Limulus Amebocyte Lysate (LAL)
QCL-1000®

Catalog Number: 50-647U, 50-648U

TABLE OF CONTENTS

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TITLE

PAGE

Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Explanation of Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Reagents Supplied and Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Materials and Equipment Not Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Specimen Collection and Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Reagent Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Test Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Performance Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Calculation Of Endotoxin Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Product Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Colored Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Correlation With Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

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QCL-1000 ®
Limulus Amebocyte Lysate
U.S. License No. 1775
For the rapid, chromogenic quantitation of bacterial endotoxin.
IMPORTANT
Read Entire Brochure Before Performing Test
INTENDED USEThis product is intended as an In Vitro end-product endotoxin test for human andanimal parenteral drugs, biological products, and medical devices. This product is notintended for the detection of endotoxin in clinical samples or as an aid in the diagnosisof human disease. This method utilizes a modified Limulus Amebocyte Lysate and asynthetic color producing substrate to detect endotoxin chromogenically.
In December, 1987, the United States Food and Drug Administration (FDA) published the “Guideline on the Validation and Use 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 for pharmaceuticals andmedical devices, 2) validating the use of LAL as an end-product 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 end point chromogenic assays have been published and are updated regularly in the United States Pharmacopeia.9
WARNINGFor In Vitro Diagnostic Use Only. Not for the In Vitro Determination of Endotoxemia
in Man. The LAL test may be substituted for the USP Rabbit Pyrogen Test when usedaccording to the FDA Guideline for the end-product testing of human and animal parenteral drugs, biological products, and medical devices.8
EXPLANATION OF TESTThe Chromogenic Limulus Amebocyte Lysate (LAL) Test is a quantitative test forgram-negative bacterial endotoxin. A sample is mixed with the LAL supplied in the test
kit and incubated at 37°C (±1°C) for 10 minutes. A substrate solution is then mixed with
the LAL-sample and incubated at 37°C (±1°C) for an additional 6 minutes. The reaction
is stopped with stop reagent. If endotoxin is present in the sample, a yellow color willdevelop. The absorbance of the sample can be determined spectrophotometrically at
405-410 nm. Since this absorbance is in direct proportion to the amount of endotoxinpresent, the concentration of endotoxin can be calculated from a standard curve.

3

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 intra-vascular 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 a suitable anticoagulant for
Limulus blood, Levin and Bang4 prepared a lysate from washed amebocytes which was
an extremely sensitive indicator of the presence of endotoxin. Solum5,6 and Young, Levinand Prendergast7 have purified and characterized the clottable protein from LAL andhave shown the reaction with endotoxin to be enzymatic.
The present LAL method utilizes the initial part of the LAL endotoxin reaction to activate an enzyme which in turn releases p-nitroaniline (pNA) from a synthetic substrate,
producing a yellow color.
PRINCIPLE
Endotoxin
1 Proenzyme
EnzymeEnzyme2 Substrate + H2O
Peptide + pNA
Gram-negative bacterial endotoxin catalyzes the activation of a proenzyme in the
Limulus Amebocyte Lysate (LAL)7. The initial rate of activation is determined by the concentration of endotoxin present. The activated enzyme catalyzes the splitting of pNA
from the colorless substrate Ac-lle-Glu-Ala-Arg-pNA. The pNA released is measured
photometrically at 405-410 nm after the reaction is stopped with stop reagent. The correlation between the absorbance and the endotoxin concentration is linear in the 0.1-1.0
EU/ml range. The concentration of endotoxin in a sample is calculated from theabsorbance values of solutions containing known amounts of endotoxin standard.
REAGENTS SUPPLIED AND STORAGE CONDITIONS
Kit part number Lysate vials Endotoxin vials Substrate vials
50-647U

5

1

2

50-648U

5

2

5

LAL Reagent
Water vials
2___

Limulus Amebocyte Lysate (LAL)
The LAL reagent contains lyophilized lysate prepared from the circulating amebocytes of
the horseshoe crab Limulus polyphemus.
Reconstitute immediately before use with LAL Reagent Water per following table:
Kit part
numberLAL Reagent
Water required

50-647U
50-648U

1.4 ml/vial lysate
3.0 ml/vial lysate

If the contents of more than one vial are required, pool two or more vials before use.
Swirl gently to avoid foaming. Lyophilized LAL is to be stored at 2-8°C. Protect from longterm exposure to light.
Reconstituted LAL Reagent should be used promptly. Reconstituted lysate can be
stored at -10°C or colder up to one week if frozen immediately after reconstitution.
Thaw and use only once.

