Differential blocking of coagulation activating pathways of Limulus amebocyte lysate

JOURNALOFCLINICALMICROBIOLOGY, June1994,p. 1537-1541 Vol.32,No.6 0095-1137/94/$04.00+0

Copyright© 1994, American Societyfor Microbiology

Differential Blocking of

Coagulation-Activating

Pathways of

Limulus

Amebocyte Lysate

GUI-HANG ZHANG,1 LEIF BAEK,2 OLE BUCHARDT,3 ANDCLAUS KOCHI*

Department ofImmunology, State

Serum

Institute, DK-2300 Copenhagen

S,1

Department of Microbiology, Herlev Hospital, Herlev

Ringvej,

DK-2730Herlev,2andDepartment ofChemistry, H.C. 0rsted Institute,

Universityof Copenhagen, DK-2100 Copenhagen 0,3 Denmark

Received 15December 1993/Returned for modification 25 January 1994/Accepted1 March 1994

Thecoagulation of Limulus amebocyte lysate(LAL) can beactivated through two pathways, one initiated by endotoxin and theother by,-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be

differentially

inhibited by two methods in a Limulusenzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10differentendotoxin preparations could be inhibited by the former method by a factor of104to 106and could be blocked almost

totally

by the latter method, irrespective of the source of endotoxin. The

sensitivity

of the assay was

approximately 50 pg/ml both for curdlan fromAlkaligenesfaecalis and for laminarin fromLaminariadigitata. Wealso foundthat thej-glucan-activated pathway could be totally blocked by laminarin (>1 ,ug/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISAwith adetection limit of 0.005 endotoxin unit per ml. The use of uninhibited and

differentially

inhibited ELISAs demonstrated that different LALpreparations showed much greater variation in assaying

I-glucans

thaninassayingendotoxins. The LAL

reactivity

ofnormal human plasma was found to be due to the activation

ofthe

P-glucan

pathway, but not theendotoxin pathway, of LAL.

Limulus amebocyte lysate (LAL) is known to contain a

coagulation system that can be activated by a minute amount of bacterialendotoxin(lipopolysaccharide [LPS]). The coagu-lation cascade consists of three proenzymes (factor C, factor B, andproclotting enzyme) and one clottable protein

(coagulo-gen)(7, 14).LPSactivates factor C, which then activates factor B, which in turn converts proclotting enzyme to clotting enzyme.Clottingenzymecleavescoagulogen toyield an insol-ublecoagulin gel.Theformationofgelclotand measurement ofclottingenzymeactivity bymeans ofasynthetic chromoge-nic substrate constitute, respectively, the bases of the gel clot andchromogenic LAL assays for thedetection of endotoxins

(8, 15).Wehaverecentlydeveloped sensitiveendotoxin assays basedon theimmunochemical detection ofLimulus

coagulo-genorits cleavedfragment,peptide C,withdetectionlimitsas lowas 0.001 endotoxin unit(EU)perml (2, 24,25).

Because of its extreme sensitivity to endotoxins, the LAL test has been considered a

promising

means of

detecting

endotoxin in clinical settings

(9).

However, its

specificity

for

endotoxin appears to be

compromised by

certain

,B-glucans,

originatingfromfungi

(5,

6,

10)

or

hemodialysis

membranes

(3,

23),whicharealso LAL

reactive,

although

these

glucans

are at least 1,000-fold less active than endotoxin.

Fortunately,

the

activation of LAL by

3-glucan

occurs

through

an

initiating

factordifferent from that for

endotoxin,

namely,

factor G

(16).

Separation

ofthetwo

pathways

by

fractionation and

reconsti-tution of the relevant factors has been

reported recently

(12,

19). Addition of curdlan, a

carboxymethylated

3-glucan,

to LALathigh concentrations

(>1

mg/ml)

has also been shown toblock the

,B-glucan

pathway

(11).

Another

study

shows that the surfactant Zwittergent interferes with LAL

reactivity

to

*Corresponding author. Mailing address: Statens Seruminstitut,

Division of Immunology, 5, Artillerivej, DK-2300 Copenhagen S, Denmark.Phone: +453268 3719. Fax: +45 3268 3149.

,B-glucan (20). However, the reconstituted LAL fractions are notstable, Zwittergent also affects lipid A to some extent, and curdlan tends to autopolymerize to form an insoluble gel at high concentrations after storage for 3 months, even at 4°C

(unpublished observation).

