Pharma&Biotech

Limulus Amebocyte Lysate (LAL)

PYROGENT™-5000

U.S. Licence No. 1775. Translated versions available at www.lonza.com

Content

Section Page No. Section Page No.

1 Intended Use  2 7 Performance Characteristics  17

1 Warning  2 7 Calculation of Endotoxin Concentration  18
1 Explanation of Test  3 8 POWERCURVE™  21
2 Principle  4 9 Product Inhibition  23
2 Reagents Supplied and 9 Limitations and Indications  26
Storage Conditions  5
9 Turbid Samples  26
3 Materials and Equipment Not Provided  6
9 Archived Standard Curve  27
4 Sample Collection and Preparation  8
10 Correlation with Other Methods 28
4 Types of PYROGENT™-5000 Assays  9
10 A Note for Our International Customers 28
5 Reagent Preparation 11
10 References 28
6 Test Procedure 14
Important: Read Entire Brochure Before Performing Test Explanation of Test
1
PYROGENT™-5000 is a quantitative, kinetic assay for the detection of
Intended Use Gram-negative bacterial endotoxin. A sample is mixed with the recon-
This product is intended as an in vitro end-product endotoxin test for stituted LAL reagent, placed in the incubating microplate reader, and
human and animal parenteral drugs, biological products, and medical automatically monitored over time for the appearance of turbidity. The
devices. This product is not intended for the detection of endotoxin time required before the appearance of turbidity (Reaction Time) is
in clinical samples or as an aid in the diagnosis of human disease. inversely proportional to the amount of endotoxin present. That is, in
This test utilizes a preparation of Limulus Amebocyte Lysate (LAL), the presence of a large amount of endotoxin the reaction occurs rapidly;
in combination with an incubating microplate reader and appropriate in the presence of a smaller amount of endotoxin the reaction time is
software, to detect endotoxin photometrically. increased. The concentration of endotoxin in unknown samples can be
calculated from a standard curve.
The Pharmacopeia outlines procedures that are considered necessary
for: The use of LAL for the detection of endotoxin evolved from the observa-
1.  Establishing endotoxin limits for pharmaceuticals and medical tion by Bang1 that a Gram-negative infection of Limulus polyphemus,
devices the horseshoe crab, resulted in fatal intravascular coagulation. Levin
2. Validating the use of LAL as an end-product endotoxin test and Bang2,3 later demonstrated that this clotting was the result of a
reaction between endotoxin and a clottable protein in the circulating
3.  Developing a routine testing protocol9
amebocytes of Limulus. Following the development of a suitable anti-
The procedures described herein are based on the Pharmacopeial coagulant for Limulus blood, Levin and Bang4 prepared a lysate from
guidelines. washed amebocytes, which was an extremely sensitive indicator of
the presence of endotoxin. Solum5,6 and Young, Levin and Prendergast8
Warning have purified and characterized the clottable protein from LAL and have
For In Vitro Diagnostic Use Only. The PYROGENT™-5000 Assay is shown the reaction with endotoxin to be enzymatic. Teller and Kelly7
not intended to detect endotoxemia in man. The LAL Test may be demonstrated that the endotoxin activation of lysate could be monitored
substituted for the USP Rabbit Pyrogen Test when used according to photometrically.
the Pharmacopeial guidelines for end-product testing of human and
animal parenteral drugs, biological products, and medical devices9.