4E. coli Endotoxin
Each vial contains approximately 15-40 EU lyophilized endotoxin. Reconstitute byadding 1.0 ml of LAL Reagent Water warmed to room temperature. The actual concentration of the vial will be determined by the value stated on the enclosed Certificate ofAnalysis. For example, if the value of the vial is 24 EU, when reconstituted with 1.0 ml
water it will yield a concentration of 24 EU/ml. Shake vigorously for at least 15 minutes,
preferably with a vortex mixer.
Lyophilized endotoxin is to be stored at 2-8°C. Reconstituted stock endotoxin isstable for four weeks at 2-8°C. Prior to use, the solution must be warmed to room temperature and vigorously mixed for 15 minutes. This is important because the endotoxin tends to attach to glass.
This endotoxin is provided for the user’s convenience. Other endotoxin preparations
may be used to prepare the standards; however, their performance in the chromogenic
assay relative to the Reference Standard Endotoxin (RSE) must be determined.
WARNING: Contains human source material
Chromogenic Substrate
Each vial contains approximately 7 mg lyophilized substrate. Reconstitute byadding 6.5 ml of LAL Reagent Water to yield a concentration of ~2mM.
Lyophilized chromogenic substrate is to be stored at 2-8°C. Once reconstituted, thesubstrate solution is stable for four weeks at 2-8°C, if not contaminated with microorganisms or endotoxin.
PROTECT SUBSTRATE FROM LONG-TERM EXPOSURE TO LIGHT
LAL Reagent Water
Each bottle contains 30 ml and is used in the reconstitution of all reagents and as
a negative control (blank). LAL Reagent Water should be stored at 2-8°C. (Note: Not included in 50-648U kit.)
MATERIALS AND EQUIPMENT NOT PROVIDED
1.
For 50-648U, LAL Reagent Water #W50-640 (30 ml), #W50-100 (100 ml), #W50-500(500 ml), or equivalent.
2. Stop reagent (e.g. Acetic acid, 25% v/v glacial acetic acid in water; or sodium
dodecylsulfate (SDS) solution, 10g/100 ml in water).
3. Sodium hydroxide, 0.1N, dissolved in LAL Reagent Water, for pH adjustment.
4. Hydrochloric acid, 0.1N, diluted in LAL Reagent Water, for pH adjustment.
5. Disposable endotoxin-free glass dilution tubes (13x100 mm, #N207 orequivalent).
6. Individually wrapped measuring pipettes.
7. Automatic hand-held pipettes with sterile, individually wrapped or racked tips(#25-415, #25-416, #25-417 or equivalent).
8. FOR TUBE METHOD Disposable endotoxin-free glass assay tubes (10x75 mm,#N201, #N205, or equivalent).

5

9.

10.
11.
12.
13.
14.
15.
16.

FOR MICROPLATE METHOD Disposable sterile microplates.
NOTE: 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.
8 channel pipettor.
Reagent reservoirs (#25-364 or equivalent).
Dry Bath/ Multi-Blok Heater at 37°C ± 1.0°C.
Stopwatch.
Vortex mixer.
Spectro- or filterphotometer with 405-410 nm filter.
Microplate reader (ELx808TM IU Reader, #25-315; or equivalent).
If using a nonincubating reader:
Tube block for heater, 1 each.
Microplate adapter for heater (#25-038).

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-free by heating at
250°C for 30 minutes. Appropriate precautions should be taken to protect depyrogenated materials from subsequent environmental contamination.
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.
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.
If the container of diluent used to rehydrate the reagents has been opened previously or was not supplied by Lonza, the diluent alone must be tested for endotoxin
contamination
REAGENT PREPARATION
In each series of determinations, four standard endotoxin solutions should be used.
The table below suggests a dilution scheme for the construction of these standards
from the endotoxin supplied in the kit. Alternative dilution schemes can be used as wellas other endotoxins not supplied in this kit. The initial dilution from the endotoxin stock
is 1/X, where X equals the concentration of the endotoxin vial. This yields an endotoxin solution containing 1.0 EU/ml. For example, if the potency is 23 EU/ml, the initial dilution is 1/23 or 0.1 ml of endotoxin stock into 2.2 ml of LAL Reagent Water.

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Endotoxin
Concentration
EU/ml

Endotoxin
Stock Solution

Endotoxin
Std. Solution
1 EU/ml

1.0
0.1 ml0.5
0.5 ml0.25
0.5 ml0.1
0.1 mlX = endotoxin concentration of the vial
1.

