Jpn. J. Infect. Dis., 54, 1311136, 2001

Original Article
A quantitative ln Ⅵtro Assay fわr Detecting Biological Activityof Endotoxin Using Rabbit Peripheral Blood
Masaki Ochiai*, Michiyo Kataoka, Hiromi TbyoIZumi,
Akihiko Yamamoto and Yoshinobu Horiuchi

Department ofSafetyResearch on Biologics, National Institute oflnfectious Diseases,
Gakuen 4‑7‑1, Musashimurayama, Too)0 208‑0011, Japan(Received July 4, 2001. Accepted August 3 1, 2001)

SUMMARY: The pyrogen test or the endotoxin test has been playlng a Crucial role in detectlng endotoxin inparenteral dmgs. The current test methods, however, have disadvantages such as requlrlng a relatively highnumber of animals or an inadequacy in direct evaluation of in vivo activlty. We made an attempt to establish a
new in vitro assay method that can overcome the shortcomlngS Of the current assay methods. We standardized
the system of tumor necrosis factor‑alpha (TNF‑ a ) induction from the peripheral blood of rabbits for assaying
endotoxin activity. This in vitro assay showed a linear dose‑response regression between 0. 1 and 5.0 endotoxin units

per milliliter of endotoxin and a definite homogeneltyof variance by logarithmically transformlng the endotoxin
and TNF‑a concentrations in the reaction mixtures at 5 h of incubation at 37oC. The assay showed a definite
co汀elation with the pyrogen test but not with the endotoxin test when endotoxins丘.om various bacteria were tested.

the pyrogen test in cases in which pyrogenic response tolNTRODUCTION

endotoxin was enhanced synergistically by a product (1 2).
Therefore the endotoxin test has an obviously limited efficacy

Lipopolysaccharide (LPS) of gram‑negative bacteria, also

refTerred to as endotoxin, isknown to perfom variousbiologicalin regulating the biological activity of endotoxin in such prod‑

activities that cause harmful physical effects on humans evenucts.

in very small amounts (1 ,2). Contamination ofendotoxin has

In the present study, We attempted to utilize the system ofbeen, therefore, a serious threat to the safetyofparenteral drugs.

tumor necrosis factor‑alpha (TNF‑a) induction (1 3,14)from

The rabbit pyrogen test (3) has been effectively applied for

peripheral blood of rabbits f♭r assaylng endotoxin activlty.

The results of standardization and evaluation of the assay

testing for endotoxin contamination.

method are presented in comparison with the endotoxin test.

The bacterial endotoxin test which was丘rst developed by

Levin and Bang (4,5) is based on the highly sensitive clotting

or amebocyte lysate of the horseshoe crab (LAL) by endo‑

MATERIALS AND METHODS

toxin. The test was markedly improved, thereafter, to allow
its practical application to a wide range of parenteral dmgs

LPS : Japanese national reference standard endotoxin (RSE)

including biological products. Particularly, removal or suppres‑sion of the reactivity of LAL reagents to (1‑3)‑BID‑glucqn(6) remarkably proPeeded the wide application of the test ln
Japan by allowlng lt tO avoid the possible confusion caused

extracted from Escherichia colt UKTIB strain ( 1 3,000 EU/vial) (I 5), USP reference standard endotoxin (EC16) derived
from E. coli Ol 13 strain (10,000 EU/vial) and commercial
LPSs; E. colt Olll:B4, E. coli 055:B5, E. colt 127:B8,by reaction with (1‑3)‑β‑D‑glucan or other non‑pyrogenic

Salmonella妙hosa 090 1 (Difco Laboratories, Detroit, Mich.,

substances.

USA), S. abortus equi, Pseudomonas aeruginosa Serotype
10, Klebsiella pneumoniae and Vibrio cholerae Serotype

The endotoxin test was approved in the early 1980s in theevaluatlng the end product or parenteral human drugs andlnaba 569B (Sigma Chemical Co., St. Louis, Mo., USA)strains extracted by the Westphal 's phenol water method wereUnited States (7) as a replacement of the pyrogen test forthis approval has been fわllowed by other nations (8,9). The

used. The commercial LPSs were suspended in pyrogen‑freepyrogen test for interferon Injections and some blood products
was replaced by the endotoxin test in Japan in l993 (10).

distilled water for injection (Otsuka Pharmaceutical Co., Ltd.,
Tbkyo) at a concentration of 1 ･0 mg/ml and kept at 40C until

Application or the endotoxin test to antibiotic products started

use. Without detectable change in LAL activating activity(LALin 1995 and was fわllowed by its expanded application in 1998

actlVity).