2 3
Principle Swirl gently to avoid foaming.
Proenzyme Endotoxin Coagulase
Important: Prior to use, wait about two (2) minutes to allow any bubbles
Coagulogen Coagulase Coagulin
to rise to the top of the vial. 2
Gram-negative bacterial endotoxin catalyzes the activation of a
proenzyme in the LAL8. The initial rate of activation is determined by the Store lyophilized (unreconstituted) PYROGENT™-5000 LAL under
concentration of endotoxin present. The activated enzyme (Coagulase) refrigeration at 2–8°C. Avoid exposure to temperatures in excess of
hydrolyzes specific bonds within a clotting protein (Coagulogen) also 37°C. Avoid exposure to bright light. Discard lysate which has turned
present in LAL. Once hydrolyzed, the resultant coagulin self-associates yellow or become insoluble. Reconstituted PYROGENT™-5000 LAL is
and forms a gelatinous clot. The turbidimetric LAL assay measures the stable up to 8 hours at 2–8°C.
increase in turbidity (optical density) that precedes the formation of
the gel clot. PYROGENT™-5000 LAL Reconstitution Buffer
(B50-300) Blue-Labeled Vial (B50-600) Green-Labeled Vial
This buffer must be used to rehydrate the PYROGENT™-5000 LAL
Reagents Supplied and Storage Conditions Reagent. Allow buffer to warm to room temperature before use.
PYROGENT™-5000 LAL Reagent
(T50-300, T50-600) Yellow-Labeled Vial Store PYROGENT™-5000 LAL Reconstitution Buffer at 2–8°C.
The LAL reagent contains a lysate prepared from the circulating Note: Not included but required for lysate only kits.
amebocytes of the horseshoe crab, Limulus polyphemus. Reconstitute
before use with PYROGENT™-5000 LAL Reconstitution Buffer, per the E. coli O55:B5 Endotoxin (7460) Red-Labeled Vial
following table: The reconstitution volume of the vial is stated on the Certificate
of Analysis and is calculated to yield a solution containing 100.0 EU
PYROGENT™-5000 PYROGENT™-5000 LAL (or IU)/ml. Reconstitute with the specified volume of LAL Reagent Water.
LAL Reagent Reconstitution Buffer Required Shake vigorously for at least 15 minutes at high speed on a vortex
T50-300 5.2 ml/vial mixer. Prior to subsequent use, a stored stock solution must be warmed
T50-600 10.4 ml/vial to room temperature and vigorously vortexed for 15 minutes. This is
important because the endotoxin tends to attach to glass. The COA is
available at www.lonza.com/coa.
4 5
Store lyophilized endotoxin at 2–8°C. Reconstituted endotoxin is stable 7. Eight channel pipettor.
up to four weeks at 2–8°C. Note: Endotoxin is not included but required
for lysate only kits. 8. Reagent reservoirs (#00190035 or equivalent).

9. Microplate reader (ELx808™ IU Reader, #25-315 or equivalent).

This endotoxin is provided for the user’s convenience. Other endotoxin
preparations may be used to prepare the standards; however, their per- 10. WinKQCL™ Software.
formance in the turbidimetric assay relative to the Reference Standard 3
Endotoxin (RSE) must be determined. 11. Timer.

12. Vortex mixer.

Materials and Equipment NOT Provided 13. For kits without endotoxin: Endotoxin Standard (Control Standard
1. LAL Reagent Water (#W50-640, #W50-100, #W50-500, or equiv­ Endotoxin that has been matched with the LAL).
alent). LAL Reagent Water is equivalent to Water for Bacterial
Endotoxins Test (BET). 14. For kits without buffer: PYROGENT™-5000 LAL Reconstitution Buffer.

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 × 100 mm, #N207

or equivalent).

4. Individually wrapped serological pipettes.

5. Automatic hand-held pipettes with sterile, individually wrapped or

racked tips.

6. Disposable sterile microplates. Note: Prior to routine use, micro-

plates should be pre-qualified 9 (#25-340 or equivalent).

6 7
Sample Collection and Preparation Types of PYROGENT™-5000 Assays
Careful technique must be used to avoid microbial or endotoxin The incubating microplate reader and WinKQCL™ Software are an integral
contamination. All materials coming in contact with the sample or part of the turbidimetric LAL assay. It is important to become familiar
test reagents must be endotoxin-free. Clean glassware and materials with the operation of the incubating microplate reader and the features
may be rendered endotoxin-free by heating at 250°C for 30 minutes. of the WinKQCL™ Software. Please refer to the incubating microplate
Appropriate precautions should be taken to protect depyrogenated reader and WinKQCL™ Software Manuals or Help for more detailed
materials from subsequent environmental contamination. information.