In this study, we have attempted to develop specific LAL testsfor endotoxin and

P-glucan

bydifferentially blocking the two activation pathways of LAL, using our newly developed Limulus peptide C enzyme-linked immunosorbent assay

(ELISA) to detect reactivity. We have also assessed the influence ofdifferentformulations ofLAL onthe

quantifica-tion ofendotoxin or

P-glucan

activities of various laboratory

specimens.

MATERIALS

AND METHODS

Allglasswarewasrendered pyrogen freebybeingheated to 250°C for at least 3 h. Sterile,

pyrogen-free tips

and

micro-plateswere

purchased

from

Eppendorf, Hamburg,

Germany,

andNunc,Roskilde,Denmark,

respectively.

TheLALreagent waterusedwasconfirmedtohaveless than 0.001

EU/ml by

the LALtest.

LALpreparations.CommercialLAL

preparations

included Pyrotell(lot42-99-541) fromAssociates of

Cape

Cod

(ACC),

Woods Hole, Mass., and LAL

(lot

2L0860)

from Whittaker

Bioproducts

(WB),

Walkersville,

Md. The

Tachypleus

amebo-cyte lysate

(TAL)

was

prepared

from the horseshoe crab

Tachypleus tridentatus without chloroform

extraction,

as de-scribed

previously

(25).

LPS. The controlstandard endotoxinwas

NP-3,

whichis a purified

preparation

of LPS from Salmonella abortus

equi

purchased from Pyroquant

Diagnostik, Walldorf,

Germany,

and its potencywasconfirmed tobe 10

EU/ng

in

comparison

withUSP reference standard endotoxin EC-5. The

following

LPS

preparations,

obtained

by

phenol

extraction,

were

pur-chasedfrom

Sigma

Chemical

Co.,

St.

Louis,

Mo.: Escherichia

1537

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coli0111:B4(determinedpotency, 2.5EU/ng),E.coli 055:B5,

E. coli 0127:B8, E. coli 0128:B12, Klebsiella pneumoniae, Pseudomonas aeruginosa,S.abortusequi, Salmonellaenteritidis, Salmonella minnesota, Salmonella typhimurium, and Shigella flexneni.

Glucan preparations. Laminarin isolated from Laminaria digitatawasobtained fromSigma.Highlypurified curdlanfrom Alcaligenesfaecaliswaspurchasedfrom Wako Pure Chemical

Industries, Osaka, Japan. Both laminarin and curdlan were

dissolvedin0.2NNaOHat5mg/mlandincubatedat56°Cfor 6hto inactivatecontaminating endotoxin (25).

Polymyxin B. Polymyxin B sulfate was purchased from Calbiochem,LaJolla, Calif. Theendotoxin and ,-glucan-like activities found in polymyxin Bwere removed by adsorption with activated charcoal (Merck, Darmstadt, Germany); 0.1 g wasmixedwith 10 ml ofpolymyxinB solution (20mg/ml) at 37°C for1h,and thiswasfollowedbycentrifugationtoremove the charcoal.

MAb against factor C. Partially purified factor C was obtained from TAL by dextran sulfate-Sepharose CL-6B chromatographyasdescribedbyNakamura etal.(17)andwas

furtherpurified bysodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) carried out with a 12% gel, essentiallyasdescribedbyLaemmli(13).Theband at120kDa

wascut outand eluted with an electroelutor(Bio-Rad Labo-ratories, Richmond, Calif.).Purified factor Cwasadsorbedto aluminium hydroxide suspension (2 mg/ml in normal saline) and injected intraperitoneally (0.5 ml, containing 25 ,ug of factor C, per mouse) into five female CF1 x BALB/c mice. Boosterdosesweregivenat2-weekintervals,andanimalswere

testbled 10days aftereach boost. When high antibodytiters

were found by ELISA in factor C-coated microtiter plates, spleen cells were fused by a standard procedure (4) with myeloma cells from line X63/Ag8.6.5.3. Selected monoclonal antibodies(MAbs) againstfactorCwerepurifiedfrom

hybrid-oma supernatants by affinity chromatography on protein

A-Sepharose CL-4B columns(Pharmacia, Uppsala, Sweden). One of the MAbs (MAb-B2) was found to react with both Tachypleus factorC andLimulusfactor C, sinceitrecognized the 120-kDa band of the partially purified factor C prepara-tionsfrom both LALand TAL inSDS-PAGE immunoblotting (unpublished observation), and was chosen for use in the

present study. The endotoxin contamination of preparations containing 1 mgofMAb-B2permlwasabout 100ng/ml and wasremoved by TritonX-114extraction (1).

