Limulus Amebocyte Lysate (LAL)PYROGENT¡-5000

Catalog Numbers: N383, N384, N588, N688

LIMULUS AMEBOCYTE LYSATEPYROGENT® -5000
U.S. License No. 1775
Catalog No. N383, N384, N588, N688
IMPORTANT
Read Entire Brochure Before Performing Test
INTENDED USEThis product is intended as an In Vitro end-product endotoxin test for humanand animal parenteral drugs, biological products, and medical devices. This product is not intended for the detection of endotoxin in clinical samples or as an aid
in the diagnosis of human disease. This test utilizes a preparation of Limulus
Amebocyte Lysate (LAL), in combination with an incubating photometer andappropriate software, to detect endotoxin photometrically.
In December 1987, the United States Food and Drug Administration (FDA)
published the “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”.8 This guideline outlines those procedures which the FDA considers necessary for: 1) establishing endotoxin limits forpharmaceuticals and medical devices, 2) validating the use of LAL as an endproduct endotoxin test, and 3) developing a routine testing protocol.
The procedures described herein are based on those described in the FDA
Guideline. Similar performance requirements for kinetic-turbidimetric assays havebeen published and are updated regularly in the United States Pharmacopeia.10

2

WARNING
For In Vitro Diagnostic Use Only. Not for the In Vitro Determination ofEndotoxemia in Man. The LAL Test may be substituted for the USP Rabbit
Pyrogen Test when used according to the FDA Guideline for end-product testingof human and animal parenteral drugs, biological products, and medical devices8.
EXPLANATION OF TESTPYROGENT®-5000 is a quantitative, kinetic assay for the detection of Gram
negative bacterial endotoxin. A sample is mixed with the reconstituted LAL
reagent, placed in the photometer, and automatically monitored over time for theappearance of turbidity. The time required before the appearance of turbidity(Reaction Time) is inversely proportional to the amount of endotoxin present. Thatis, in the presence of a large amount of endotoxin the reaction occurs rapidly; inthe presence of a smaller amount of endotoxin the reaction time is increased. The
concentration of endotoxin in unknown samples can be calculated from a standard curve.
The use of LAL for the detection of endotoxin evolved from the observation by
Bang1 that a Gram negative infection of Limulus polyphemus, the horseshoe crab,
resulted in fatal intravascular coagulation. Levin and Bang2,3 later demonstrated
that this clotting was the result of a reaction between endotoxin and a clottable
protein in the circulating amebocytes of Limulus. Following the development of asuitable anticoagulant for Limulus blood, Levin and Bang4 prepared a lysate fromwashed amebocytes which was an extremely sensitive indicator of the presenceof endotoxin. Solum5,6 and Young, Levin and Prendergast9 have purified and characterized the clottable protein from LAL and have shown the reaction with endotoxin to be enzymatic. Teller and Kelly7 demonstrated that the endotoxin activationof lysate could be monitored photometrically.

3

PRINCIPLE
Proenzyme
Coagulogen

Endotoxin
Coagulase

Coagulase
Coagulin

Gram negative bacterial endotoxin catalyzes the activation of a proenzyme in
the Limulus Amebocyte Lysate9. The initial rate of activation is determined by theconcentration of endotoxin present. The activated enzyme (Coagulase) hydrolyzes
specific bonds within a clotting protein (Coagulogen) also present in Limulus
Amebocyte Lysate. Once hydrolyzed, the resultant coagulin self-associates andforms a gelatinous clot. The turbidimetric LAL assay measures the increase in turbidity (optical density) that precedes the formation of the gel clot.
REAGENTS SUPPLIED AND STORAGE CONDITIONSPYROGENT®-5000 LAL Reagent
The LAL reagent contains a lysate prepared from the circulating amebocytesof the horseshoe crab, Limulus polyphemus. Reconsititute before use withPYROGENT®-5000 LAL Reconstitution Buffer, per the following table:

Kit part number
N383
N384
N588
N688

PYROGENT®-5000
LAL Reagent
2 vials/kit
2 vials/kit
45 vials/kit
45 vials/kit

PYROGENT®-5000 LAL
Reconstitution Buffer
required
5.2 ml/vial
10.4 ml/vial
5.2 ml/vial
10.4 ml/vial

Swirl gently to avoid foaming.
IMPORTANT: Prior to use, wait about two (2) minutes to allow any bubbles to4

rise to the top of the vial.
Store lyophilized (unreconstituted) PYROGENT®-5000 LAL under refrigeration
at 2-8oC. Avoid exposure to temperatures in excess of 25oC. Avoid exposure tobright light. Discard lysate which has turned yellow or become insoluble.
Reconstituted PYROGENT®-5000 LAL is stable up to 8 hours at 2-8°C.

