Limulus Amebocyte Lysate (LAL)PYROGENT TM Plus

Catalog Numbers: N283-06, N283-125
N284-25, N288-25
N294-03, N294-06, N294-125
N494-03, N494-06, N494-125
Certificate of Analysis at www.lonza.com/coa

62656 N283-06

8/26/04

8:37 AM

Page 2

MULTI-TEST
LIMULUS AMEBOCYTE LYSATEPYROGENT TM Plus
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 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. The Limulus Amebocyte Lysate (LAL) test is aqualitative test for Gram-negative bacterial endotoxin. Limulus Amebocyte Lysate
as supplied is to be reconstituted with LAL Reagent Water and then mixed in equalparts with the solution being tested. After incubation, and in the presence of endotoxin, gelation occurs; in the absence of endotoxin, gelation does not occur.
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”10. This guideline outlines those procedures which the FDA considers necessary for: 1) establishing endotoxin limits for2

62656 N283-06

8/26/04

8:37 AM

Page 3pharmaceuticals 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 conform with those described in the FDA
Guideline. Similar performance requirements for gel clot assays have been published and are updated regularly in the United States Pharmacopeia.11
WARNINGFor 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 testing of humanand animal parenteral drugs, biological products, and medical devices10.
EXPLANATION OF TESTThe use of LAL for the detection of endotoxin evolved from the observation by
Bang1 that Gram-negative infection of Limulus polyphemus resulted in fatal
intravascular coagulation. Levin and Bang2,3 later demonstrated that this clotting
was a result of the action between endotoxin and a clottable protein in the circulating amebocytes of Limulus blood. Following the development of a suitable anticoagulant for Limulus blood, Levin and Bang4 prepared a lysate from washedamebocytes which was an extremely sensitive indicator of the presence of endotoxin. Solum5,6 and Young, Levin, and Prendergast7 have purified and characterized the clottable protein from LAL and have shown the reaction with endotoxin to
be enzymatic.
PRINCIPLE
Proenzyme
Coagulogen

Endotoxin
Coagulase

Coagulase
Coagulin3

62656 N283-06

8/26/04

8:37 AM

Page 4Gram-negative bacterial endotoxin catalyzes the activation of a proenzyme in
the Limulus Amebocyte Lysate7. 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.
REAGENTS SUPPLIED AND STORAGE CONDITIONSLimulus Amebocyte Lysate (LAL), Lyophilized
A lysate prepared from the circulating amebocytes of the horseshoe crab
(Limulus polyphemus) standardized to detect the labeled concentration (EU/ml) of
the FDA Reference Standard Endotoxin.
Contains buffered mono and divalent cations. Lysate is lyophilized and sealed
under vacuum and is to be reconstituted with LAL Reagent Water. Do not rehydrate until immediately prior to use.
Lyophilized (unreconstituted) Limulus Amebocyte Lysate should be stored
under refrigeration at 2-8°C. Care should be taken to avoid exposing the lysate totemperatures in excess of 37°C. Lysate which has been exposed to prolonged
periods of temperatures above 37°C or to bright light may turn yellow and/or
become insoluble. Lysate which exhibits such characteristics should be discarded.
Reconstituted lysate may be stored at 2-8°C for 24 hours. For longer storage,
reconstituted lysate can be stored at -10°C or colder. Freeze and thaw only once. The
lysate should be protected from exposure to light during storage. Use within fourweeks after reconstitution.