Preparation and pretreatment of plasma. Blood from

hu-man donorswasdrawn intopyrogen-free glass tubes contain-ing pyrogen-free heparin (final concentration, 4 IU/ml).

Plasmawasseparatedbycentrifugationat500 xgfor 15min.

Perchloric acid (PCA) precipitation of plasma to remove

factors that interfere withLALwas carriedoutessentially as

described byObayashi (18) but withoneminor modification:

theneutralizedsupernatantwasfurther dilutedtwofold in the

LALreagent water.

Limulus peptide C ELISA. Unless otherwise stated, the

Limulus peptide C ELISA was performed as previously

de-scribed (25). When MAb-B2 was used as a blocking agent,

stock solution (1 mg/ml) was added to diluted LAL and

incubatedat4°Cfor 20minbeforethe ELISAwasperformed.

Whendimethyl sulfoxide(DMSO) andpolymyxinBwereused

as blocking agents, the ordinary LAL bufferwas replaced by buffercontaining 20%(vol/vol)DMSO,100,ugof polymyxinB perml, 60 mM MgCI2, and 100 mM Tris-HCl, pH8.

1.4 1.2

1

0.8 0.6 0.4 0.2

0

OD490-650

-3 -2 -1 0 1 2 3 4 5

ConcentrationofLPSorCurdlan, log ng/mI

FIG. 1. Differential inhibition of the LPS-activated pathway of LALbyMAb-B2 (10,ug/ml). OD490-650, optical densityat490nm

corrected byopticaldensityat650nm.

RESULTS

Differential blocking oftheendotoxin-activated LAL

path-way. MAb-B2 is amurine MAb raised

against

factor Cfrom TALandwasfoundtocross-reactwithfactorC from LAL

(see

Materials andMethods). Figure 1showsthe effect ofMAb-B2

onthereactivityof LALtoLPS

(from

E. coli

O111:B4)

andto curdlan. LAL(fromACC)became almostunreactivetoLPS in the presenceof MAb-B2 at 10

,ug/ml,

while thereactivity of LAL to curdlan was unaltered. The same effect was also

observedwith other LPSpreparations,asshown in Table 1. Figure 2 shows the dose-dependent inhibition ofthe endo-toxin-activated pathway ofLALby MAb-B2. When the con-centration of the antibody inLAL was at or above 10

,ug/ml,

thereactivityof LALtoLPSwasalmost totallyinhibited. No further inhibitionwasobserved when the MAb-B2 concentra-tionwasincreased above 20,ug/ml.Theresidual LALreactivity

of LPS atconcentrations of >100,ug/ml waspresumedto be duetothecontamination of the LPSpreparationswith

3-glu-can-like substances, since it could not be abolished even by treatmentwith 0.2NNaOH at 60°Cfor24h.

TABLE 1. Inhibitionof the LAL-LPS reaction by DMSO plus polymyxin B(D-P)orby MAb-B2

Detection limit(ng/ml)with: Source of LPS

LALonly LAL+ D-Pa LAL +

MAb-B2b

E.coli055:B5 0.001 100 >10,000

E.coli0127:B8 0.001 2,500 >10,000 E.coli0128:B12 0.0005 1,000 >10,000

Kpneumoniae 0.001 1,250 >10,000

P.aeruginosa 0.001 250 >10,000

S. abortusequi 0.002 20 >10,000

S. enteritidis 0.002 250 >10,000

S. minnesota 0.002 100 >10,000

S. typhimurium 0.003 1,000 >10,000

Shigella flexneni 0.005 2,500 >10,000

aThe LALbuffercontaining 20%DMSO, 100 ,ug ofpolymyxin B per ml, 60

mMMgCl2,and 100 mM Tris-HCI (pH 8) was used to dilute LAL in the Limulus

pepFtide

C ELISA.

The concentrationof MAb-B2 in LAL was 10Kg/ml.