From experience, most sterile, individually wrapped, plastic pipettes There are four (4) basic types of turbidimetric LAL assays, each of
4
and pipette tips are endotoxin-free. However, these materials should which is designed to perform a different aspect of LAL testing.
be tested before regular use.
1. Routine
It may be necessary to adjust the pH of the sample to within the range A Routine assay calculates the concentration of endotoxin in
6.0–8.0 using endotoxin-free sodium hydroxide or hydrochloric acid. unknowns by comparison to the performance of a series of
Always measure the pH of an aliquot of the bulk sample to avoid endotoxin standards.
contamination by the pH electrode. Do not adjust unbuffered solutions.
As part of a Routine assay, the user has the option to include a
Samples to be tested must be stored in such a way that all bacterio­ Positive Product Control (PPC) as a monitor for product inhibition
logical activity is stopped or the endotoxin level may increase with or enhancement (section 2 below). A PPC is a sample of product
time. For example, store samples at 2–8°C for less than 24 hours and to which a known amount of endotoxin spike has been added.
frozen for periods greater than 24 hours. It is the responsibility of the The WinKQCL™ Software automatically calculates the amount of
end-user to validate the proper container and storage conditions for endotoxin recovered in the PPC, allowing for a comparison to the
their samples. known amount of endotoxin spike.

2. Inhibition/Enhancement
The LAL reaction is enzyme mediated and, as such, has an opti-
mal pH range and specific salt and divalent cation requirements.

8 9
 Occasionally test samples may alter these optimal conditions to an 4. 
Initial Qualification
extent that the lysate is rendered insensitive to endotoxin. Negative An Initial Qualification assay is designed according to the require-
results with samples which inhibit the LAL test do not necessarily ments described in the Pharmacopeia9. This assay is required as
indicate the absence of endotoxin. part of the validation of the LAL assay and is also to be performed
with each new lot of PYROGENT™-5000.
An Inhibition/Enhancement assay is designed to determine what
level of product dilution overcomes inhibition or enhancement. Each The Initial Qualification assay performs a log/log linear correlation
product dilution must be accompanied by a Positive Product Control of the individual Reaction Time values for each replicate of each
(PPC). The WinKQCL™ Software automatically calculates the amount endotoxin standard. The other assays use the average Reaction
of endotoxin recovered in the PPC for comparison to the known Time of all the replicates of each standard.
amount of endotoxin spike. In this manner it can be determined 
which product dilutions are non-interfering. The Initial Qualification assay does not provide for the inclusion of 5
any samples.
3. RSE/CSE
An RSE/CSE assay is designed to determine the potency of a Control Reagent Preparation
Standard Endotoxin (CSE) in terms of the concentration units of the Allow reagents to equilibrate to room temperature prior to use.
Reference Standard Endotoxin (RSE).
In order to calculate endotoxin concentrations in unknown samples
The assay requires a single series of RSE dilutions and one or more each turbidimetric LAL assay must be referenced to a valid standard
sets of dilutions of the CSE. Depending on the concentration units curve.
of the CSE, the WinKQCL™ Software automatically computes mean
potency values in terms of EU/ng or EU/ml. The user also has the Because of the large concentration range over which endotoxin values
option to enter units other than EU or ng. can be determined, it is possible to adjust the quantitative range of any
given assay by adjusting the concentration of endotoxin standards
used to generate the standard curve. A minimum of three standards
is required.