4

62656 N283-06

8/26/04

8:37 AM

Page 5

E. coli Endotoxin O55:B5, Lyophilized
A lyophilized preparation of purified endotoxin from E. coli strain 055:B5.
Each vial when prepared according to the instructions below provides theuser with a Control Standard Endotoxin (CSE) whose potency has been
established using the current FDA Reference Standard Endotoxin (RSE)and the enclosed lot of lysate according to the procedures described herein. The
appropriate RSE/CSE ratio and resultant CSE potency is provided on the Certificate
of Analysis.
This CSE preparation, with established potency, is an acceptable substitute for
the FDA RSE in all aspects of quality control provided the laboratory is using thedesignated lysate lot according to the procedures specified in this insert.
Store vial at 2-8°C prior to reconstitution. Reconstitute with 5.0 ml LAL Reagent
Water. Potency (in EU/ml) is calculated from RSE/CSE ratio as in the following example:
Potency (EU/ml) = RSE/CSE Ratio (in EU/ng) X___ng/vial ÷ 5.0 ml/vial.
Store reconstituted vial at 2-8°C for up to 4 weeks. Prepare 1.0 EU/ml dilution
from this vial only in quantities as needed. See section on Reagent Preparation.
Do not store or use diluted endotoxin preparations for more than 1 day.
WARNING: Contents pyrogenic. Not to be administered to humans.
KIT CONFIGURATION
Cat. No. N283 contains Limulus Amebocyte Lysate 4 x 16 test vials(1.8 ml/vial) and 1 vial E. coli Control Standard Endotoxin (CSE)
Cat. No. N284 and N294 contains Limulus Amebocyte Lysate 4 x 50 test vials(5.2 ml/vial) and 1 vial E. coli Control Standard Endotoxin (CSE)
Cat. No. N288 and N494 contains Limulus Amebocyte Lysate 80 x 50 test vials(5.2 ml/vial) and 20 vials E. coli Control Standard Endotoxin (CSE)5

62656 N283-06

8/26/04

8:37 AM

Page 6

MATERIALS AND EQUIPMENT NOT PROVIDED
1.
LAL Reagent Water which should not cause gelation of reconstituted lysate
after 24 hours incubation at 37°C ± 1°C (#W50-640 or equivalent).
2.
Pipettes, 1.0 ml, 5.0 ml, 10.0 ml and 100 microliter, endotoxin-free.
3.
10 x 75 mm glass reaction tubes, endotoxin-free (see Specimen Collection
and Preparation for sterilizing procedure) (#N201, #N205 or equivalent).
4.
13 x 100 mm glass dilution tubes, endotoxin-free (#N207 or equivalent).
5.
Sodium hydroxide, 0.1N, or Hydrochloric acid, 0.1N dissolved in LAL
Reagent Water, for pH adjustment of sample if necessary.
6.
Heating block or non-circulating hot water bath (37°C ± 1°C).
7.
Test tube rack.
8.
Timer.
9.
Vortex Mixer.

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.8,9 Always measure
the pH of an aliquot of the bulk sample to avoid contamination by the pH elec-

662656 N283-06

8/26/04

8:37 AM

Page 7

trode. 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 and frozen for periods greater than 24hours. It is the responsibility of the customer to validate the proper container andstorage conditions for their samples.
If the container of diluent used to rehydrate the Limulus Amebocyte Lysate(LAL) has been opened previously or was not supplied by Lonza, the diluent alone
must be tested for endotoxin contamination.
REAGENT PREPARATION
1.

2.

Allow reagents to equilibrate to room temperature prior to use.
Preparation of Limulus Amebocyte Lysate.
Caution: Do not rehydrate until immediately prior to use.
A. Reconstitute lyophilized lysate by adding 1.8 ml LAL Reagent Water tothe 16-test vial or 5.2 ml to the 50-test vial. Swirl gently but thoroughly for at least 30 seconds. Do not shake as contents will foam.
B. Reconstituted lysate can be stored for up to 24 hours at 2-8°C without
loss of sensitivity. Reconstituted lysate can be divided into more convenient volumes and stored below -10°C for up to four weeks. Frozen
liquid lysate should be thawed immediately before use. Freeze andthaw only once.
Preparation of E. coli Control Standard Endotoxin (CSE).
Note: Plastic tubes are not recommended for making endotoxin dilutions.
A. Reconstitute the vial of endotoxin with 5.0 ml LAL Reagent Water.
B. Vortex the vial of endotoxin for at least 15 minutes.
C. Dilute the endotoxin with LAL Reagent Water to a concentration of 1
EU/ml.
This is accomplished by diluting the reconstituted endotoxin to 1/X,

7

62656 N283-06

D.

8/26/04

8:37 AM

Page 8where X is the CSE potency in EU/ml as specified on the Certificate
of Analysis.
Using X as defined above, the general formula is 0.1 ml reconstituted
endotoxin diluted with 0.1 (X-1) ml LAL Reagent Water.
Example for X = 21 EU/ml:
Dilute 0.1 ml endotoxin with 0.1 (21-1) = 2.0 ml LAL Reagent Water.
Vortex 60 seconds before proceeding.
Using the 1 EU/ml endotoxin solution, prepare a serial two-fold dilution series that brackets the sensitivity of lysate as shown in the following example. Each dilution should be vortexed for 60 seconds prior toproceeding to the next dilution.