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BLOCKING OF LIMULUS COAGULATION 1539 OD490-650

1.4 1.2

0.8 0.6 0.4 0.2 0

OD490-650

-3 -2 -1 0 1 2 3 4 5 6

Concentration of LM, CLorLPS, log ng/ml

-3 -2 -1 0 1 2 3 4 5 6 FIG. 4. Differential inhibition ofthe 3-glucan-activated pathwayof LALby laminarin (LM)orcurdlan(CL). #, Concentrationof LM in

Concentration of LPS, log ng/ml LAL was 5

,Lg/ml;

*, concentration of CL in LAL was 1 mg/ml.

FIG. 2. Dose-dependent inhibitionof the LPS-activated pathway of OD490-650,optical densityat 490 nmcorrected byoptical densityat LALby MAb-B2at0(L1), 2.5(+),5 (*), 10(-), and 20(X) ,ug/ml. 650 nm.

OD490-650, optical density at490nmcorrectedbyoptical densityat

650nm.

Figure 3 shows the effect of the combined use of 20%

DMSO andpolymyxin B(100,ug/ml)onthereactivity of LAL

toLPS (from E.coli0111:B4).The reaction of LAL with LPS

was inhibited by a factor of more than 105 (increasing the detection limit from2.5pg/mlto1 jLg/ml),but the reactivity of

LAL to curdlan was not significantly altered. The inhibitory effect ofDMSO andpolymyxinB onthe reactivity of LALto

otherLPSpreparationswasvariable, ranging from afactor of

104 to 106', asshown in Table 1.

Differential blocking of the j-glucan-activated LAL

path-way. Figure 4 shows the reaction curves of LAL with LPS,

laminarin, and curdlan andtheeffects of laminarinand curdlan

2

1.5

0.5

0

OD490-650

LPSonly

-curdlan only LPS +D-P -curdlan +D-P

-3 -2 -1 0 1

Concentration of LPSorcurdlan, FIG. 3. Differential inhibition of the LPS-a LALbythecombineduseof 20% DMSO and 14

per ml (D-P). OD490-650, optical density at i optical densityat650nm.

onthe 3-glucan-activated pathway. The concentration ranges

inwhich laminarin and curdlan reacted with LALwerefrom

0.05 ng/ml to 1 .ig/ml and from 0.1 ng/ml to 500 Fg/ml, respectively. Addition oflaminarin at5 jig/ml orcurdlan at I

mg/ml toLALtotally blocked the ,B-glucan-activated pathway withoutcompromising the reactivity ofLALtoLPS.

The reactionsofTALwith LPS and curdlanwerefound to

be similar to those of LAL, but TAL did not react with laminarin atanyconcentration from 1 pg/ml to 1 mg/ml (25).

Addition of laminarin toTALataconcentrationaslowas0.1

OD490-650

X/ assayfor

assay for

LPS(LM)#

-CL(MAb-B2)*

0.1

0.01

0 0.006 0.012 0.025 0.05 0.25

Concentration ofLPS (EU/mI)orcurdlan (CL,ng/ml) 1.25

2 3 4 FIG. 5. StandardcurvesofspecificLimuluspeptideCELISAsfor

log ng/ml LPSorcurdlan.#,theconcentrationoflaminarin(LM)inLALwas

n9/ml ,ug/ml. *,the concentration of MAb-B2inLALwas 10 ,ug/ml.**,the activated pathway of LALbuffercontaining20%DMSO,100 p.gofpolymyxinBperml,60

00 ,ugofpolymyxinB mMMgCl2,and 100 mM Tris-HCI (pH8)was usedto dilute LALin

490 nm corrected by the LimuluspeptideC ELISA.OD490-650, opticaldensityat 490nm

correctedbyopticaldensityat 650nm.