PYROGENT®-5000 LAL RECONSTITUTION BUFFER
This buffer must be used to rehydrate the PYROGENT®-5000 LAL Reagent.
Allow buffer to warm to room temperature before use.
Kit part number
N383
N384
N588
N688

PYROGENT®-5000 LAL
Reconstitution Buffer
2 vials/kit
2 vials/kit
45 vials/kit
45 vials/kit

Store PYROGENT®-5000 LAL Reconstitution Buffer at 2-8oC.
E. coli O55:B5 Endotoxin
The N383 and N384 kits contain 1 vial of lyophilized endotoxin per kit;
the N588 and N688 kits contain 10 vials per kit. The reconstitution volume ofthe vial is stated on the Certificate of Analysis, included with each kit, and is
calculated to yield a solution containing 100 EU(or IU)/ml. Reconstitutewith the specified volume of LAL Reagent Water. Shake vigorously for at least
15 minutes at high speed on a vortex mixer. Prior to use, a stored stock solution
must be warmed to room temperature and vigorously vortexed for 15 minutes.

5

Store lyophilized endotoxin at 2-8oC. Reconstituted endotoxin is stable up to
four weeks at 2-8oC.
This endotoxin is provided for the user’s convenience. Other endotoxin preparations may be used to prepare the standards; however, their performance in theturbidimetric assay relative to the Reference Standard Endotoxin (RSE) must be
determined.
MATERIALS AND EQUIPMENT NOT PROVIDED
1. LAL Reagent Water (#W50-640 or equivalent).
2. Sodium hydroxide, 0.1N, or Hydrochloric acid, 0.1N, dissolved in LAL
Reagent Water, for pH adjustment of sample if necessary.
3. Disposable endotoxin-free glass dilution tubes (13 x 100 mm,#N207 or equivalent).
4. Individually wrapped measuring pipettes.
5. Automatic hand-held pipettes with low endotoxin, individually wrapped or
racked tips.
6. Disposable sterile microplates. Prior to routine use, microplates should be
pre-qualified for each lysate lot by the acceptable performance of the linearity test as described under PERFORMANCE CHARACTERISTICS.
7. Eight channel pipettor.
8. Reagent reservoirs (#25-364 or equivalent).
9. Microplate reader (Kinetic-QCL® Reader, #25-141B; ELx808TM IU
Reader, #25-315; ELx808TM RIU Reader, #25-315R. )10. WinKQCL Software.
11. Timer and vortex mixer.

6

SPECIMEN COLLECTION AND PREPARATION
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-freeby heating at 250°C for 30 minutes. Appropriate precautions should be taken toprotect depyrogenated materials from subsequent environmental contamination.
From experience, most sterile, individually wrapped, plastic pipettes andpipette 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 thepH of an aliquot of the bulk sample to avoid contamination by the pH electrode.
Do not adjust unbuffered solutions.
Samples to be tested must be stored in such a way that all bacteriologicalactivity 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 24hours should be frozen.
TYPES OF TURBIDIMETRIC LAL ASSAYS
The incubating microplate reader with the WinKQCL® Software may be used to
run the turbidimetric LAL assay. It is important to become familiar with the operation of the incubating microplate reader and the features of the WinKQCL®
Software. Please refer to the appropriate software manual for more detailed information.
There are four (4) basic types of turbidimetric LAL assays, each of which isdesigned to perform a different aspect of LAL testing.

7

1.

ROUTINE
A 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
Product Control (PPC) as a monitor for product inhibition or enhancement.
A PPC is a sample of product to which a known amount of endotoxin spikehas been added. The WinKQCL ® Software automatically calculates theamount of endotoxin recovered in the PPC, allowing for a comparison to the
known amount of endotoxin spike.

2.

INHIBITION/ENHANCEMENT (INH/ENH)
The Limulus Amebocyte Lysate 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 lysate is rendered insensitive to endotoxin. Negative results withsamples which inhibit the LAL test do not necessarily indicate the absenceof endotoxin.
An INHIBITION/ENHANCEMENT assay is designed to determine what levelof product dilution overcomes inhibition or enhancement. Each product dilution must be accompanied by a Positive Product Control (PPC). The software calculates the amount of endotoxin recovered in the PPC for comparison to the known amount of endotoxin spike. In this manner it can be determined which product dilutions are non-inhibitory.