The bacterial endotoxin test: The suspensions of LPSs

(1 1). ln the course of implementing the endotoxin test fb∫

biological products, special care was taken to set the allowance

were serially diluted with pyrogen‑free distilled water atlimits so as to take the in vivo synerglStic effect between
endotoxinand the products into account for an effective regula‑tion ofpyrogenicity, because some products such as interferQn‑

fourfold intervals. A 50 JLl‑volume each of appropnate dilu‑
tions was mixed with an equal volume of an endotoxin‑specific
LAL reagent (Endospecy, Seikagaku Corp., Tokyo) whose

β showed marked enhancePent of endotoxin pyrogeniclty

reactivity to (1‑3)‑β‑D一glucan yas removed･ The rate ofcolor

in rabbits ( 1 2)･ Furthermor.e, ln spite of its e.xcellent sensitivity

development was measured uslng a Specially equlpped micro‑

and accuracy, the endotoxln test Shows an lnCOnSistency withplate reader (Well Reader SK601 , Seikagaku Corp.) (16). The*Corresponding author: Tel: +8 1‑42156110771 , Fax: +81‑42‑565‑

to the parallel line assay method uslng logarithmically trans‑

endotoxin content of the samples was calculated according

33 1 5, E‑mail: masakio@nih.goJp

formed dose and color‑development rate, and was expressed131as endotoxin units per milliliter (EU/ml).

the treatment fわr TNトα induction, herea氏er.

Invitro assay on TNF‑a induction uslng rabbit periph‑

TNF‑a induction by 0. 1 m1‑volume each of serial fourfold

eral blood: Heparinized (Heparin, Hoechst Marion Roussel,

dilutions of RSE from 20 to 0.0781 EU/ml was assayedTokyo) bloodfrom fTemale rabbits of the Japanese white strain

repeatedly uslng blood samples from a total of more than 15(Kitayama Labes Co., Ltd., Nagano or Japan Laboratory

rabbits. The measurement of each dose was performed in

Animals, In°., Tokyo) was used fわr the TNF‑α induction

duplicate in each assay as described in Materials and Methods.
Homogeneityof variance is essential for applying the parallel

assay within 2 h after bleeding. An approprlate dilution ofsamples in pyrogen‑free saline for injection (0. 1 ml) (Otsuka

line assay method, and its achievement might be hampered

ized rabbit blood were gently mixed in a pyrogen‑fTree centn‑

when magnltude of variance would be affected by themagnitude of response. The effect of magnitude of response

fuge tube containlng 0.75 ml of pyrogen‑free saline. The

on variance was evaluated by a co汀elation analysts uSlng thePharmaceutical Co･, Ltd･, Tokyo) and O･ 15 ml of the hepariり‑mixture in duplicate was incubated at 370C in a humidified

duplicated data of TNトα Concentration obtained in theatmosphere or 5% CO2/95% air fわr the desired length ortime.

repeated assays. A significant correlation was seen between

Supematants were isolated from themixtures by centrifuging

the mean and variance of TNF‑α concentrations as shownat 500 × g for2 min and stored fTrozen a卜300C until use. The

in Fig. 2a. However, logaritlmic transformation of the TNF‑asupematants were diluted serially at five fold intervalswith

concentrations diminished the co汀elation as shown in Fig. 2b.

distilled water containing 25% ofblocking reagent (Block Ace,
Dainippon Pharmaceutical Co., Ltd., Osaka) and TNF一α

Concentration of the dilutions measured by an enzyme‑linked

iTmunosorbent assay (ELISA)I The ELISA was performed
uslng antiィabbit TNF‑α goat imun0‑globulin G (lgG), biotinlabeled anti‑rabbit TNトα goat lgG (Research Diagnostics

lnc･, Flanders, N･ J･, USA) and horseradish peroxidase conju‑(luJ 6U)PLNト芸qetJ

gated streptavidin (Chemicon Intemational, Inc., Temecula,

Calif., USA) in wells ofa 96‑well microIPlate (Narge Nunc
International K.K., Tokyo). TNF‑a concentration in the

1s叩ematantS Was Calculated according to血e parallel line assay

method referring to the standard rabbit TNF‑a (Research
Diagnostics Inc.) using logarithmically transformed doses andll‑r‑I‑1[

absorbance values to express the results as nanograms per
milliliter (ng/ml).