1.4

1.2

0.8

0.6

0.4

0.2

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TABLE 2. Determination of LPS andI-glucan by using different formulations ofLALandTAL

Content of LPSorp-glucan (ng/ml)a

LALor TAL inspecimenno.b

formulation

1 2 3 4 5 6

LPS and,B-glucan reactive

LAL(ACC) 29.8 25.7 25.1 21.4 2.43 0.024

LAL(WB) 22.9 29.8 33.4 58.0 5.13 0.038

TAL 15.5 13.7 13.5 10.7 0.99 <0.005

CV (%)' 31.5 36.3 41.6 82.6 73.3

LPSreactive

LAL(ACC) +LMd 13.2 7.5 10.1 <0.1 <0.1 <0.005 LAL(WB)+LM 16.2 6.4 8.4 <0.1 <0.1 <0.005 TAL +LM 12.6 4.4 6.7 <0.1 <0.1 <0.005

CV(%) 13.8 25.7 20.2

,-Glucanreactive

LAL(ACC)+ 98.5 1,566 1,003 1,825 250 2.42 MAb-B2e

LAL(ACC)+D-Pf 90.6 1,740 960 1,204 186 1.82

CV(%) 5.9 7.4 3.1 28.9 20.6 20.0

a Standardsfor LPS and,B-glucanwereNP-3 andcurdlan,respectively.The dataare meanvalues oftriplicate determinations.Fortheformulationsreactive with both LPS and,B-glucan,values are LPSequivalents.

bSpecimens 1,2,and 3 werenaturallycontaminated humanimmunoglobulin preparations.Specimen4 was aculture medium(brainheartinfusion). Specimen 5 was a culture supernatantof Candida albicans. Specimen 6was apoolof normalhumanplasma(fromsixdonors)pretreated bythe PCA method.

cCoefficientof variation of the resultsobtainedwith differentformulations of LALandTAL.

dTheconcentration of laminarin(LM)in LAL was 5,ug/ml.

eTheconcentration of MAb-B2in LALwas10,ug/ml.

fD-P,DMSO-polymyxinB.The LALbuffercontaining20%DMSO,100 ,ugof polymyxinB100 perml,60 mMMgCl2,and 100 mMTris-HCI(pH 8)wasused todilute theLALinthe LimuluspeptideC ELISA.

,ug/mlwassufficienttoblock the

f-glucan

pathway

activated

by

curdlan.

Standard curves of Limuluspeptide C ELISAs specific for LPS or

0-glucan.

Figure 5 showsa linear relationship (on a doublelogarithmicplot) of the LALreaction with LPS when laminarin (5 ,ug/ml) was addedto LAL toblock the

P-glucan-activatedpathway and also shows alinearrelationship ofthe reaction with ,B-glucan when either MAb-B2 or DMSO and

polymyxinB were addedtoblock theLPS-activated pathway.

The detection limit for LPS in the LPS-specific ELISA was 0.005EU/ml, while the detection limit for curdlanorlaminarin in the ,B-glucan-specific ELISA was about 50 pg/ml. Curdlan wasfoundto be unstable in solutionat concentrations of >1 mg/ml, since it autopolymerized to formaninsoluble gel after storage at 4°C for 3 months. Laminarin was observed to be stableat 1 mg/mlin 50 mMNaOH at4°C for at least 1year. Comparison of different formulations of LAL and TAL for the determination of LPS and

13-glucan

in specimens. As shown inTable 2, the conventional LAL or TAL preparations, which react with both LPS and ,B-glucan, gave much more variable results (the coefficient of variation was more than double) for specimens that contained primarily

3-glucans

(samples4and5) than for specimens that contained both LPS and,B-glucans (samples 1, 2, and 3). The LAL or TAL made specific for LPS by the addition of laminarin (5 ,ug/ml) reacted little with the specimens containing primarily

3-glucans

and produced less variation than the conventional preparations. The LAL made specific for

,3-glucan

by the addition of

MAb-B2 gave results comparable to those obtained by the

addition of DMSO and polymyxin B. PCA-treated normal human plasma exhibited some ,B-glucan-like activity, but no endotoxin activity, when LAL preparations were used and diluted fourfold.

DISCUSSION

The LAL test iswidely used as a sensitive assay for endo-toxin. It is now known that the coagulation of LAL can be induced not only byLPS but alsoby certain 3-glucans, which act through different initiating factors. LPS activates factor C and 3-glucan activates factor G, although the latter has not been fully characterized (14). MAb-B2, used in thisstudy, is a MAb raised against Tachypleus factor C but is foundto react with and inhibit both Limulus factor C and Tachypleus factor C. Thisindicates the closesimilarityof the factors C from these species, which have also been demonstrated tohave thesame molecular size and functions (22). While the exact mechanism of MAb-B2 inhibition of the activation of factor Cby LPS is not clear, itis possible that thebinding of factor CtoMAb-B2 prevents itsbinding to LPS and consequent activationthrough an allosteric effect or one of steric hindrance by MAb-B2, as suggestedby the factthatpremixingof the antibodywith LAL or TAL at 4°C for about 15 to 30min is required to ensure effective blocking of the LPS-activated pathway.