8

3.

RSE/CSEAn RSE/CSE assay is designed to determine the potency of a Control
Standard Endotoxin (CSE) in terms of the concentration units of the USP
Reference Standard Endotoxin (RSE), or equivalent.
The assay requires a single series of RSE dilutions and one or more setsof dilutions of the CSE. Depending on the concentration units of the CSE,
the WinKQCL® Software automatically computes mean potency values interms of EU/ng or EU/ml. The user also has the option to enter units otherthan EU or ng.

4.

INITIAL QUALIFICATION (INITIAL 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 andAnimal Parenteral Drugs, Biological Products, and Medical Devices”.8 This
assay is required as part of the initial qualification and requalification of
the laboratory analysts and is also to be performed with each new lot of
PYROGENT®-5000.
The INITIAL QUALIFICATION assay performs a log/log correlation of theindividual Reaction Time values for each replicate of each endotoxin standard. The other assays use the average Reaction Time of all the replicates ofeach standard.
The INITIAL QUALIFICATION assay does not provide for the inclusion of any
samples.

9

REAGENT PREPARATION
In order to calculate endotoxin concentrations in unknown samples each turbidimetric LAL assay 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 assay byadjusting the concentration of endotoxin standards used to generate the standard
curve. A minimum of three standards is required.
The PYROGENT®-5000 assay has been optimized to be linear from 0.01 EU/ml
to 100 EU/ml. However, the individual user may choose to truncate the standard
curve depending on specific product requirements. Data indicates that truncating
a turbidimetric LAL standard curve may improve the accuracy of predicted endotoxin values for test samples. It is recommended that the user be familiar with the
FDA interim guidance requirements for kinetic LAL techniques prior to establishing a turbidimetric LAL standard curve range to be used for routine testing ofproduct samples.11
The table that follows suggests a dilution scheme for constructing a series ofendotoxin dilutions from the endotoxin supplied in the kit. Not all dilutions must
be used to generate a standard curve. Alternative dilution schemes can be used aswell as other endotoxins not supplied in this kit. If the endotoxin used is not supplied in the kit, an RSE/CSE test to determine the CSE potency may be required,
per the FDA Guideline.8

10

Endotoxin
Concentration(EU/ml)
10
1
0.10
0.01
1.

2.

3.

4.

Volume of
LAL Reagent
Water
0.9 ml0.9 ml0.9 ml0.9 mlVolume of Endotoxin
Solution added to
LAL Reagent Water
0.1 ml of 100 EU/ml solution
0.1 ml of 10 EU/ml solution
0.1 ml of 1 EU/ml solution
0.1 ml of 0.10 EU/ml solution

Prepare a solution containing 10 EU/ml endotoxin by adding 0.1 ml of the
100 EU/ml endotoxin stock into 0.9 ml of LAL Reagent Water. This solution
should be vigorously vortexed for at least 1 minute before proceeding.
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.
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 0.10 EU/ml. This solution should bevigorously vortexed for at least 1 minute before proceeding.
Transfer 0.1 ml of the 0.10 EU/ml endotoxin solution into 0.9 ml of LAL
Reagent Water in a suitable container and label 0.01 EU/ml. This solution
should be vigorously vortexed for at least 1 minute before proceeding.

TEST PROCEDURE
IMPORTANT: Refer to the microplate reader and WinKQCL® Software manuals
for more detailed information concerning the operation of the software andreader. The reader must be located in an area free of excessive vibration (ex. centrifuges, shakers, etc.) during the actual running of the test.

11Refer to the microplate reader and WinKQCL® Software manuals for moredetailed information on performing a PYROGENT®-5000 test.
1.

2.

3.
4.
5.

6.

7.
8.

Create a specific TEMPLATE for the test to be run. A TEMPLATE contains thename of the analyst, type of assay, lot numbers of reagents, the number andconcentration of endotoxin standards, number of replicates, and how standards and samples will be organized on the microplate.
The Type of Assay must be set as PYROGENT®-5000 . The default Template
Parameters that follow must not be changed:
Delta t (seconds)
060
Measurement Filter (nm)340
Delta mOD
030
Number of Reads
100
Print the TEMPLATE for use as a guide in placing standards and samples
into the microplate.
“Run” the TEMPLATE, following the WinKQCL® Software prompts.
Carefully dispense 100 Ml of the LAL Reagent Water blank, endotoxin standards, product samples, positive product controls (see pages 20-22 for positive product control instructions), etc. into the appropriate wells of the
microplate. Bubbles must be avoided!
Place the filled plate in the microplate reader and close the lid. If using theKinetic-QCL® Reader, press the UP directional key on the reader to position
the plate in the incubation chamber.
Pre-incubate the plate for r10 minutes at 37oC ± 1oC.
Near the end of the pre-incubation period reconstitute each of the appropriate number of the PYROGENT®-5000 Reagent vials with PYROGENT®-5000