2.

3.

4.

LAL Reagent
Water(X-1)/10 ml
0.5 ml
1.5 ml0.9 mlPrepare a solution containing 1.0 EU/ml endotoxin by diluting 0.1 ml of the endotoxin stock solution with (X-1)/10 ml of LAL Reagent Water in a suitable container, where X equals the endotoxin concentration of the vial. This solution should bevigorously vortexed for at least 1 minute before proceeding. For example, if X =23 EU/ml, then dilute 0.1 ml of the endotoxin stock solution with 2.2 ml, (231)/10, LAL Reagent Water.
Transfer 0.5 ml of this 1.0 EU/ml solution into 0.5 ml of LAL Reagent Water in asuitable container and label 0.5 EU/ml. This solution should be vigorously vortexed for at least 1 minute before use.
Transfer 0.5 ml of the 1.0 EU/ml solution into 1.5 ml of LAL Reagent Water in asuitable container and label 0.25 EU/ml. This solution should be vigorously vortexed for at least 1 minute before use.
Transfer 0.1 ml of the 1.0 EU/ml solution into 0.9 ml of LAL Reagent Water in asuitable container and label 0.1 EU/ml. This solution should be vigorously vortexed for at least 1 minute prior to use.

TEST PROCEDURE
The addition of all reagents in the LAL assay must be consistent. All tubes or
microplate wells must be treated in exactly the same manner in order to determine theproper endotoxin concentration. It is suggested that, in a series of tests, reagentsshould be pipetted in the same order from tube to tube or well to well, and at the samerate. The table below outlines the test procedure.
Sample50 µl

Test sample or standard at 20-25°C
LAL Reagent Water
LAL
Mix and incubate at 37°C ± 1°C
Substrate solution at 37°C ± 1°C
Mix and incubate at 37°C ± 1°C
Stop reagent
Mix immediately

50 µl
10 min.
100 µl6 min.
100 µl

7

Blank
50 µl
50 µl
10 min.
100 µl6 min.
100 µlTest Tube Method
1. Carefully dispense 50 µl of sample or standard into the appropriate endotoxin-freereaction tube in a 37°C ± 1°C block or waterbath.
Each series of determinations must include a blank plus the four endotoxin standards run in duplicate. The blank tubes contain 50 µl of LAL Reagent Water
instead of sample. All reagent additions and incubation times are identical.
2. At time T = 0, add 50 µl of LAL to the reaction vessel. Begin timing as LAL is added
to the first reaction vessel. It is important to be consistent in the order of reagent
addition from vessel to vessel and in the rate of pipetting. Thorough mixing of thetwo solutions is essential, but do not vortex.
3. At T = 10 minutes, add 100 µl of substrate solution (prewarmed to 37°C ± 1°C).
Pipette the substrate in the same order as in Step 2. Maintain a consistent pipetting rate. Assure thorough mixing of solutions.
4. At T = 16 minutes, add 100 µl of stop reagent. Maintain the same pipetting orderand rate as in Steps 2 and 3. Mix well.
5. Read the absorbance of each reaction tube at 405-410 nm using distilled water toadjust the photometer to zero absorbance.
Microplate Method
1. Pre-equilibrate the microplate at 37°C ± 1°C in the heating block adapter.
(NOTE: Do not use a cabinet-style incubator to run this test.)
2. While leaving the microplate at 37°C ± 1°C, carefully dispense 50 µl of sample
or standard into the appropriate microplate well.
Each series of determinations must include a blank plus the four endotoxin standards run in duplicate. The blank wells contain 50 µl of LAL Reagent Water insteadof sample. All reagent additions and incubation times are identical.
3. At time T = 0, add 50 µl of LAL to the first microplate well, or first column ofmicroplate wells if using a multi-channel pipettor and reagent reservoir. Begin timing as the LAL is added. It is important to be consistent in the order of reagent
addition from well to well or row to row, and in the rate of pipetting. Once the LAL
has been dispensed into all microplate wells containing samples or standards,
briefly remove the microplate from the heating block adapter and repeatedly tap
the side of the plate to facilitate mixing. Return the plate to the heating block
adapter and replace cover.
4. At T = 10 minutes, add 100 µl of substrate solution (prewarmed to 37°C ± 1°C).
Pipette the substrate solution in the same manner as in Step 3. Maintain a consistent pipetting rate. Once the substrate solution has been dispensed into all
microplate wells, briefly remove the microplate from the heating block adapter
and repeatedly tap the side of the plate to facilitate mixing. Return the plate to theheating block adapter and replace cover.