The use of laminarin to block the 3-glucan-activated path-way has advantages overthe use of curdlan in that laminarin is stable in solution and is effective at a concentration at least 100-foldlower than that of curdlan. Commercial curdlan and laminarin, in solutions of 5 mg/ml, contain about 1 ng of endotoxin per ml. Thecontaminatingendotoxincanbetotally

destroyed by heating to 50 to60°C in 0.2NNaOH for about 6 h, withoutaffecting the LAL reactivities of curdlan and lami-narin. However, the curdlan solutions subjected tothis treat-ment seem tobe moresusceptibletogelling, probablybecause ofautopolymerization, than untreatedsolutions(gellingwithin 3 monthsafter treatment versus 1year). It is desirable to use as differential blocking agents those ,B-glucans that have no reactivitywith LAL at anyconcentration and thatcanproduce

total inhibition of the

3-glucan-activated

pathway without

affecting the LPS-activated pathway. Tanaka et al. (21) have recently reported the inhibition ofhigh-molecular-weight

(1-3)-p-D-glucan-dependent

activation of Limulus

coagulation

factor G by laminaran oligosaccharides and curdlan degrada-tion products.

Inhibition of the LPS-activated pathway by the combined use of DMSO and polymyxin B is based on the LPS-neutral-izing effect of polymyxin B and the inhibitory effect of DMSO on factor C (unpublished observation). The inhibition of the LALreactivity of LPS by polymyxin B alone at 100 ,ug/ml or by 20% DMSO alone is limited, increasing the detection limit of the assay to about 10 ng of LPS per ml, but combining the two reagents produces a synergistic effect.

Greater variations occur among different LAL and TAL preparations in quantifying ,-glucans than in quantifying LPS, andthis probably stems from the different reactivities of these preparations to curdlan, which is used as a control standard for

3-glucan,

and to other naturally occurring

3-glucans.

For

example, the TAL prepared in our laboratory and the LAL from WB used in this study are amebocyte lysates that have not been subjected to chloroform treatment, and these prepara-tions have been found to react with curdlan but not with laminarin, while the LAL from ACC, which is a chloroform-treated preparation, reacts with curdlan and laminarin with almost equal sensitivities (25). The great variation of different LAL and TAL preparations in their reactivities with

3-glucans

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BLOCKING OF LIMULUS COAGULATION 1541

may

complicate

the standardization of a Limulus

,3-glucan

assay.

Normalhuman

plasma pretreated

with PCA exhibits some

3-glucan-like

activity

when the LAL is diluted more than

twofold but shows no endotoxin

activity

regardless of the

dilutionoftheLAL.The greaterthedilution ofLALused, the morethe

,-glucan

activity

isobserved. A similarphenomenon

has been observed when normal human

plasma

ispretreated

by

the

diluting-heating

method

(the plasma

is diluted 10-fold in water and heatedto

75°C

for 5

min).

Whetherthe observed

f-glucan-like activity

is a

genuine

property of normalplasma or is caused

by

denaturation of

plasma

constituents during

PCAorheat treatment remainstobe

investigated.

In

conclusion,

this

study

has established

specific

Limulus

peptide

C ELISAmethods for determination ofeither endo-toxin or

,B-glucan by differentially

blocking

the

coagulation-activating pathways

of LAL. The

blocking

agents

developed

in this

study

may in

principle

be

applied

to other

quantitative

LAL assays,suchasthe

chromogenic

andturbidimetricassays,

although

their effective concentrationsmaywellbe differentin view ofthe factthat the LAL isdilutedatleast fourfold in the

Limulus

peptide

C ELISA.

Optimization

studies and assess-ment of the clinical

applications

of these assays for the

diagnosis

of endotoxemia and

fungemia

are

being

carriedout. ACKNOWLEDGMENTS

Thisstudywassupported by the Danish BiotechnologyCentre(a grant to G.-H. Zhang) and the Danish Blood Donor Research Foundation.

WeareindebtedtoLarsOttoUttenthal forhis valuablesuggestions

during preparation

of themanuscript.

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VOL.32, 1994

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