Rabbit pyrogen test: Female rabbits of the Japanese whitestrain weighing approximately 3 kg were housed in cages
separately ln an aiトCOnditioned animal room. Three animals

0

5

10

were allocated for each treatment. They were intravenously

1

5

20

25

Hours

ln)eCted with 1 ml/kg of a dilution of test samples. Rectal

Fig. I. Kinetics ofTNF‑α release from rabbit peripheral blood after

temperature of the animals was monitored fわr 3 h uslng an

stimulatlng With RSE. TNF‑α concentratioll i一l the supematant orelectric thermometer (Scanner Unit Xl 1 5 with High Accurate

thc blood samples was monitored up to 24 h after stimulation with

5.0 EU/ml (+), 1.25 EU/mI (A) ofRSE or Saline (A) at 37oC. The

Data Logger K730, TECHNOL SEVEN, Kanagawa)･ The

assay was carried out twice independently using blood samples frompyrogeniclty Of a test sample was calculated using the average
maximum rise in rectal temperature of the three rabbits during

different rabbits. Each point represents the mean of two independent
aSSayS･

3 h (AT) after sample injection.

Statistical analysis: Analysis Of the parallel line assays
5

was carried out according to Finney's method (17). Signifi‑(b)cance and validitytests were made at a level ofP ‑ 0.05. The′= ‑0.021

pyrogeniclty Or TNF‑α induction index of each LPS was(P= 0.849)

4calculated according to the parallel line assay method refeming○

to the activlty OfRSE which was assumed as 100.
3

(:D

2

RESULTS

♂

.壁
L‑

>d2

oO

The kinetics of TNF‑a release from rabbit blood stimulatedoowith 0.1 m1‑volume each of5.0 and 1.25 EU/ml ofRSE at00

1

370C was monitored up to 24 h. The blood treated with saline.0もin parallel was also monitored as the control.

While the saline treatment did not stimulate TNF‑ cHelease,0

RSE showed a marked effect as shown in Fig. 1. TNF‑a

0

2

4

6

8

10

12

MBan

concentration in the supematant of the blood samples stimu‑

壱00500.00｡ 6才｡048d&盛.35
ー1.5

‑1.0

‑0.5

0.0

0.5

1.0

Moan

lated with RSE started to increase at approximately 1 h andFig･ 2･ Correlation between mean and variance of arithmetic or logarith‑

reached a maximum about 4 or 6 h･ No significant change was

mic TNトα concentrations. Homogcnclty Of variance is essential fわrapplyingthc parallel line assay method. The homogcneityofvariancc

noted, thereaRer, up to 8 h of incubation. TNF‑α Concentration

wi11 not be attained when there is a correlation betwccn magnitudesof the RSE groups seemed to have slightly declined by 10 h,of response and variance. A correlation was seen bctwecn mean andthen stayed at the same level without changlng until 24 h ofvariancc of arithmetic TNF‑α.concentrations (a) but it disappearedby the logarithmic transfbmatlOn (b).

incubation. Accordingly, 5 h of incubation was chosen fわr132

and a typical example of the measurement results is depicted

Fig･ 3b and Table I ･ A straight linear dose‑response regres‑sion line for RSE was attained from 0.0781 to 5.0 EU/ml bymeansof logarithmic transfomation. Consequently, hereaRer

in Fig. 3a, b.

in the present study, the TNF‑α‑inducing activlty Of a test

TNF‑α‑inducing activlty Of EC‑6 in a test sample was

assayed relatively to that ofRSE in five repeated experiments,

RSE and the resultant TNF‑α Concentrations showed asample was determined by refeming to RSE activityby means

straight linear dose‑response relationship from 0.078 1 up toof the parallel line assay method uslng the logarithmicallyl･25 EU/ml of RSE as seen in Fig. 3a. EC‑6 measured

transform‑ed dose and TNF‑a concentrations.

together with RSE also showed a straight linear dose十reSpOnSeFigure 4 shows the results of five repeated measurements

but with a much smaller regression coefficient, therefore

orthe TNF‑α‑inducing activlty Of EC‑6. The level ofTNF‑

suggesting the applicability of the method ofanalysis fb∫ slope

a induced by RSE or EC‑6 varied to some extent in different

ratio assays. However, logarithmic transfbmation of endo‑

experiments. The slope of the dose‑response regression also

toxin and TNF‑α Concentrations was fわund to glVe parallel

showed considerable variation in different experiments.