Dilution Series for Use With Lysate of 0.125 EU/ml Sensitivity
Water
Endotoxin
Tube#
(ml)
Volume Added to Water
Concentration
1
1.0
1.0 ml from 1 EU/ml
0.5 EU/ml
2
1.0
1.0 ml from Tube 1
0.25 EU/ml
3
1.0
1.0 ml from Tube 20.125 EU/ml
4
1.0
1.0 ml from Tube 30.06 EU/ml
51.0
1.0 ml from Tube 40.03 EU/ml
TEST PROCEDURE AND INTERPRETATION
Each assay should include serial two-fold dilutions of the Control Standard
Endotoxin (CSE) which brackets the labeled lysate sensitivity, dilutions of the test
sample, and LAL Reagent Water to serve as a negative control. To avoid microbial
or endotoxin contamination, carefully transfer 0.10 ml of standard, sample, or
water into the appropriate 10 x 75 mm reaction tube.

862656 N283-06

8/26/04

8:37 AM

Page 9Add 0.10 ml of the reconstituted lysate to each tube beginning with the blank thenmoving from lowest to highest concentration of endotoxin. Immediately following theaddition of the lysate to each tube, the contents should be mixed thoroughly and thetube placed in a 37°C + 1°C non-circulating hot water or dry heat bath. This procedure
should be followed for each dilution of the endotoxin. The unknown test sample mustbe run in parallel with the control standard endotoxin. The assay may be done either
as a yes/no test at a single dilution or as a quantitative test via a dilution series.
The incubation time should be determined from the time each tube is placed in the
37°C ± 1°C bath. Assay tubes should not be removed from incubation or disturbed prior to the time specified for reading the test. After 60 minutes (±2 minutes)
of incubation, carefully remove each tube and invert 180°.
1.
2.

3.

A positive reaction is characterized by the formation of a firm gel thatremains intact momentarily when the tube is inverted.
A negative reaction is characterized by the absence of a solid clot after
inversion. The lysate may show an increased turbidity or viscosity. This isconsidered a negative result.
The reaction in each tube should be recorded in columns as either positiveor negative.

CONFIRMATION OF LABEL CLAIM
Each vial of LAL is labeled with the lysate sensitivity obtained using the FDA
Reference Standard Endotoxin, and is expressed in Endotoxin Units.
As part of an initial in-house validation, each user should reverify the labeled
lysate sensitivity using an endotoxin standard whose potency is known.
Prepare serial two-fold dilutions of the Control Standard Endotoxin whichbracket the labeled lysate sensitivity. Each dilution, as well as a negative water
control, should be assayed in quadruplicate. After the one hour incubation period,

962656 N283-06

8/26/04

8:37 AM

Page 10the positive and negative results are recorded. The endpoint dilution is determinedas the last dilution of endotoxin which still yields a positive result.
Assay Results - Gel Clot Method

Labeled Lysate Sensitivity = 0.125 EU/ml

Replicate
1
2
3
4

0.50
+
+
+
+

Endotoxin Dilution (EU/ml)
0.25
0.125
0.06
0.03
+
+
+
+
+
+
+
+
+
-

H20
-

Endpoint
0.125
0.125
0.06
0.125

The lysate sensitivity is calculated by determining the geometric mean of theendpoint. Each endpoint value is converted to log10. The individual log10 values areaveraged and the lysate sensitivity is taken as the antilog10 of this average log
value.
Calculation of Geometric Mean Endpoint
Endpoint(EU/ml)
0.125
0.125
0.06
0.125

Log10
Endpoint-0.903-0.903-1.222-0.903
Mean = -0.983
Antilog10 Mean = 0.10 EU/ml

1062656 N283-06

8/26/04

8:37 AM

Page 11

Acceptable variation is one half to two times the labeled lysate sensitivity.
DETERMINATION OF ENDOTOXIN IN AN UNKNOWN

To determine the endotoxin concentration of an unknown solution, test serial
two-fold dilutions of sample until an endpoint is reached. Calculate the geometricmean dilution as before and multiply by the labeled lysate sensitivity.
Determination of Endotoxin Concentration in an Unknown
Labeled Lysate Sensitivity = 0.125 EU/ml

Replicate1
2
Endpoint Dilution
1 / 8 ( 0 .1 2 5 )1 / 1 6 ( 0 .0 6 2 5 )

