Limulus Amebocyte Lysate (LAL)
Kinetic-QCL®

Catalog Number: 50-650U, 50-650NV, 50-650H

BLANKTABLE OF CONTENTS
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 . . . . . . . . . . . . . . . . . . . . . . . . .5
TYPES OF KINETIC-QCL ASSAYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
REAGENT PREPARATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
TEST PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
PERFORMANCE CHARACTERISTICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
CALCULATION OF ENDOTOXIN CONCENTRATION . . . . . . . . . . . . . . . . . . . . .10
POWERCURVE™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
PRODUCT INHIBITION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
LIMITATIONS AND INDICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
COLORED SAMPLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
ARCHIVED STANDARD CURVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
CORRELATION WITH OTHER METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
A NOTE FOR OUR INTERNATIONAL CUSTOMERS . . . . . . . . . . . . . . . . . . . . .16
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
PATENT INFORMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

2

Kinetic-QCL®
U.S. License No. 1775

—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 test utilizes a preparation of Limulus Amebocyte Lysate (LAL),
in combination with an incubating photometer and appropriate software, to detect endotoxin photometrically.
In December 1987, the United States Food and Drug Administration (FDA) published the “Guideline on the Validation of the Limulus Amebocyte Lysate Test as an EndProduct Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products,
and Medical Devices”7. This guideline outlines those procedures which the FDA considers necessary for: 1) establishing endotoxin limits for pharmaceuticals and medical
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 kinetic-chromogenic assays have been publishedand 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 end-product testing of human and animal parenteral drugs, biological products, and medical devices7.
EXPLANATION OF TEST
Kinetic-QCL® is a quantitative, kinetic assay for the detection of Gram-negative bacterial endotoxin. A sample is mixed with the LAL/SUBSTRATE reagent, placed in an
incubating plate reader, and automatically monitored over time for the appearance of ayellow color. The time required before the appearance of a yellow color (Reaction Time)is inversely proportional to the amount of endotoxin present. That is, in the presence ofa large amount of endotoxin the reaction occurs rapidly; in the 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.

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 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 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,
Levin, and Prendergast8 have purified and characterized the clottable protein from LAL
and have 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 from a synthetic substrate, producing a yellow color.
PRINCIPLE
Endotoxin
Proenzyme
EnzymeEnzymeSubstrate + H2O
Peptide + pNA
Gram negative bacterial endotoxin catalyzes the activation of a proenzyme in the
Limulus Amebocyte Lysate (LAL). The initial rate of activation is determined by the concentration of endotoxin present. The activated enzyme catalyzes the splitting of pnitroaniline (pNA) from the colorless substrate Ac-Ile-Glu-Ala-Arg-pNA. The pNA
released is measured photometrically, at 405 nm continuously throughout the incubation period. The concentration of endotoxin in a sample is calculated from its Reaction
Time by comparison to the Reaction Time of solutions containing known amounts ofendotoxin standard.

REAGENTS SUPPLIED AND STORAGE CONDITIONS
Kit part number

Kinetic-QCL®
LAL Reagent

Endotoxin

50-650U

8 vials/kit

2 vials/kit

50-650NV

85 vials/kit

15 vials/kit

50-650H

100 vials/kit

10 vials/kit

LAL Reagent
Water
3 vials
____
____

®

Kinetic-QCL Reagent
Each vial contains a co-lyophilized mixture of lysate prepared from the circulating
amebocytes of the horseshoe crab, Limulus polyphemus, and chromogenic substrate.
Reconstitute immediately before use with 2.6 ml of LAL Reagent Water per vial. If
the contents of more than one vial are required, pool two or more vials before use. Swirlgently to avoid foaming. Lyophilized Kinetic-QCL® Reagent is to be stored at 2-8°C.
Protect from long term exposure to light.
Reconstituted Kinetic-QCL® Reagent should be used promptly. Reconstituted KineticQCL® Reagent is stable for 8 hours at 2-8°C. Reconstituted reagent can be stored at-10°C or colder for up to two weeks. Freeze and thaw only once.