doseィesponse regression lines of RSE and EC‑6 as seen in

However, so血r as the activityofEC‑6 was calculated relatively

i‑ i.〜20(l∈P5U)t>jNト

864

buotodO>葛JOtJ601

2

1 1 0

0

2 0 8

(一∈P5u)D･LNト601
1

2

3

4

5

Experi ment
0 0 0 0 6 4 2 0

Fig. 4. Reproducibility of the TNF‑ α induction assay. Weighted meanof logarithmic potency of TNF‑α‑inducing activityof a test sample

was calculated relatively to that ofRSE. The vertical bars show 95%

confidencc intervals. X2 test showed homogeneltyof the relative
potency values, suggesting the assay's strong reproducibility.

0.1

1

LAL activ〜 (EU/ml)

to that of RSE in each experiment, the TNF‑α induction assay

Fig. 3. Comparison orarithmetic and logarithmic dose‑responses of

was shown to be highly reproducible as seen in Fig. 4, showing

TNF‑ α induction by cndotoxins. TNF‑α‑inducing activlty Of EC‑

no significant deviation of the result of each measurement

6 (▲) was mcasurcd as a test sample referring tothe activityofRSE

from the weighted mean.

The relationship between LAL activlty and biologlCal(●)･ Each point showsthc rヮcan value ofduplicated mcasurc.mcnts

in an cxpcrimcnt. ArithmctlC concentrations (a) of cndotoxln andactivities such as pyrogeniclty and the TNF‑α induction

TNF‑α showed a lincar dose‑rcsponsc relationship up to a TNF‑αconccntration or around 8 mg/ml, suggesting the applicability orthc

activityof LPSs of the bacteria listed in Materials and Methods

sloperatio analysis. Logarithmic transformation (b) ofendotoxin andwas examined. RSE and other LPSs were diluted to have an

TNF‑α Concentrations was fわund to allow application or the parallel

equal level ofLAL activlty, and the pyrogenlClty Ofeach oflime assay mcthod.

their serial threefold dilutions were assayed using three rabbits

to calculate the activity ofLPSs relative to the activity ofRSE
uslng the parallel line assay method. The TNF‑α induction

Tablc 1. Analysis of variance table

activlty Of the approprlate'Serial fourfold dilutions of each
FactorSumofsquaredf.

McansquareF2

Preparation 0. 043 3

1

0.04337.427

239.3253

Rcgrcssion 1.3963

1

1.3963

Paralleli sm 0.0097

1

0.0097

1.668

Lincarlty 0.0367

3

0.01222.098

Between doses I.4861
Error

0.

Total

040

8

1.526960.2477

7

0.005

1

LPS was also assayed according to the parallel line assay

lnethod. Figures 5a and 5b depict TNF‑a‑inducing activlty
and pyrogeniclty Of the LPSs, respectively.
TNF‑α‑inducing activity and the pyrogenicity of the LPSs

with equlValent LAL activity ranged from I.5% to 141.7%

and from 6.6% to 480.6% of the activities ofRSE, respec‑

42.495･ltively. The results suggested that there is no co汀elation between

8

LAL activlty and both of the biologlCal activities of the LPSs

3

so far tested.

Analysts On the data dcpictcd in Figure 3b.

On the other hand, when co汀elation between the pyrogenic‑

I df: degrees of freedom.

2F: Fvalue.

ityand the TNF‑a‑inducing activityof the LPSs was analyzed,

3Significant at P < 0,05.

it was found that there was a fine correlation between these133

〜: Ⅷ 乃 5｡ 25(%)甲]31n.tJ0 山IOSJ10 >葛一BJ倉>苛く

JoQ'PJoth据♂匂蝉♂榔♂樺♂
Fig. 5. Rclationshjps betwccn LAL activity and TNF‑α‑inducing activity or pyrogcnicity. TNF‑α‑inducing activity(a) andpyrogenicity (b) were. measured on LPSs of various bacterial strains adjusted to have equivalent LAL activity･ In each assay

thc biologICal activities Of the LPSs were comparcdwith those of RSE which was assumed to bc lOO%･ Both activities
showed considerable variation among different LPSs to suggest a discrepancy between these activities and LAL activity.