10 11
The PYROGENT™-5000 Assay has been optimized to be linear from 1. Prepare a solution containing 10.0 EU/ml endotoxin by adding
0.01 EU/ml to 100.0 EU/ml. However, the individual user may choose 0.1 ml of the 100.0 EU/ml endotoxin stock into 0.9 ml of LAL
to truncate the standard curve depending on specific product Reagent Water in a suitable container and label 10.0 EU/ml. This
requirements. Data indicates that truncating a turbidimetric LAL solution should be vigorously vortexed for at least 1 minute before
standard curve may improve the accuracy of predicted endotoxin proceeding.
values for test samples. It is recommended that the user be familiar
with the Pharmacopeial requirements for kinetic LAL techniques prior 2. Transfer 0.1 ml of the 10.0 EU/ml endotoxin solution into 0.9 ml of
to establishing a turbidimetric LAL standard curve range to be used for LAL Reagent Water in a suitable container and label 1.0 EU/ml. The
routine testing of product samples9. solution should be vigorously vortexed for at least 1 minute before
proceeding.
The following table suggests a dilution scheme for constructing a series
of endotoxin dilutions from the endotoxin supplied in the kit. Not all 3. Transfer 0.1 ml of the 1.0 EU/ml endotoxin solution into 0.9 ml of 5
dilutions must be used to generate a standard curve. Alternative dilution LAL Reagent Water in a suitable container and label 0.10 EU/ml. This
schemes can be used as well as other endotoxins not supplied in this solution should be vigorously vortexed for at least 1 minute before
kit. If the endotoxin used is not supplied in the kit, an RSE/CSE test to proceeding.
determine the CSE potency may be required.
4. Transfer 0.1 ml of the 0.10 EU/ml endotoxin solution into 0.9 ml of
Note: Plastic tubes are not recommended for making endotoxin LAL Reagent Water in a suitable container and label 0.01 EU/ml. This
dilutions. solution should be vigorously vortexed for at least 1 minute before
proceeding.
Endotoxin Concentration Volume of LAL Volume of Endotoxin Solution
(EU/ml) Reagent Water Added to LAL Reagent Water
10.0 0.9 ml 0.1 ml of 100.0 EU/ml solution
1.0 0.9 ml 0.1 ml of 10.0 EU/ml solution
0.10 0.9 ml 0.1 ml of 1.0 EU/ml solution
0.01 0.9 ml 0.1 ml of 0.10 EU/ml solution

12 13
Test Procedure 3. Print the Template for use as a guide in placing standards and
Important: The reader must be located in an area free of excessive samples into the microplate.
vibration (ex. centrifuges, shakers, etc.) during the actual running of
the test. 4. “Run” the Template, following the WinKQCL™ Software prompts.

Refer to the microplate reader and WinKQCL™ Software Manuals for more 5. Carefully dispense 100 μl of the LAL Reagent Water blank, endotoxin
detailed information on performing a PYROGENT™-5000 Test. standards, product samples, positive product controls (see pages
23 – 25 for positive product control instructions), etc. into the
1. Create a specific Template for the test to be run. A Template contains appropriate wells of the microplate.
the name of the analyst, type of assay, lot numbers of reagents, Note: Bubbles must be avoided!
the number and concentration of endotoxin standards, number of
replicates, and how standards and samples will be organized on 6. Place the filled plate in the microplate reader and close the lid.
the microplate.
7. Pre-incubate the plate for ≥10 minutes at 37°C ± 1°C.
2. The Assay Type must be set as PYROGENT-5000. The default 6
Template Parameters that follow should not be changed without 8. Near the end of the pre-incubation period reconstitute each of the
prior qualification: appropriate number of the PYROGENT™-5000 Reagent vials with
PYROGENT™-5000 Reconstitution Buffer (5.2 ml for the T50-300
Delta t (seconds) 60 vials and 10.4 ml for the T50-600 vials). Mix gently but thoroughly.
Measurement filter (nm) 340 Allow any bubbles to rise to the top of the vial before use.
Delta mOD 30 Note: Do not vortex the lysate.
Number of Reads 100
9. Pool the reagents into a reagent reservoir and mix by gently rocking
the reservoir from side to side.

14 15
10. Using an eight channel pipettor dispense 100 μl of the PYROGENT™- Performance Characteristics
5000 Reagent into all wells of the microplate beginning with the Linearity
first column (A1-H1) and proceeding in sequence to the last column The linearity of the standard curve within the concentration range
used. Add reagent as quickly as possible. used to determine endotoxin values should be verified. No less than 3
Note: Avoid causing bubbles! endotoxin standards, spanning the desired concentration range, and
an LAL Reagent Water blank should be assayed at least in triplicate
11. Immediately click on the OK button in the WinKQCL™ Software to according to the test parameters of an Initial Qualification assay.
initiate the test. Additional standards should be included to bracket each log interval
Note: The PYROGENT™-5000 Assay is performed with the microplate over the range of the standard curve.
cover removed.
The absolute value of the correlation coefficient (r) of the calculated
standard curve should be ≥0.980.

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
7
mean and is usually expressed as a percent. The %C.V. of the reaction
times for the replicates should be less than 10%. With experience,
values of 3–4% should be attainable.