4E. coli O55:B5 Endotoxin
Each vial contains 50-200 EU lyophilized endotoxin. The reconstitution volume ofthe vial is stated on the Certificate of Analysis, included with each kit, and is calculated toyield a solution containing 50 EU(or IU)/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 use, the solution must be warmed to room temperature and vigorously
vortexed for 15 minutes. This is important because the endotoxin tends to attach toglass.
Lyophilized E. coli O55:B5 Endotoxin is to be stored at 2-8°C. Reconstituted stock
endotoxin is stable for four weeks at 2-8°C.
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.
LAL Reagent Water
Each vial contains 30 ml of LAL Reagent Water. This water is to be used to rehydrate the Kinetic-QCL® Reagent and E. coli Endotoxin and to prepare endotoxin and sample dilutions.
LAL Reagent Water is to be stored at 2-8°C. (Note: not included in 50-650H oror 50-650NV kits)
MATERIALS AND EQUIPMENT NOT PROVIDED
1. Disposable endotoxin-free glass dilution tubes (13 x 100 mm, #N207 or equivalent).
2. Individually wrapped 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 for each lysate lot by the acceptable performance of the linearity test
as described under PERFORMANCE CHARACTERISTICS.
5. Reagent reservoirs (#25-364 or equivalent).
6. Eight-channel multi-pipettor.
7. Sodium hydroxide, 0.1N, or Hydrochloric acid, 0.1N, dissolved in LAL Reagent
Water, for pH adjustment of sample if necessary.
8. Microplate reader (Kinetic-QCL® Reader, #25-141B; ELx808TM IU Reader,#25-315; or ELx808TM RIU Reader, #25-315R).
9. WinKQCL® Software
10. Timer and Vortex Mixer.
.
11. For 50-650H and 50-650NV: LALReagent Water (#W50-640, #W50-100, #W50-500,
or equivalent).
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

5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 Kinetic-QCL® reagent has been
opened previously or was not supplied by Lonza , the diluent alone must be tested forendotoxin contamination.
TYPES OF KINETIC-QCL® ASSAYS
The incubating microplate reader and WinKQCL® Software are an integral part of theKinetic-QCL® 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
incubating microplate reader and WinKQCL® Software Manuals for more detailed information.
There are four (4) basic types of Kinetic-QCL® assays, each of which is designed toperform a different aspect of LAL testing.
1.

ROUTINE
A ROUTINE assay calculates the concentration of endotoxin in unknowns by comparison 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 asample of product to which a known amount of endotoxin spike has been added.
The WinKQCL® Software automatically calculates the amount 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 with samples which inhibit the
LAL test do not necessarily indicate the absence of endotoxin.

6

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 of endotoxin spike. In this manner it can be determinedwhich product dilutions are non-inhibitory.
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 Reference Standard
Endotoxin (RSE).
The assay requires a single series of RSE dilutions and one or more sets of dilutions of the CSE. Depending on the concentration units of the CSE, the WinKQCL®
Software automatically computes mean potency values in terms of EU/ng or
EU/ml. The user also has the option to enter units other than EU or ng.

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”.7 This assay is required as part of thevalidation of the LAL assay and is also to be performed with each new lot ofKinetic-QCL®.
The INITIAL QUALIFICATION assay performs a log/log linear 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 of each standard.
The INITIAL QUALIFICATION assay does not provide for the inclusion of any samples.

REAGENT PREPARATION
In order to calculate endotoxin concentrations in unknown samples each KineticQCL® 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 theconcentration of endotoxin standards used to generate the standard curve. A minimumof three standards is required.
The Kinetic-QCL® assay has been optimized to be linear from 0.005 EU/ml to 50.0
EU/ml. However, the individual user may choose to truncate the standard curve depending on specific product requirements. Data indicates that truncating a chromogenic LAL

7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 chromogenic LAL standard
curve range to be used for routine testing of product samples.10
The table below suggests a dilution scheme for constructing a series of endotoxin
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 as well 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 Guidelines 7.
Endotoxin
Concentration(EU/ml)
5
0.5
0.05
0.005
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 50 EU/ml solution
0.1 ml of 5 EU/ml solution
0.1 ml of 0.5 EU/ml solution
0.1 ml of 0.05 EU/ml solution

Prepare a solution containing 5 EU/ml endotoxin by adding 0.1 ml of the 50 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 5 EU/ml endotoxin solution into 0.9 ml of LAL Reagent
Water in a suitable container and label 0.5 EU/ml. The solution should be vigorously vortexed for at least 1 minute before proceeding.
Transfer 0.1 ml of the 0.5 EU/ml endotoxin solution into 0.9 ml of LAL Reagent
Water in a suitable container and label 0.05 EU/ml. This solution should be vigorously vortexed for at least 1 minute before proceeding.
Transfer 0.1 ml of the 0.05 EU/ml endotoxin solution into 0.9 ml of LAL Reagent
Water in a suitable container and label 0.005 EU/ml. This solution should be vigorously vortexed for at least 1 minute before proceeding.