紬棚卸抑Ⅷ

this synerglStic effect and could be effective in regulating not
only endotoxin contamination but also overall pyrogenlClty
including any possible synerglSm. The pyrogen test also has
limitations, however,inits utilitydue to itsinsu氏cient ac.ctmcy

and its inferior sensitivityas compared to the endotoxln test.

xopul倉o!uo6oJQ

Furthermore, it requlreS a relatively large number of animals･We made an attempt to establish an assay method that
requlreS a minimal number of animals and has levels or
sensitivity and accuracy similar to those of the endotoxin test

and can even effectively detect the biologlCal activlty Of
endotoxin as can the pyrogen test. TNF‑α is known as theearliest detectable innammatory cytokine released frommacrophages or monocytes by stimulation with endotoxin(13,14,18). ln the present study, We attempted to utilize the
0

30

60

90

1

20

1

in vitro system ofTNF‑a induction kom peripheral blood of50

rabbits fわr detecting the biological activity or endotoxin.

TNF‑cL induction Index

The concentration of TNF‑α released in the supematantFig. 6. Correlation between TNF‑a‑inducing activityand pyrogenicity

of fresh rabbit blood treated with endotoxin was found toof LPSs. The biologlCal activities of the LPSs wcrc shown as pyr0‑

reach a peak at around 5 h of incubation at 370C as seen in

gcnlClty index and TNF‑α induction index that were calculated

Fig. 1. The most sensitive detection orendotoxin activity was

rcfcrring to RSE assumlng Its activities to be loo. Each point shows
the activlty OreaCh LPS relative to that orRSE. A clear co汀elation

found to be possible, therefore, at 5 h of incubation. Although

was seen bctwccn TNF‑α ‑inducing activity and pyrogcnicity.

a straight linear dose‑response regression could be attained
between concentrations of TNF‑α and endotoxin to suggest

the applicabilityof the slope ratio analysis method, the range
activities, showing a correlation coefncient ofO.893 (P < 0.0 1 )of the concentrations of TNF‑α and endotoxin fわr the straightas seen in Fig. 6. The in vitro assay method for endotoxin

linear dose‑response relationship seemed to be limited without

uslng rabbit blood was suggested to allow an assay that closely

any transformation (Fig. 3a). A logarithmic transfomationreflects the in vivo biologlCal activlty Ofendotoxin.

of the values was shown to be adequate for achievlng homo‑

geneity of variance and parallelism of the regression lines so
as to allow analysis by the parallel line assay method (Figs.

DISCUSS10N

2b, 3b and Table 1 ). Thus, the transformed dose and response

Although the bacterial endotoxin test has been playlng aenabled the assay to have comparable levels of accuracy andcrucial role in regulating endotoxin c6ntamination in parenteral

sensitivity as those of the endotoxin test (Table I)･ The assey
method could detect approximately 0. 1 EU/ml of endotoxln
in a test sample or,therefore, 0.01 EU/ml of that in the reaction

drugs, it has a limited efficacy ln eValuatlng the harmful bio‑

logical activities ofendotoxincontainedindrugsinthe case wherea drug enhanced the biological activityof endotoxin syner*‑

mixture and showed a straight linear dose‑response relation‑tically (12). The pyrogen test would be c叩able of detectlng

ship up to 5 EU/ml or 0.5 EU/ml of test sample or reaction134

mixture, respectively (Fig. 3b).

difference (25) from i.ts corresponding rLabbit interferon･Relationships of the endotoxin test, the pyrogen test andAlthough the rabbit in vltrO TNF‑ α induction assay was fわundthe in vitro rabbit TNF‑α induction test were examined uslng

to co汀elate well with the pyrogen test, there was an endotoxin

LPSs derived from various bacterial strains listed in Materials
and Methods. It has been widely accepted that results of thethat showed a significant discrepancy in the two tests. The

pyrogen test and the endotoxin test show reasonable correlation

explained in the present study.

in routin? quality control tests (1 9,20)･ However, when LPSs
from various differing bacterial strains were compared, thecontamination to guarantee the same level of safety as that ofresults of both tests showed considerable mutual discrepancy

the pyrogen test. Although the endotoxin test is specific foras shown in Fig･ 5b. A similar discrepancy was also seendetectlng endotoxin, it cannot detect other pyrogens suchreason for the differing results of the two assays could not be
It is important for a new method for testlng endotoxin

between the results of the endotoxin test and the in vitro rabbit

as peptidoglycan that constitute the cell walls of both gram‑TNトα induction test as shown in Fig. 5a. Although nonegative and gram‑positive bacteria. It might be interestlngco汀elation was fわund between LAL activity and pyrogenicity

to examine the possibility of detectlng Other pyrogens suchor in vitro rabbit TNF‑α induction activlty, the results of theas peptidoglycan by the present in vitro rabbit TNトα induc‑in vitro rabbit TNF‑α induction test was fわund to co汀elatetion assay method.

well with those of the pyrogen test as shown in Fig. 6.