16 17
Calculation of Endotoxin Concentration Linear Correlation
Continuously throughout the assay, the microplate reader/WinKQCL™ Example Calculations
Software monitors the absorbance at 340 nm of each well of the Mean Reaction Log Log Mean
microplate. Using the initial absorbance reading of each well as its own Standards Concentration Time (Sec) Concentration Reaction Time

blank, the reader determines the time required for the absorbance to Neg. Control — Unreactive — —

increase 0.03 absorbance units. This time is termed Reaction Time. S1 0.010 EU/ml 3103 -2.000 3.492

The WinKQCL™ Software automatically performs a log/log linear cor- S2 0.100 EU/ml 1484 -1.000 3.171

relation of the Reaction Time of each standard with its corresponding S3 1.000 EU/ml 808 -0.000 2.907

endotoxin concentration. The standard curve parameters are printed on S4 10.000 EU/ml 485 1.000 2.686

the report printout. If the absolute value of the correlation coefficient S5 100.000 EU/ml 312 2.000 2.494

(r) is ≥0.980, a polynomial model can be used to construct a standard Samples

curve and in turn predict endotoxin concentrations of test samples. This 1 — 1576 — 3.198

polynomial curve-fitting model (POWERCURVE™) is an important feature 2 — 943 — 2.975

of the WinKQCL™ Software (see POWERCURVE™, page 21).

( )
Slope = Sy r
Sx
Linear Regression Y-intercept = ∑y / N – (∑x / N × slope)
The information that follows is an example of how the the WinKQCL™
Software performs the log/log linear correlation and computes r = N∑xy – (∑x)(∑y) 7
N(N–1)SxSy
endotoxin concentrations in unknowns. It is not necessary to perform
these calculations independently. For each sample of each product, the
Endotoxin concentration = antilog
slope [
log Mean Reaction Time – Y int.
]
WinKQCL™ Software calculates the corresponding endotoxin concentra- x = log10 Endotoxin concentration in EU/ml.
tion from the Reaction Time for that sample. The software automatically
y = log10 Mean Reaction Time.
adjusts the final Test Result value to account for any product dilution.
N = Number of standards used.

∑x = Summation of log10 concentration of standards used in EU/ml.

∑y = Summation of log10 Reaction Time.

∑xy = Summation of the log10 standard concentrations times log10 Mean Reaction Time.

18 19
Sx = Standard deviation of x =

√N∑xN(N-1)
- (∑x)
2 2
POWERCURVE™
If the absolute value of the correlation coefficient (r) is ≥0.980, a
Sy = Standard deviation of y =

√ N∑y2 - (∑y)2
N(N-1)
polynomial model can be used to construct a standard curve and predict
endotoxin concentrations of test samples. It has been determined
Calculations using Example Data: that this polynomial model (POWERCURVE™) improves the accuracy
N=5 of predicting endotoxin concentrations over the entire (5-log) endo­
∑x = 0.000 = (-2.000 - 1.000 + 0.000 + 1.000 + 2.000) toxin range. The use of the POWERCURVE™ Model requires the use of
∑y = 14.75 = (3.492 + 3.171 + 2.907 + 2.686 + 2.494)
WinKQCL™ Software.
∑xy = - 2.481 = (-2.000 × 3.492) + (-1.000 × 3.171) + (0.000 × 2.907) +
(1.000 × 2.686) + (2.000 × 2.494) When using POWERCURVE™, a standard curve is generated using the
Sx = 1.581 log10 Reaction Time values and their corresponding log10 endotoxin con-
Sy = 0.394 centration to define a polynomial equation. The order of the poly­nomial
equation used to generate the regression curve is determined by the
r = 5(-2.481) - (0.000)(14.75) = -0.996
5(5 - 1)(1.581)(0.394) number of endotoxin standards in the assay. The order of the poly­nomial
will always be one less than the number of endotoxin standards, with
Slope = 0.394 × -0.996 = -0.248
1.581 a maximum of a fourth-order polynomial for assays with five or more
[ ]
Y-intercept = 14.75 - 0.000 × (-0.248) = 2.950 - [(0) × (-0.248)] = 2.950
5 5
endotoxin standards and a minimum of a second order polynomial for
assays with three standards.
Sample 1
Endotoxin Conc. EU/ml = antilog
[
3.198 - 2.950
-0.248 ] 8
= antilog (-1.000)
= 0.100 EU/ml