12

Reconstitution Buffer (5.2 ml for the N383 or N588 kits and 10.4 ml for theN384 and N688 kits). Mix gently but thoroughly. Allow any bubbles to riseto the top of the vial before use.
9. Pool the reagents into a reagent reservoir and mix by gently rocking thereservoir from side to side.
10. If using the Kinetic-QCL® reader, open the lid and press the DOWN directional key to remove the microplate from the incubation position.
11. Using an 8 channel multipipettor dispense 100 Ml of the PYROGENT®-5000
Reagent into all wells of the microplate beginning with the first column (A1H1) and proceeding in sequence to the last column used. Add reagent as
quickly as possible. Bubbles must be avoided!
12. Immediately click on the OK button in the dialog box to initiate the test.
NOTE: The PYROGENT®-5000 assay is performed with the microplate cover
removed.
PERFORMANCE CHARACTERISTICS
Linearity
The linearity of the standard curve within the concentration range used todetermine endotoxin values should be verified. No less than 3 endotoxin standards, spanning the desired concentration range, and an LAL Reagent Water blankshould be assayed at least in triplicate according to the test parameters of an INITIAL QUALIFICATION assay. Additional standards should be included to bracket
each log interval over the range of the standard curve.
The absolute value of the coefficient of correlation (r) of the calculated standard curve should be r0.980.

13

Reproducibility
Replicate samples should be run in order to establish good technique and low
coefficient of variation. The coefficient of variation (C.V.) equals the “sample”
standard deviation of the reaction times divided by the mean and is usually
expressed as a percent. The %C.V. of the reaction times for replicates of a sample
should be less than 10%. With experience, values of 3-4% should be attainable.
CALCULATION OF ENDOTOXIN CONCENTRATION
Continuously throughout the assay, the microplate reader / WinKQCL® Software
monitors the absorbance at 340 nm of each well of the microplate. Using the initial absorbance reading of each well as its own blank, the reader determines thetime required for the absorbance to increase 0.03 absorbance units. This time istermed Reaction Time. The WinKQCL® Software automatically performs a log/log
linear correlation of the Reaction Time of each standard with its corresponding
endotoxin concentration. The standard curve parameters are printed on the reportprintout. If the absolute value of the correlation coefficient (r) is r0.980, a polynomial model can be used to construct a standard curve and in turn predict endotoxin concentrations of test samples. This polynomial curve-fitting model (POWERCURVE£) requires the use of the WinKQCL® Software (see POWERCURVE£, page 19).
Linear Regression
The information that follows is an example of how the the WinKQCL® Software
performs the log/log linear correlation and computes endotoxin concentrations in
unknowns. It is not necessary to perform these calculations independently. For
each sample of each product, the WinKQCL® Software calculates the corresponding

14

endotoxin concentration from the Reaction Time for that sample. The software
automatically adjusts the final TEST RESULT value to account for any productdilution.

Standards
Neg. Control
S1
S2
S3
S4
S5
Samples
1
2

LINEAR CORRELATION
EXAMPLE CALCULATIONS
MeanLog
Concentration Reaction Time (Sec) Concentration

Log Mean
Reaction Time±
0.010 EU/ml
0.100 EU/ml
1.000 EU/ml
10.000 EU/ml
100.000 EU/ml