TEST PROCEDURE
Refer to the microplate reader and WinKQCL® Software manuals for more detailed
information on performing a Kinetic-QCL® test.
1.

2.

Create a specific TEMPLATE for the test to be run. A TEMPLATE contains the nameof the analyst, type of assay, lot numbers of reagents, the number and concentration of endotoxin standards, number of replicates, and how standards and samples will be organized on the microplate.
The Type of Assay must be selected as KQCL. The Default Template Parametersthat follow must not be changed:8Delta t (seconds):
150
Measurement filter (nm):405
Delta mOD:
200
Number of Reads:
40
3. Print the TEMPLATE for use as a guide in placing standards and samples into themicroplate.
4. “Run” the TEMPLATE, following the WinKQCL® Software prompts.
5. Carefully dispense 100 Ml of the LAL Reagent Water blank, endotoxin standards,
product samples, positive product controls (see pages 13 and 14 for positive
product control instructions), etc. into the appropriate wells of the microplate.
6. Place filled plate in the microplate reader and close the lid. If using the KineticQCL® Reader, press the UP directional key to position the plate in the incubationchamber.
7. Pre-incubate the plate for r10 minutes at 37°C ± 1oC.
8. Near the end of the preincubation period reconstitute each of the appropriate
number of Kinetic-QCL® Reagent vials with 2.6 ml LAL Reagent Water per vial. Mix
gently but thoroughly.
9. Pool the reagents into a reagent reservoir and mix by gently rocking the reservoir
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 chamber.
11. Using an 8-channel multipipettor dispense 100 Ml of the Kinetic-QCL® Reagent into
all wells of the microplate beginning with the first column (A1-H1) and proceeding in sequence to the last column used. Add reagent as quickly as possible. Avoid
causing bubbles!
12. Immediately click on the OK button on the computer keyboard to initiate the test.
NOTE: The Kinetic-QCL® assay is performed with the microplate cover removed.
PERFORMANCE CHARACTERISTICS
Linearity
The linearity of the standard curve within the concentration range used to determine
endotoxin values should be verified. No less than 3 endotoxin standards, spanning thedesired concentration range, and an LAL Reagent Water blank should be assayed atleast 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 ofthe standard curve.
The absolute value of the correlation coefficient (r) of the calculated standard curveshould be r0.980.

9

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 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.
CALCULATION OF ENDOTOXIN CONCENTRATION
Continuously throughout the assay, the microplate reader/WinKQCL® Software monitors the absorbance at 405 nm of each well of the microplate. Using the initialabsorbance reading of each well as its own blank, the reader determines the timerequired for the absorbance to increase 0.200 absorbance units. This time is termed
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 report printout. If the absolutevalue 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™) is an important feature of
the WinKQCL® Software (see POWERCURVE™, page 12).
Linear Regression
The information provided below is an example of how 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 eachsample of each product, the WinKQCL® Software calculates the corresponding endotoxin concentration from the Reaction Time for that sample. The software automaticallyadjusts the final TEST RESULT value to account for any product dilution.

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

LINEAR CORRELATION
EXAMPLE CALCULATIONS
MeanLog
Concentration Reaction Time (Sec) Concentration}
Unreactive}
0.005 EU/ml
4351-2.3010.05 EU/ml
2496-1.301
0.5 EU/ml
1406-0.301
5.0 EU/ml
895
0.699
50.0 EU/ml
561
1.699
}
}

1576
943

10

}
}

Log Mean
Reaction Time
}
3.639
3.397
3.148
2.952
2.749
3.198
2.975

11

TM

POWERCURVE
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 the entire (5 log) endotoxin
range. The use of the POWERCURVE TM model requires the use of WinKQCL ®
Software.
When using POWERCURVETM, a standard curve is generated using the log 10 Reaction
Time values and their corresponding log10 endotoxin concentration to define a polynomial equation. The order of the polynomial equation used to generate the regression
curve is determined by the number of endotoxin standards in the assay. The order ofthe polynomial will always be one less than the number of endotoxin standards, with amaximum of a 4th order polynomial for assays with five 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 asolution to a polynomial equation using the same set of data from the linear correlation
example on page 10.

