15 minutes at high speed on a vortex mixer. Prior to futureuse, the solution must be warmed to room temperature and vigorously vortexed for 15
minutes.
The following table suggests a dilution scheme for constructing a series ofendotoxin dilutions from the endotoxin supplied in the kit.

9

TESTING PROCEDURE
General testing procedure
1.

2.
3.
4.

The PyroGene endotoxin assay uses a 96-well plate assay format. Add 100 Ml ofthe blank, endotoxin standards, and samples to appropriate wells of the
microplate. Duplicate or triplicate assays are generally used.
To spike the samples with endotoxin, add 10 Ml of the 1 EU/ml solution to thedesignated Positive Product Control wells.
Pre-incubate the plate in the reader at 37°C + 1°C for a minimum of 10 minutes.
During the incubation period, prepare the working reagent. The rFC enzyme
solution, assay buffer and substrate are mixed in a 1:4:5 ratio, respectively, toform the working reagent.
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10

Carefully dispense 100 Ml of the working reagent to each well.
Read fluorescence at time zero.
Incubate the reaction for one hour and read the plate at elapsed time one-hour.
The difference of time one-hour and time zero readings are corrected with theblank.
9.
The log net $RFU is then plotted against log endotoxin concentration in a linear
regression curve.
10. An example curve is shown below:
5.
6.
7.
8.

&OEPUPYJO4UBOEBSE$VSWFJOUIFS'$BTTBZ
1e+6'MVPSFTDFODF 3'6

1e+5

1e+4

1e+3

1.

Name: Enter the template name. The template name can be a maximum of 20
characters long, and no two templates can have the same name.

2.

Analyst: The Analyst ID of the user is automatically entered and cannot bechanged.

3.

Test Type: Click on the down arrow button then click on Initial Qualification.

4.

Type of Assay: Select the PyroGene radio button.

5.

Parameters: The reader parameter values for a template may be verified orchanged by clicking on Parameters.

1e+2
Log y = 0.946 Log x + 3.588
1e+1

r=1.000

1e+0
0.001

0.01

0.1

1

10

100

&OEPUPYJO &6NM®

11. Calculate endotoxin concentration of samples according to the standard curve.

When using FLx800 and WinKQCL Software
TM®

The fluorescence reader FLx800 and WinKQCL Software can be used inthis assay. Turn on the reader and launch the software. In the software, create a template
for the rFC assay using Templates / New. Several types of assays may be programmed.
Consult the Software User Manual for more information. The example that follows describes
setting up an Initial Qualification Assay.
TM®

Settings pictured are the defaults used in the assay.

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12

6.

Lot Numbers: Selecting the Lot No./Exp Dates button will bring up the following
screen:Enter all appropriate information.
7.

Standards: Select the number of standards by clicking on the up or down arrow
button. A minimum of 3 and a maximum of 12 standards are allowed. Fourstandards are typically run with the PyroGene® assay.

8.

Standards Setup: Click on Standards Setup to enter the concentrations and unitsof the standard curve.

9.

Layout:Defines the positioning of the blank, standards and samples. Number of
replicates is selectable from 2 to 6. Click OK after completing the Standard Layout
Setup dialog box.
10. Save the template. From the list of the templates an assay is initiated by selecting
Run. Follow on screen prompts to begin the test. Consult Software User Manual
for more details.
11. Place the microplate (loaded with blank and standards) in the reader and pre-incubate
the plate for a minimum of 10 minutes at 37°C + 1°C.
12. During the 10 minute incubation, prepare working reagent by mixing the rFC
enzyme solution, the assay buffer and the fluorogenic substrate in a 1:4:5 ratio,
respectively. To ensure enough working reagent is prepared for the assay andexcess is minimal, determine the number of wells requiring working reagent, add
4 additional wells, and calculate the appropriate amount for each component tomake the working reagent. Allow reagents to equilibrate to room temperature
before mixing. The order of mixing should remain consistent. Add enzyme last
to the combined buffered substrate. Mix gently but thoroughly. Do not vortex. Referto the following table:
NOTE: Once prepared, the working reagent cannot be stored.

Click OK after completing the Standards Setup dialog box.

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14

Volume ( l) of reagent required per total number of wells
To assure slight excess, assume volume prepared is
Total number of
wells
6
12
24
36
42
48
54
60
66
72
78
84
90
96

13.

rFC enzyme
solution
100
160
280
400
460
520
580
640
700
760
820
880
940
1000

rFC
assay

Fluorogenic
substrate
500
800
1400
2000
2300
2600
2900
3200
3500
3800
4100
4400
4700
5000

400
640
1120
1600
1840
2080
2320
2560
2800
3040
3280
3520
3760
4000

Total Volume
1000
1600
2800
4000
4600
5200
5800
6400
7000
7600
8200
8800
9400
10000

When prompted by the software at the end of the incubation period, dispense
100 Ml of working reagent into the appropriate wells using an eight channel pipettor.

14. Immediately click OK. The software will initiate read one (time zero reading)
immediately and read two an hour later.