We also examined human (21) and C3H/HeN mPuse (22)

REFERENCES

blood fわr their utility in the in vitro TNトα inductlOn assay(data not shown)･ Human blood was shown to rea.ct with

1 ･ Sasaki, S･ (1979): Biolog.ical activity oflipopolysaccha‑

endotoxin to release TNF‑α. However, ltS reaCtivlty Was

rides isolatedfrom bactena in human periodontal lesions.

affected considerably depending on the source individuals or

Bull. Tbkyo Dent. Col1., 20, 159‑174.

especially on their immunization state. Blood丘om those having

2. Grisman, S. E. and Homick, 良. B. (1969): Comparative

immunlty agalnSt influenza virus could not be used for the

pyrogenic reactivlty Of rabbit and man to bacterial

endotoxin. Proc. Soc. Exp. Biol. Med., 131, 1154‑1158.

effective detection of endotoxin. activityin influenza vaccin.es･

3. U.S. Phamacopeia. 24th (2000): Pyrogen test.

In addition, this procedure uslng human blood might ralSe

ethical issues or create possible biohazards. Mouse blood was
also found to be inappropriate due to its insufficient sensitivity

4. Levin, ∫. and Bang, F. B. (1964): The role ofendotoxinin the extracellular coagulation of limulus blood. Bull.

Johns Hoplins Hosp., 115, 265‑274.

even thoughuslng bloodfrom the mice whose responsivenessof the endotoxin‑dependent TNF‑a release has been enhanced

5. Levin, ∫. and Bang, F. B. (1968): Clottable protein in

by BCG‑sensitization (23,24). The in vitro TNF‑α induction

Limulus; ltS localization and kinetics of its coagulation

test uslng rabbit blood was, therefわre, suggested to be theby endotoxin. Thromb. Diath. Haemo汀h., 19, 186‑197.

most approprlate test method fわr detectlng the biologlCal

6. Morita, T., Tanaka, S. and lwanaga, S. (1981): Anew (1‑activlty Of endotoxin so far.

3)‑ β‑D‑glucan一mediated coagulation pathway fわund inlimulus amebocytes. FEBS Lett., 129, 318‑321.

The in vitro assay method uslng rabbit peripheral blood

7. U.S. Pharmacopeia. 24th (2000): Bacterial endotoxin test.
8. European Phamacopoeia. 3rd ed. (2000): supplement.
9. Japanese Phamacopoeia. 13th ed. (1995): (in Japanese).
1 0. Ministry ofHealth and Welfare (1993): MinimumRequire‑

was shown to have advantages in comparison to the othertest methods as described above. This assay method is able
to assay many test samples uslng Only one rabbit and can use

the same rabbit repeatedly, differing from the pyrogen test
which cannot use rabbits that showed positive response in aments fわr Biological Products (in Japanese).

1 1. Ministry ofHealth and Welfare (1998): MinimumRequire‑previous measurement and which requlreS three rabbits per

ments fわr Antibiotic Products of J叩an (in Japanese).

sample. LAL reagents fわr the endotoxin test are also very

12. Kawasaki, H., Moriyama, M. and Tanaka, A. (1987):

expensive and the endotoxin test costs at least 5 times thepresent assay uslng an ELISA system. The new in vitro assay

Augmentation of endotoxin fever by recombinant humanmethod uslng rabbit peripheral blood has an advantage inbetainterferon in rabbits. Infect. Immun., 55, I 121‑1 125.

A rabbit might have imunity to a pathogen that has an antigen

13. Mahony, J. R. Jr･, Beutler, B. A., Trang, N. L･, Vine, W･,
Ikeda, Y, Kawakami, M. and Cerami, A. (1985):
Lipopolysaccharide‑treated RAW 264･7 cells produce acommon with a vaccine under test. The test might be hampered

mediator that inhibits lipoprotein lipase in 3T3‑L 1 cells･

regarding cost.

However, this assay method still has possible limitations.