12

POLYNOMIAL (POWERCURVETM) MODEL
Y = A + BX + CX2 + DX3 + EX4
A = 3.08374
B = -0.20432
C = 0.02894
D = -0.00596
E = -0.00503
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 in those cases
to comply with FDA regulations. 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 Kinetic-QCL® 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 each specific sample, either undiluted or at an appropriate dilution.
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.
It is recommended that the endotoxin spike result in a final endotoxin concentrationin the sample equal to 0.5 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 5.0 EU/ml.
In an INHIBITION/ENHANCEMENT assay, the spiked solution (PPC) is assayedalong with the unspiked sample, and their respective endotoxin concentrations, as wellas the endotoxin recovered in the spiked sample are automatically calculated. The endotoxin recovered should equal the known concentration of the spike within 50 – 200%.9
A spiked aliquot of the test sample (or dilution) may be prepared as in one of thefollowing examples:
Tube method:
Transfer 50 Ml of the 50 EU/ml solution into 4.95 ml of test sample (or dilution). Thissolution contains an endotoxin concentration of 0.5 EU/ml in test sample (or dilution).

13

This sample should be vigorously vortexed for one minute prior to use.
Plate method #1:
Transfer 10 Ml of the 5 EU/ml solution into each of the PPC wells in the 96-well 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 0.5 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 Ml of the 5 EU/ml solution. Eachwell will now contain a 0.5 EU/ml solution. Mix gently.
If the test sample (or dilution) is found to be inhibitory to the Kinetic-QCL® reaction,
the sample may require further dilution until the inhibition is overcome.
Example:Determination of a Non-Inhibitory Dilution
Sample Dilution
1/10

ENDOTOXIN RECOVERED
0.125
Inhibitory

1/20

0.212
Inhibitory

1/40

0.550
Non-Inhibitory

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 AND INDICATIONS
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.
COLORED SAMPLES
Since the initial absorbance reading of each well is used as its own blank, samples
which possess significant color on their own do not present a special problem. If the
background color is r1.5 absorbance units, the sample should be diluted and reassayed.

14

ARCHIVED STANDARD CURVEThe WinKQCL® Software may be run using an archived standard curve. Provided thatthe current reagent identifications for the Kinetic-QCL® Reagent, LAL Reagent Water,
Endotoxin, and microplate reader parameters match those used to generate thearchived 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 curveshould 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 50 to 0.005 EU/ml,
a standard-control equal to 0.5 EU/ml should be assayed.
log 50=1.6990log 0.005=-2.3010-------------------------log average=-0.3010antilog -0.3010=
0.5
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 assistance in standardizing LAL testing. The FDA reference LAL is not readily used in this test since it has
not been formulated for the Kinetic-QCL® 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 chromogenic method. The
endotoxin standard supplied in this kit has been compared to the FDA ReferenceStandard Endotoxin (RSE) using the Kinetic-QCL® assay, and the potency is 50 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 0.005 to 50
Endotoxin Units/ml relative to the RSE. It should be remembered, however, that the tra-15

ditional gel test is standardized by two-fold dilutions, so that variations will appear quite
large in comparison to those in the Kinetic-QCL®‚ test where standardization is continuous and variations are minimal.
A NOTE FOR OUR INTERNATIONAL CUSTOMERSOther regulatory agencies may adopt other performance standards which will need
to be satisfied in order to be in compliance in their jurisdictions.
TRADEMARKS
ELx808TM is a trademark of BioTek Instruments, Inc.
Unless otherwise noted, all trademarks herein are marks of the Lonza Group or its
affiliates.

16

REFERENCES
1.
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PATENT INFORMATION
Various components of this product are protected under the following U.S. patents:
43222174510241
5310657

17

NOTES

18

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