1. ROUTINEA ROUTINE assay calculates the concentration of endotoxin in unknowns bycomparison to the performance of a series of endotoxin standards.
As part of a ROUTINE assay, the user has the option to include a Positive ProductControl (PPC) as a monitor for product inhibition or enhancement. A PPC is a sample
of product to which a known amount of endotoxin spike has been added. The WinKQCLSoftware automatically calculates the amount of endotoxin recovered in the PPC,
allowing for a comparison to the known amount of endotoxin spike.

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2. INHIBITION/ENHANCEMENT (INH/ENH)
The PyroGene reaction is enzyme mediated and, as such, has an optimal pH range
and specific salt and divalent cation requirements. Occasionally, test samples may alter
these optimal conditions to an extent that the rFC is rendered insensitive to endotoxin.
Negative results with samples which inhibit the PyroGene test do not necessarily
indicate the absence of endotoxin.
An INHIBITION/ENHANCEMENT assay is designed to determine what level ofproduct dilution overcomes inhibition or enhancement. Each product dilution must be
accompanied by a Positive Product Control (PPC). The WinKQCL Software calculates
the amount of endotoxin recovered in the PPC for comparison to the known amount ofendotoxin spike. In this manner it can be determined which product dilutions are noninhibitory.
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3. RSE/CSE
An RSE/CSE assay is designed to determine the potency of a Control Standard
Endotoxin (CSE) in terms of the concentration units of the Reference Standard
Endotoxin (RSE).
The assay requires a single series of RSE dilutions and one or more sets of dilutionsof the CSE. Depending on the concentration units of the CSE, the WinKQCL Software
automatically computes mean potency values in terms of EU/ml or EU/ng. The user also
has the option to enter units other than EU or ng.
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15. After the assay is completed, the data is automatically saved to the hard drive.
TYPES OF PYROGENE ASSAYS
The incubating fluorescence microplate reader and WinKQCL Software are an
integral part of the PyroGene assay. It is important to become familiar with the operationof the incubating microplate reader and the features of the WinKQCL Software. Pleaserefer to the incubating microplate reader and WinKQCL Software Manuals for moredetailed information.
There are four (4) basic types of PyroGene assays. Each type is designed to performa different aspect of endotoxin testing.
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4. INITIAL QUALIFICATION (INT. QUAL)An INITIAL QUALIFICATION assay is designed according to the requirements
described in the FDA’s “Guideline on Validation of the Limulus Amebocyte Lysate Test
as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological
Products, and Medical Devices”.12 This assay is required as part of the validation of the
LAL assay and is also to be performed with each new kit lot. PyroGene testing evolved
from the LAL test and Lonza suggests a similar approach be utilized.
The INITIAL QUALIFICATION assay performs a log/log linear correlation of theindividual Net $RFU values for each replicate of each endotoxin standard. The otherassays use the average Net $RFU of all the replicates of each standard.
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The INITIAL QUALIFICATION assay does not provide for the inclusion of any
samples.

A spiked aliquot of the test sample (or dilution) may be prepared as in one of thefollowing examples:

PERFORMANCE CHARACTERISTICS
Tube Method:
Linearity*
The linearity of the standard curve within the concentration range used to determine
endotoxin values should be verified. Assay no less than 3 endotoxin standards,
spanning the desired concentration range, and a LAL Reagent Water blank at least in
triplicate. Additional standards should be included to bracket each log interval over therange of the standard curve. The value of the correlation coefficient (r) of thecalculated standard curve should be ≥ 0.980.
*See Further data considerations under Other Technical Issues section.

Transfer 50 µl of the 10 EU/ml solution into 4.95 ml of test sample (or dilution).
This solution contains an endotoxin concentration of 0.1 EU/ml in test sample (ordilution). This sample should be vigorously vortexed for one minute prior to use.
Plate Method #1:
Transfer 10 µl of the 1 EU/ml solution into each of the PPC wells in the 96-well plate,as directed by the assay template. To these wells add 100 µl of test sample (or dilution).
Each well will now contain a 0.1 EU/ml solution. Mix gently.
Plate Method #2:

PRODUCT INHIBITION
Product inhibition occurs when substances in the test sample interfere with the
enzyme reaction. Inhibition results in a lower final 6RFU in a sample reading, indicating
lower levels of endotoxin than what may actually be present in the test sample. The lackof product inhibition should be determined for each specific sample, either undilute orat an appropriate dilution.
To verify the lack of product inhibition, an aliquot of a test sample (or a dilution ofa test sample) is spiked with a known amount of endotoxin. It is recommended that theendotoxin spike result in a final endotoxin concentration in the sample equal to 0.1
EU/ml. For samples which may contain a background endotoxin level > 0.1 EU/ml, theendotoxin spike should result in a final endotoxin concentration of 1.0 EU/ml. The
spiked solution is assayed along with the unspiked samples and their respective
endotoxin concentrations are determined. The differences between these two
calculated endotoxin values should equal the known concentration of the spike within
the range of 50-200%.13

Place 100 µl of test sample (or dilution) into the PPC wells in the 96-well plate, as
directed by the assay template. To these wells, add 10 µl of the 1 EU/ml solution. Eachwell will now contain a 0.1 EU/ml solution. Mix gently.
If the test sample (or dilution) is found to be inhibitory to the enzyme reaction, thesample may require further dilution until the inhibition is overcome.