Sample 2
[
Endotoxin Conc. EU/ml = antilog 2.975 - 2.950
-0.248 ]
= antilog (-0.101)
= 0.793 EU/ml

20 21
Finding solutions to these polynomial equations is readily accomplished Product Inhibition
using the WinKQCL™ POWERCURVE™ Software. The information provided Product inhibition occurs when substances in the test sample interfere
below is an example of a solution to a polynomial equation using the with the LAL reaction. In the turbidimetric LAL assay, this inhibition
same set of data from the linear correlation example on page 19. results in a longer Reaction Time, indicating lower levels of endotoxin
than may actually be present in the test sample. The lack of product
Polynomial (POWERCURVE™) Model inhibition should be determined for each specific sample, either
Y = A + BX + CX2 + DX3 + EX4 undiluted or at an appropriate dilution.
A = 2.90741
B = -0.24066 To verify the lack of product inhibition, an aliquot of test sample (or a
C = 0.02111
dilution of test sample) is spiked with a known amount of endotoxin.
D = -0.00219
It is recommended that the endotoxin spike result in a final endotoxin
E = 0.00007
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
The standard curve parameters are printed on the report printout. The should result in a final endotoxin concentration of 1.0 EU/ml.
WinKQCL™ POWERCURVE™ Software uses these parameters to calculate
the corresponding endotoxin concentration from the Reaction Time of In an Inhibition/Enhancement assay, the spiked solution (PPC) is
each sample. The software automatically adjusts the final Test Result assayed along with the unspiked sample and their respective endotoxin
value to account for any product dilution. concentrations, as well as the endotoxin recovered in the spiked sample
are automatically calculated. The endotoxin recovered should equal the
It is important to note that the POWERCURVE™ Polynomial Model CANNOT known concentration of the spike within 50% to 200%9.
be used for Initial Qualification assays. Linear regression must still be
used in those cases. Additionally, the POWERCURVE™ Polynomial Model 9
has only been evaluated for the Kinetic-QCL™ and PYROGENT™-5000
Reagents supplied by Lonza.

22 23
A spiked aliquot of the test sample (or dilution) may be prepared as in Plate Method #2
one of the following examples: 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
Tube Method 10 μl of the 1.0 EU/ml solution. Each well will now contain a 0.1 EU/ml
Transfer 50 μl of the 10.0 EU/ml solution into 4.95 ml of test sample solution. Mix gently by tapping the side of the plate.
(or dilution). This solution contains an endotoxin concentration of
0.1 EU/ml in test sample (or dilution). This solution should be vigorously If the test sample (or dilution) is found to be inhibitory to the turbidi-
vortexed for one minute prior to use. metric LAL reaction, the sample may require further dilution until the
inhibition is overcome.
Transfer 100 μl of this solution into the 96-well plate as directed by
the assay template. Example: Determination of a Non-Inhibitory Dilution
Sample Dilution Endotoxin Recovered
Plate Method #1 1/10 0.015 Inhibitory
Transfer 10 μl of the 1.0 EU/ml solution into each of the PPC wells 1/20 0.042 Inhibitory
in the 96-well plate, as directed by the assay template. To these wells 1/40 0.110 Non-Inhibitory
add 0.1 ml of test sample (or dilution). Each well will now contain a
0.1 EU/ml solution. Mix gently by tapping the side of the plate. 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.