8

5.

6.

At T = 16 minutes, add 100 µl of stop reagent. Maintain the same pipetting order
as in Steps 3 and 4. Once the stop reagent has been dispensed into all microplate
wells, remove the plate and repeatedly tap the side of the plate.
Read the absorbance of each microplate well at 405-410 nm using distilled waterto adjust the photometer to zero absorbance.
NOTE: The performance characteristics of certain microplate readers are optimal
with sample volumes less than 300 µl. The final reaction volume per well can be
reduced by adding only 50 µl of the above suggested stop reagents without
adversely affecting the test results.

PERFORMANCE CHARACTERISTICS
Linearity
The linearity of the standard curve within the concentration range used to predictendotoxin values should be verified with each new lot of reagents (known as an Initial
Qualification). No less than 4 endotoxin standards spanning the desired concentration
range should be assayed along with a blank, in quadruplicate. Under standard assay
conditions, endotoxin standards ranging from 0.1 to 1.0 EU/ml may be prepared as
described in REAGENT PREPARATION.
The coefficient of correlation, r, for the individual mean $ absorbance of the standards (at least 16 points) vs. their respective endotoxin concentration (see CALCULATION OF ENDOTOXIN CONCENTRATION, Calculator Method) should be r0.980.
Reproducibility
Replicate samples should be run in order to establish good technique and low coefficient of variation. The coefficient of variation (C.V.) equals 100 times the standard deviation of a group of values divided by the mean and is expressed as a percent. The C.V.
absorbance should be less than 10%. With experience, values of 3-4% should be attainable when measured on the uncorrected absorbance for the 1 EU standard during thequalification assay.

B.

Calculator Method
A calculator equipped with linear regression capability can be used. Enter themean 6 absorbance and the corresponding concentrations of the four standards.
Determine the corresponding endotoxin concentration of the samples from theirabsorbance by linear regression.

Tube
Well

Sample

1
2
3
4
5
6
7
8
9
10
11
12
13
14

LAL Reagent
Water (Blank)
0.1 EU/ml
Standard
0.25 EU/ml
Standard
0.5 EU/ml
Standard
1.0 EU/ml
Standard
Product#1
Product#2

Example Data
AbsorbanceMean
at 405 nm
Absorbance
0.080
0.084
0.160
0.180
0.309
0.325
0.570
0.557
1.052
1.012
0.372
0.392
0.916
0.912

CALCULATION OF ENDOTOXIN CONCENTRATION
Under the standard conditions, the absorbance at 405-410 nm is linear in the concentration range of 0.1 to 1.0 EU/ml endotoxin (See PERFORMANCE CHARACTERISTICS). There are several methods to determine the endotoxin concentration of samples.
Subtract the mean absorbance of the blank from the mean absorbance value of the standards and samples to calculate mean $ absorbance.
A. Graphic Method
Plot the mean $ absorbance for the four standards on the y-axis vs. the corresponding endotoxin concentration in EU/ml on the x-axis. Draw a best fit straightline between these points and determine endotoxin concentrations of samples
graphically.

9

10

Mean 6
Absorbance

0.082

–

0.170

0.088

0.317

0.235

0.564

0.482

1.032

0.950

0.382

0.300

0.914

0.832

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To verify the lack of product inhibition, an aliquot of test sample (or a dilution of test
sample) is spiked with a known amount of endotoxin (e.g. 0.4 EU/ml). The spiked solution is assayed along with the unspiked samples and their respective endotoxin concentrations are determined. The difference between these two calculated endotoxin values
should equal the known concentration of the spike ± 25%.
A spiked aliquot of the test sample (or dilution) may be prepared as follows:1.

2.

Prepare a 1.0 EU/ml endotoxin solution in the test sample (or dilution) by diluting
the endotoxin stock solution 1/X, where X is the endotoxin concentration of stockin EU/ml. Use the test sample (or dilution) as the diluent. This solution should bevigorously vortexed for one minute before proceeding. For example, if the concentration of the endotoxin stock solution is 24 EU/ml, the initial dilution is 1/24 or
0.1 ml of endotoxin stock solution into 2.3 ml of test sample (or dilution).
To prepare a 0.4 EU/ml endotoxin solution in test sample (or dilution), dilute the
1.0 EU/ml solution 1/2.5 using the test sample (or dilution) as the diluent. Thiscan be done by combining 1.0 ml of the 1.0 EU/ml solution in test sample (or dilution) with 1.5 ml of test sample (or dilution). This solution should be vigorously
vortexed for 1 minute prior to use.