1/2
+
+

Sample Dilution
1/4
1/8 1/16
+
+
+
+
+

1/32
-

1/64
-

Log10 Endpoint- 0 .9 0 3- 1 .2 0 4
Mean =-1.054
Antilog 10 Mean =0.088 = 1/11.4

Endotoxin Concentration= lysate sensitivity x endpoint dilution= 0.125 EU/ml x 11.4 = 1.4 EU/ml

11

62656 N283-06

8/26/04

8:37 AM

Page 12PRODUCT INHIBITION
The Limulus Amebocyte Lysate reaction is enzyme mediated and, as such, hasan optimal pH range, and specific salt and divalent cation requirements.
Occasionally test samples may alter these optimal conditions to an extent that thelysate is rendered insensitive to endotoxin. Negative results with samples which
inhibit the LAL test do not necessarily indicate the absence of endotoxin.
Initially, each type of sample should be screened for product inhibition.
Prepare a series of two-fold dilutions of endotoxin in LAL Reagent Water and asimilar series of endotoxin dilutions using sample as diluent. Assay each series inparallel using standard procedures. At the end of the incubation period, record
positive and negative results and calculate the geometric mean endpoint for both
series of endotoxin dilutions. Products are said to be free of product inhibition if
the geometric mean endpoint of endotoxin in product is within 1/2 to 2 times thelabeled lysate sensitivity. See the following example.

Endotoxin
in Water

1)
2)
3)
4)

Product Inhibition Testing
Labeled Lysate Sensitivity = 0.125 EU/ml
Endotoxin Dilution (EU/ml)
0.50
0.25
0.125
0.06
+
+
+
+
+
+
+
+
+
+
+
+
+
geometric mean endpoint = 0.10 EU/ml

12

0.03
-

62656 N283-06

8/26/04

8:37 AM

Page 13in Product A

1)
2)
3)
4)

+
+
+
+
+
+
+
+
+
+
+
geometric mean endpoint = 0.15 EU/ml
non-inhibitory-

in Product B

1)
2)
3)
4)

+
+
+
+
geometric mean endpoint = 0.50 EU/ml
inhibitory-

The easiest way to overcome product inhibition is through dilution. The
dilution factor must be taken into account when calculating the total endotoxin concentration in a test sample. As a quick screen to determine a non-inhibitory dilution of product, prepare a series of increasing dilutions of the product containing an endotoxin spike equal in concentration to twice the lysate sensitivity.
Assay each spiked product dilution using standard procedures. Positive results
indicate when product inhibition has been overcome. Products which are
extremely acidic or basic may require pH adjustment as well as dilution in order
to completely overcome product inhibition.
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.

13

62656 N283-06

8/26/04

8:37 AM

Page 14

REFERENCES
1.
Bang, F.B. (1956) Bull. Johns Hopkins Hosp. 98:325-351.
2.
Levin, J. and F.B. Bang (1964) Bull. Johns Hopkins Hosp. 115:337.
3.
Levin, J. and F.B. Bang (1964) Bull. Johns Hopkins Hosp. 115:265-274.
4.
Levin, J. and F.B. Bang (1968) Thrombos. Diath. Haemorrh. 19:186-197.
5.
Solum, N.O. (1970) Thrombos. Diath. Haemorrh. 23:170-181.
6.
Solum, N.O. (1973) Throm. Res. 2:55-70.
7. Young, N.S., J. Levin and R.A. Prendergast (1972) J. Clin. Investig. 51:17901797.
8.
Nachum, R., A. Lipsey and S.E. Siegel (1973) N. Eng. J. Med. 289:931-934.
9.
Cooper, J.F., H.D. Hochstein and E.B. Seligman, Jr. (1972) Bull. Parent. Drug
Assoc. 26:153-162.
10. 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).
11. Chapter <85> Bacterial Endotoxins. Rockville, MD: United States
Pharmacopeia.
TRADEMARKS
Unless otherwise noted, all trademarks herein are marks of the Lonza
Group or its affiliates.

14

62656 N283-06

8/26/04

8:37 AMNOTES

15Page 15

62656 N283-06

8/26/04

8:37 AM

Page 168830 Biggs Ford Road ⏐ Walkersville, MD 21793
301-898-7025 ⏐ www.lonza.com

08289
PN283/N284/N288-8
01/11