Determination of a Non-Inhibitory Dilution with
0.1 EU/ml Endotoxin Spike
SampleRaw Endotoxin (EU/ml)
Spike
SampleEndotoxin
Dilution Unspiked Spiked Difference Recovery
Inhibition (Raw x DF)
1/10
0.075
0.100
0.025
25%
Inhibitory
N/A
1/20
0.044
0.088
0.044
44%
Inhibitory
N/A
1/40
0.025
0.105
0.080
80%
Non-Inhibitory 1 EU/ml

Initially, Lonza recommends screening for product inhibition by testing ten-folddilutions of the test sample. Once the approximate non-inhibitory dilution isdetermined, the exact dilution can be found by testing two-fold dilutions around thisdilution.

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REFERENCESPATENT INFORMATION

1.

Various components of PyroGene® are protected under the following patents:
US 4,745,051; US 4,879,236; US 5,716,834; US 5,712,144; Australia 581,174;
Canada 1,222,213; Colombia 24,556; Denmark 172,401; EP 127,839; Ireland
58,011; Israel 71,906; Japan 2,129,487 & 2,644,447; Korea 51,077; Mexico 164,250;
Norway 173,944; New Zealand 208,259; Philippines 25,395; South Africa 843,914;
Spain 532,825; Taiwan 50,740. Additional Patents Pending.

2.
3.
4.
5.

6.

7.

8.
9.

10.

11.
12.

13.

Levin, J. and F.B. Bang. The Role of endotoxin in the extracellular coagulation ofLimulus blood. Bull. Johns Hopkins Hosp. 115: 265 (1964).
Levin, J. and F.B. Bang. A description of cellular coagulation in the Limulus. Bull.
Johns Hopkins Hosp. 115: 337 (1964).
Levin, J. and F. B. Bang. Clottable protein in Limulus: its localization and kinetics
of its coagulation by endotoxin. Thromb. Diath. Haemorrh. 19: 186 (1968).
Iwanaga S. The Limulus clotting reaction. Curr. Opin. Immunol. 5: 74 (1993).
Nakamura T., Morita T., Iwanaga S. Lipopolysaccharide-sensitive serine-protease
zymogen (factor C) found in Limulus hemocytes. Eur. J. Biochem. 154: 511(1986).
Muta T., Miyata T., Misumi Y. et al. Limulus Factor C: an endotoxin-sensitive
serine protease zymogen with a mosaic structure of complement-like, epidermal
growth factor-like, and lectin-like domains. J. Biol. Chem. 266: 6554 (1991).
Tokunaga F., Nakajima H., Iwanaga S. Further studies on lipopolysaccharidesensitive serine protease zymogen (factor C): its isolation from Limulus
polyphemus hemocytes and identification as an intracellular zymogen activated byalpha-chymotrypsin, not by trypsin. J. Biochem. 109: 150 (1991).
Morita, T. et al. A new (1-3) beta-D glucan mediated coagulation pathway found in
Limulus amebocytes. FEBS Letts. 129: 318 (1981).
Ding J.L., Navas M.A.A., Ho B. Two forms of Factor C for the amebocytes ofCarcinoscorpius rotundicauda: purification and characterization. Biochim.
Biophys. Acta 1202: 149 (1993).
Ding J.L., Navas M.A.A., Ho B. Molecular cloning and sequence analysis of Factor
C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda. Mol.
Mar. Biol. Biotechnol. 4: 90 (1995).
Ding J.L. and Ho B. New era in pyrogen testing. Trends in Biotechnology, 19: 277(2001).
U.S. Department of Health and Human Service, Food and Drug Administration,“Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product
Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, andMedical Devices” (1987).
Chapter <85> Bacterial Endotoxins Test. Rockville, MD: United States
Pharmacopeia.

19

OTHER TECHNICAL ISSUES
Improving spike recovery
By using a different data treatment procedure (for example, a polynomial curve fit using
POWERCURVE), calculation of the recovery of the positive product control can be
improved.
Truncation of standard curve
In our experience, a truncated range of endotoxin standards, 0.01 – 1 EU/ml or 0.110 EU/ml, can provide better linearity than the whole range of 0.01- 10 EU/ml.
Overcoming inhibition with a dispersing agent
PYROSPERSE®, #N190, a dispersing agent, is not compatible with the PyroGene®
assay.

TRADEMARK INFORMATIONThe FLx800TM is a trademark of BioTek Instruments, Inc.
Unless otherwise noted, all trademarks herein are marks of the Lonza Group or
its affiliates.

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NOTES

NOTES

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