J. Immuno1., 134, 1673‑1675.

for acellular pertussis vaccines if the blood was taken from a

rabbit with positive immunityagainst BoTdetella bflOnChiseptica
which might have an antlgen common tO a COrPpOnent Of

14. Beutler, B., Greenwald, D., Hulmes, J. D., Chang, M., Pan,
Y. C. E., Mathison, J., Ulevitch, R. and Cerami, A. (1985):

acellular pertussis vaccines. It would be therefわre important

ldentlty Of tumor necrosis factor and the macrophage‑secreted factor cachectin. Nature, 3 16, 552‑554.

to examine the responsiveness ofanimal blood by means of

15. Murai, T., Nakagawa, Y, Miyawaki, E., Ogawa, Y and

an interference test. lf TNF‑α induction by spiked endotoxin

uslng Such an animal which may have immunlty tO the

Horiuchi, Y. ( I 997): The endotoxin reference standard of
the National Institute ofHealth Sciences (the Japaneseorganism associated with the product. As the assay method

Pharmacopoeia endotoxin reference standard) (Control

was interfered for such a vaccine product, we need to avoid

makes use ofonly TNF‑α induction, it may show a discrep‑

971). Bull. Natl. lnst. Health S°i., 115, 202‑203 (in Japa‑

ancy with the pyrogen test especially when testlng a drugnese).

which can affect a physiologlCal step that follows TNF‑a

16. Tanaka, S. and Iwanaga, S. (1993): Limulus test for

release. Furthermore, the test may have a limitation in

detectlng bacterial endotoxins･ Methods Enzymol･, 223,
358‑364.

predicting the safetyofa drug that is suspected to have a host‑specific synerglStic effect with endotoxin such as interferon‑

17. Finney, D. ∫. (1978): Statistical Methods in Biological

Assay. 3rd ed. Charles Griffin Co., Ltd., London･

T Injection which is known to have a slgnificant structural135

18. Zee, K. J. V., Deforge, L. E., Fischer, E., Marano, M･ A･,
Kenney, J. S., Remick, D. G, Lowry, S. F. and Moldawer,

21. Foster, S. ∫., McComick, L M., Ntolosi, B. A. andCampbell, D･ (1993): Production ofTNF‑α by LPS‑

L L. (1991): IL‑8 in shock, endotoxemia, and a触r ILl

stimulated murine, rat and human bloodand its pharmacol

administration. ∫. Immuno1., 146, 3478‑3482.

logical modulation. Agents Actions, 3 8 Special Conference

19. Naito, S., Fujiwara, H.,Asakawa, S., Homma, R., Ishida,lssue, C77‑79.

S., Chino, F., Tanaka, S., Ohki, M., Tsuchiya, M. and

22. Glode, L M., Scher, 1., Osbome, B. and Rosenstreich,

Matsuura, S. ( 1 992): Application of the limulus amebocyte

D･ L･ ( 1 976): Cellular mechanism ofendotoxin uⅣespon‑test to measurement of endotoxin in therapeutic humansiveness in C3H/HeJ mice. J. Immuno1., I 16, 454‑461.

plasma protein fraction. Comparison with the rabbit

23. Peavy, D.し., Baughn, 良. E. and Musher, D. M. (1979):

Effects of BCG infection on the susceptibility of mousepyrogen test. J. Pham. Soc. Jpn., 1 12, 551‑556 (in Japa‑

macrophages to endotoxin. Infect.lmmun., 24, 59164.

nese).

20.Asakawa, S., Fujiwara, H., Naito, S., Homma, R., Ishida,

24. Ⅵ)gel, S. N., Moore, R. N., Sipe, ∫. D. and Rosenstreich,S., Chino, F., Tsuchiya, M., Matsuura, S., Tanaka, S･ andD.し. (1980): BCG‑induced enhancement of endotoxin

Ohki, M. (1 994): Application of the limulus test fわr prac‑sensitivityinC3fi/HeJmice. I. h vivo studies. Hmun01.,
124, 2004‑2009.

tical quality control on endotoxin content in commercial

human serum albumin (HSA) products. In colnParison

25. Farrar, A. M. and Schreiber, D. R. (1993): The molecularwith the rabbit pyrogen test. ∫. Pharm･ Soc暮 Jpn･, I 14,

cell biology of interferon‑r and its receptor. Annul Rev.

888‑893 (in Japanese).

Immuno1., ll, 571‑611.

136