24 25
Limitations and Indications Archived Standard Curve
The degree of inhibition or enhancement will be dependent upon the The WinKQCL™ Software may be run using an archived standard curve.
concentration of product. If several concentrations of the same product Provided that the current reagent lot numbers for the PYROGENT™-5000
are to be assayed, it is necessary to establish performance character- Reagent, LAL Reagent Water, buffer and endotoxin as well as microplate
istics for each independently. reader parameters match those used to generate a valid archived stan-
dard curve, the archived standard curve may be used instead of placing
Patterns of inhibition or enhancement different from those seen with new endotoxin standards on the 96-well plate.
the traditional LAL gelation test may be found.
If an archived standard curve is used, a single standard-control
It may be necessary to adjust the pH of the sample to within the range containing an endotoxin concentration equal to the mid-point, on a log
6.0 to 8.0 using endotoxin-free sodium hydroxide or hydrochloric acid basis, between the endotoxin concentration of the highest and lowest
to overcome inhibition. endotoxin standards in the archived standard curve should be assayed.
The predicted endotoxin concentration should be within ± 25% of its
Turbid Samples known value.
Samples that possess significant turbidity on their own may require
clarification prior to testing. Clarification may be achieved by centrifu­ For example, in an assay with a standard curve spanning from 100.0 to
gation, filtration or dilution of sample. 0.01 EU/ml, a standard-control equal to 1.0 EU/ml should be assayed.
log 100.0 = 2.0
log 0.01 = -2.0
log average = 0.0
antilog 0.0 = 1.0

In an assay with a standard curve spanning from 1.0 to 0.01 EU/ml, 9

a standard-control equal to 0.1 EU/ml should be assayed.
log 1.0 = 0.0000
log 0.01 = -2.0000
log average = -1.0000
antilog -1.0000 = 0.1
26 27
 4. Levin, J. and F.B. Bang. Clottable protein in Limulus: its localization and kinetics
Correlation with Other Methods of its coagulation by endotoxin. Thromb. Diath. Haemorrh. 19:186 (1968).
The FDA regulates the official use of LAL testing in the United States.
5. Solum, N.O. Some characteristics of the clottable protein of Limulus polyphemus
The potency of different endotoxin preparations varies in both the
blood cells. Thromb. Diath. Haemorrh. 23:170 (1970).
traditional gel test and the turbidimetric method. The endotoxin standard
supplied in this kit has been compared to the USP Reference Standard 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).
Endotoxin (RSE) using the PYROGENT™-5000 Assay, and the potency
is 100.0 EU/ml when reconstituted using the volume specified on the 7. Teller, J.D. and K.M. Kelly. A turbidimetric Limulus amebocyte assay for the
lot-specific Certificate of Analysis. The calibration curve diluted from this quantitative determination of Gram negative bacterial endotoxin. In Biomedical
Applications of the Horseshoe Crab (Limulidae), Cohen, E., Ed., A.R. Liss, Inc., New
standard will yield a range of 0.01 to 100.0 Endotoxin Units/ml relative
York, N.Y. p. 423 (1979).
to the RSE. It should be remembered, however, that the traditional
gel test is standardized by two-fold dilutions, so that variations will 8. Young, N.S., J. Levin, and R.A. Prendergast. An invertebrate coagulation system
appear quite large in comparison to those in the turbidimetric LAL test activated by endotoxin: Evidence for enzymatic mechanism. J. Clin. Invest.
51:1790 (1972).
where standardization is continuous and variations are minimal.
9. United States Pharmacopeial Convention. General Chapter <85>
A Note for Our International Customers Bacterial Endotoxins Test. United States Pharmacopeia (USP).

Other regulatory agencies may adopt other performance standards 10. European Directorate for the Quality of Medicines. Chapter 2.6.14
which will need to be satisfied in order to be in compliance in their Bacterial Endotoxins Test. European Pharmacopoeia (EP).
jurisdictions.
11. Ministry of Health, Labour, and Welfare, General Chapter 4.0.1
Bacterial Endotoxins Test. Japanese Pharmacopoeia (JP).
References
12. U.S Department of Health and Human Services, Food and Drug Administration,
1. Bang, F.B. A bacterial disease of Limulus polyphemus. Bull. Johns Hopkins Hosp.
Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers
98:325 (1956).
(June 2012).
2. Levin, J. and F.B. Bang. The role of endotoxin in the extracellular coagulation
of Limulus blood. Bull. Johns Hopkins Hosp. 115:265 (1964).

3. Levin, J. and F.B. Bang. A description of cellular coagulation in the Limulus. 10

Bull Johns Hopkins Hosp. 115:337 (1964).

28 29
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