Unreactive
3103
1484
808
485
312

±
-2.000-1.000
0.000
1.000
2.000

±
3.492
3.171
2.907
2.686
2.494

±
±

1576
943

±
±

3.198
2.975

15

16

17

18

POWERCURVETM
If the absolute value of the correlation coefficient (r) is r0.980, a polynomial
model can be used to construct a standard curve and predict endotoxin concentrations of test samples. It has been determined that this polynomial model (POWERCURVETM) improves the accuracy of predicting endotoxin concentrations over theentire (5 log) endotoxin range. The use of the POWERCURVETM model requires theuse of WinKQCL® Software.
When using POWERCURVETM, a standard curve is generated using the log 10
Reaction Time values and their corresponding log10 endotoxin concentration todefine a polynomial equation. The order of the polynomial equation used to generate the regression curve is determined by the number of endotoxin standards inthe assay. The order of the polynomial will always be one less than the number ofendotoxin standards, with a maximum of a 4th order polynomial for assays withfive or more endotoxin standards and a minimum of a second order polynomial
for assays with three standards.
Finding solutions to these polynomial equations is readily accomplished using
the WinKQCL® POWERCURVETM Software. The information provided below is an
example of a solution to a polynomial equation using the same set of data fromthe linear correlation example on page 15.
Polynomial (POWERCURVETM) Model
Y = A + BX + CX2 + DX3 + EX4
A = 2.90741
B = -0.24066
C = 0.02111
D = -0.00219
E = 0.00007

19

The standard curve parameters are printed on the report printout. The
WinKQCL® POWERCURVETM Software uses these parameters to calculate the corresponding endotoxin concentration from the Reaction Time of each sample. The
software automatically adjusts the final TEST RESULT value to account for any
product dilution.
It is important to note that the POWERCURVETM polynomial model CANNOT be
used for INITIAL QUALIFICATION assays. Linear regression must still be used inthose cases to comply with the FDA Guideline. Additionally, the POWERCURVETM
polynomial model has only been evaluated for the Kinetic-QCL® and PYROGENT®5000 reagents supplied by Lonza.
PRODUCT INHIBITION
Product inhibition occurs when substances in the test sample interfere with
the LAL reaction. In the turbidimetric LAL assay, this inhibition results in a longer
Reaction Time, indicating lower levels of endotoxin than may actually be present
in the test sample. The lack of product inhibition should be determined for eachspecific sample, either undiluted or at an appropriate dilution.
To verify the lack of product inhibition, an aliquot of test sample (or a dilutionof test sample) is spiked with a known amount of endotoxin.
It is recommended that the endotoxin 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 >1 EU/ml, the endotoxin spike should result in a final
endotoxin concentration of 1.0 EU/ml.
In an INHIBITION/ENHANCEMENT assay, the spiked solution (PPC) is assayedalong with the unspiked sample and their respective endotoxin concentrations, aswell as the endotoxin recovered in the spiked sample are automatically calculated.
The endotoxin recovered should equal the known concentration of the spike with-20

in 50% to 200%.10
A spiked aliquot of the test sample (or dilution) may be prepared as in one of
the following examples:
Tube method:
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 (or dilution). This solution 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 96well plate, as directed by the assay template. To these wells add 0.1 ml of test
sample (or dilution). Each well will now contain a 0.1 EU/ml solution. Mix gently.
Plate method #2:
Place 0.1 ml 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. Each well will now contain a 0.1 EU/ml solution. Mix gently.
If the test sample (or dilution) is found to be inhibitory to the turbidimetric LAL
reaction, the sample may require further dilution until the inhibition is overcome.

21

Example:
Determination of a Non-Inhibitory Dilution
Sample Dilution
1/10

Endotoxin Recovered
0.015
Inhibitory

1/20

0.042
Inhibitory

1/40

0.110
Non-Inhibitory

Initially, one may want to screen for product inhibition by testing 10-fold dilutions of test sample. Once the approximate non-inhibitory dilution is determined,
the exact dilution can be found by testing two-fold dilutions around this dilution.
LIMITATIONS AND INDICATIONS
The degree of inhibition or enhancement will be dependent upon the concentration of product. If several concentrations of the same product are to be
assayed, it is necessary to establish performance characteristics for eachindependently.
Patterns of inhibition or enhancement different from those seen with thetraditional LAL gelation test may be found.
It may be necessary to adjust the pH of the sample to within the range 6.0 to
8.0 using endotoxin-free sodium hydroxide or hydrochloric acid to overcome
inhibition.

22

TURBID SAMPLES
Samples that possess significant turbidity on their own may require clarification prior to testing. Clarification may be achieved by centrifugation, filtration or
dilution of sample.
ARCHIVED STANDARD CURVEThe WinKQCL ® Software may be run using an archived standard curve.
Provided that the current reagent lot numbers for the PYROGENT®-5000 LAL
Reagent, LAL Reagent Water, and Endotoxin as well as microplate reader parameters match those used to generate a valid archived standard curve, the archived
standard curve may be used instead of placing new endotoxin standards on the
microplate.
If an archived standard curve is used, a single standard-control containing an
endotoxin concentration equal to the mid-point, on a log basis, between the endotoxin concentration of the highest and lowest endotoxin standards in the archived
standard curve should be assayed. The predicted endotoxin concentration should
be within ± 25% of its known value.
For example, in an assay with a standard curve spanning from 100 to 0.01
EU/ml, a standard-control equal to 1.0 EU/ml should be assayed.
log 100=
2.0
log 0.01=-2.0-------------------------log average=
0.0
antilog 0.0
=
1.0