If the test sample (or dilution) is found inhibitory to the LAL reaction the sample may
require further dilution until the inhibition is overcome.
Example:Determination of a Non-Inhibitory Dilution

Sample Dilution
NOTE
If the concentration of endotoxin in the test sample is greater than 1.0 EU/ml, dilute
sample 5-fold in LAL Reagent Water and retest. Calculate the concentration of the diluted sample and multiply by 5 to determine the original endotoxin concentration.
PRODUCT INHIBITION
Product inhibition occurs when substances in the test sample interfere with the LAL
reaction. In the chromogenic assay, this inhibition results in a lower, final 6 absorbance,
indicating lower levels of endotoxin than what may actually be present in the test sample. The lack of product inhibition should be determined for each specific sample, either
undiluted or at an appropriate dilution.

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Calculated Endotoxin Concentration (EU/ml)
Unspiked
SpikedDifference

1/10

0.18

0.28

0.10
Inhibitory

1/20

0.11

0.36

0.25
Inhibitory

1/40

<0.1

0.44

0.44
Non-inhibitory

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Initially, one may want to screen for product inhibition by testing 10-fold dilutionsof 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
The degree of inhibition or enhancement will be dependent upon the concentrationof product. If several concentrations of the same product are to be assayed, it is necessary to establish performance characteristics for each independently.
Patterns of inhibition or enhancement different from those seen with the traditional
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.

preparations vary in both the traditional gel test and the chromogenic method. The
endotoxin standard supplied in this kit has been compared to the RSE using the chromogenic assay and the potency is provided on the enclosed Certificate of Analysis. The
calibration curve diluted from this standard will yield a range of 0.1 to 1.0 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 incomparison to those in the chromogenic test where standardization is continuous andvariations are minimal.
A NOTE FOR OUR INTERNATIONAL CUSTOMERSOther regulatory agencies may adopt other performance standards which need tobe satisfied in order to be in compliance in their jurisdictions.

COLORED SAMPLESIn the Chromogenic assay, samples which possess significant color on their own
may require special attention. Also, if using 25% acetic acid as the stop reagent, onemust be conscious of products which turn yellow in acid environments, such as certain
tissue culture media.
A quick test to determine if a product’s intrinsic color is sufficient to be of concern
is to construct a mock reaction tube. Add 50 µl of sample, 150 µl LAL Reagent Water
and 100 µl appropriate stop reagent without incubation. Read the absorbance at 405410 nm of this solution. If the absorbance is significantly greater than the absorbanceof LAL Reagent Water, then the color of the product must be taken into account.
At the time of assay, prepare a sample blank by combining 50 µl sample, 150 µl LAL
Reagent Water, and 100 µl appropriate stop reagent without incubation. In addition,
assay the product along with the appropriate standards and LAL Reagent Water blank.
To calculate 6 absorbance of the sample, subtract the absorbance of the sample blankas well as the mean absorbance of the LAL Reagent Water blank. However, use only the
LAL Reagent Water blank to calculate 6 absorbance for the endotoxin standards andnon-colored products.
If the background color is significant (>0.5 absorbance units), the sample should bediluted and reassayed. The dilution factor is then used in the final calculations for determining the concentration of endotoxin.
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 assistance in standardizing LAL testing. The FDA reference LAL is not readily used in this test since it has
not been formulated for the chromogenic method. However, the official endotoxin has
been used to standardize the endotoxin in this kit. The potency of different endotoxin

15

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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 coagulation ofLimulus 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 and kinetics
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 coagulagen of Limulus polyphemus hemocytes. A comparison of
the clotted and non-clotted forms of the molecule. Thromb. Res. 2:55 (1973).
7. 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).
8. U.S. Department of Health and Human Services, Public Health Service, Food andDrug Administration “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” (1987).
9.
Chapter <85> Bacterial Endotoxins. Rockville, MD: United States Pharmacopeia.

NOTES

PATENTS
Various components of this product are protected under the following U.S. Patents:
45102414322717

TRADEMARKS
ELx808TM 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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8830 Biggs Ford Road | Walkersville, MD 21793
301-898-7025 | www.lonza.com

08291
P50-648U-9
12/07