23

In an assay with a standard curve spanning from 1 to 0.01 EU/ml, a standardcontrol equal to 0.1 EU/ml should be assayed.
log 1.0=
0.0000log 0.01=-2.0000----------------------------log average=-1.0000
antilog -1.0000=
0.1
CORRELATION WITH OTHER METHODSThe FDA regulates the official use of LAL testing in the United States. The FDA
has official LAL and endotoxin preparations which have been of great assistancein standardizing LAL testing. The FDA reference LAL is not the LAL used in thistest since it has not been formulated for the turbidimetric LAL method. However,
the official endotoxin has been used to standardize the endotoxin in this kit. The
potency of different endotoxin preparations varies in both the traditional gel test
and the turbidimetric method. The endotoxin standard supplied in this kit has
been compared to the FDA Reference Standard Endotoxin (RSE) using the PYROGENT®-5000 assay, and the potency is 100 EU/ml when reconstituted using the volume specified on the enclosed Certificate of Analysis. The calibration curve diluted
from this standard will yield a range of 0.01 to 100 Endotoxin Units/ml relative to
the RSE. It should be remembered, however, that the traditional gel test is standardized by two-fold dilutions, so that variations will appear quite large in comparison to those in the turbidimetric LAL test where standardization is continuousand variations are minimal.

24

A NOTE FOR OUR INTERNATIONAL CUSTOMERSOther regulatory agencies may adopt other performance standards which willneed to be satisfied in order to be in compliance in their jurisdictions.
REFERENCES
1.
Bang, F.B. A bacterial disease of Limulus polyphemus. Bull. Johns Hopkins
Hosp. 98:325 (1956).
2.
Levin, J. and F.B. Bang. The role of endotoxin in the extracellular coagulationof Limulus blood. Bull. Johns Hopkins Hosp. 115:265 (1964).
3.
Levin, J. and F.B. Bang. A description of cellular coagulation in the Limulus.
Bull. Johns Hopkins Hosp. 115:337 (1964).
4.
Levin, J. and F.B. Bang. Clottable protein in Limulus: its localization andkinetics of its coagulation by endotoxin. Thromb. Diath. Haemorrh. 19:186(1968).
5.
Solum, N.O. Some characteristics of the clottable protein of Limulus
polyphemus blood cells. Thromb. Diath. Haemorrh. 23:170 (1970).
6.
Solum, N.O. The coagulogen of Limulus polyphemus hemocytes. A comparison of the clotted and non-clotted forms of the molecule. Thromb. Res.
2:55 (1973).
7.
Teller, J.D. and K.M. Kelly. A turbidimetric Limulus amebocyte assay for thequantitative determination of Gram negative bacterial endotoxin. In
Biomedical Applications of the Horseshoe Crab (Limulidae), Cohen, E., Ed.,
A.R. Liss, Inc., New York, N.Y. p. 423 (1979).
8.
U.S. Department of Health and Human Services, Public Health Service, Foodand Drug Administration, “Guideline on Validation of the Limulus
Amebocyte Lysate Test As An End-product Endotoxin Test for Human andAnimal Parenteral Drugs, Biological Products, and Medical Devices” (1987).

25

9.

Young, N.S., J. Levin, and R.A. Prendergast. An invertebrate coagulation
system activated by endotoxin: Evidence for enzymatic mechanism. J. Clin.
Invest. 51:1790 (1972).
10. Chapter <85> Bacterial Endotoxins. Rockville, MD: United States
Pharmacopeia.
11. U.S. Department of Health and Human Services, Public Health Service, Foodand Drug Administration, “Interim Guidance for Human and Veterinary Drug
Products and Biologics” (1991).
PATENT INFORMATION
Various components of this kit are protected under U.S. Patent 4322217.
TRADEMARKSThe ELx808TM is a trademark of BioTek Instruments, Inc.
Unless otherwise noted, all trademarks herein are marks of the Lonza
Group or its affiliates.

26

NOTES

27

8830 Biggs Ford Road | Walkersville, MD 21793
301-898-7025 | www.lonza.com

08295
PN383-5
12/07