JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1980, p. 644-6500095-1137/80/11-0644/07$02.00/0

Vol. 12, No. 5

Principles of a Quantitative Assay for Bacterial Endotoxins inBlood That Uses Limulus Lysate and a Chromogenic

SubstrateCHRISTOPHER J. WEBSTER

Department ofImmunology, Royal Postgraduate Medical School, Hammersmith Hospital, LondonW12 OHS, United Kingdom

Some factors affecting the use of chromogenic substrates with Limulus lysatefor assaying bacterial endotoxins in blood have been assessed. It was found thatendogenous amidases, which degrade the substrate, could be inactivated byheating serum at 60°C for 15 min. Endotoxin was found not to be removed fromserum during clotting. A potent inhibitor of the activated lysate was found to beanti-thrombin II, but specific absorption of anti-thrombin II from plasma reducedonly marginally the inhibition of lysate by plasma. The presence of specificantibody to the endotoxin was found not to affect its ability to activate lysate.Inactivation of endotoxin by serum enzymes was biphasic in unheated serum, andmost of the activity was destroyed in 3 h at 37°C or in 24 h at 5°C. The relevanceof these findings to the objective quantitation of endotoxin activities is discussed.

Amoebocytes present in the hemolymph ofcertain species of Limulidae, notably the horse-shoe crab (Limulus polyphemus) and the Japa-nese crab (Tachypleus spp.), contain granules ofa material which, in the presence of minutequantities of endotoxin from many species ofgram-negative bacteria, will form a gel (16, 17).This reaction has been used as the basis of asensitive qualitative or semiquantitative assayfor endotoxins, with the reagent used being alysate of Limulus amoebocytes (Limulus amoe-bocyte lysate [LAL]) and the endpoint being thedetection of gelation after incubation for certaintimes under standardized conditions (19, 31).The pathway of the gelation process, which hassome similarities to that of mammalian bloodcoagulation (11, 22, 28), is shown in Fig. 1. Thisassay has been applied in both the pharmaceu-tical and clinical fields either to detect or toquantitate roughly bacterial endotoxins (6, 20).Recently, it has been reported that activatedLAL will cleave certain synthetic amino acidsubstrates which act as carriers for a chromo-genic p-nitroanilide group (21), and that theintensity of the color thus generated is propor-tional to the quantity of endotoxin present (13).This paper reports the results ofan investigationof the following factors which may affect thequantitative detection of endotoxins in blood,using LAL and a chromogenic-substrate.Inhibition of LAL by serum and plasma.

The presence of substances in blood which arecapable of inhibiting the LAL enzyme is wellknown, and a number of methods for their re-

moval or diminution have been published, in-cluding extraction with chloroform (19), dilution(19), specific adsorption of endotoxin on plasticbeads (10), shifting the pH of the plasma (23),and dilution and precipitation with ammoniumsulfate, followed by boiling (9). Most laborato-ries have chosen to use plasma as the sample ofchoice when testing for endotoxemia becauseLevin et al. (19) found that there was less endo-toxin detectable by the LAL gelation assay inserum prepared from blood to which endotoxinwas added than in preformed serum "spiked"with an equivalent quantity of endotoxin. Theseworkers suggested that endotoxin either becamebound with the clotted blood or was rapidlyinactivated during clotting. However, the factthat a substantial fraction of the lost endotoxinactivity could be restored by dilution (19) indi-cated that any such inactivation was potentiallyreversible.

Effect of antibody against endotoxin. Iinvestigated the possibility that preexisting anti-bodies in sera might interfere with the detectionof endotoxin.Inactivation of endotoxin by serum fac-

tors. Endotoxin in blood not only binds toplasma proteins (27) but also is inactivated byan enzyme system which is resistant to diisopro-pylfluorophosphonate, inhibited by some diva-lent cations, heat labile at 56°C for 1 h, andunaffected by the presence of antibody againstendotoxin (25, 26). Since this could affect thedemonstration of endotoxin in the LAL assay, Iinvestigated the relative rates at which endo-

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COLORIMETRIC ASSAY OF ENDOTOXINS IN BLOOD 645

toxin is inactivated by fresh and heated (60°C,15 min) human sera.

MATERIAIS AND METHODSPryrogen-free glassware and sundries. Glass-

ware was soaked overnight in a decontaminating de-tergent solution (5% [vol/vol] Lipsol [Lip Equipmentand Services Ltd., Shipley, United Kingdom]) fol-lowed by individual internal brushing and extensiverinsing in deionized water and drying in a hot-aircabinet. Finally, lids were replaced, and all glasswarewas baked at 160°C overnight. This treatment wasshown to destroy endotoxins. Plastic pipette tips (Jen-cons Ltd., Hemel Hempstead, United Kingom) wereloosely packed into clean glass screw-topped con-tainers and also baked at 1600C overnight. Graduatedand Pasteur pipettes were preplugged with nonabsor-bent wool before baking.Chromogenic substrate. A chromogenic sub-

strate, benzyl-Val-Gly-Arg-p-nitroanilide, which issensitive to the LAL enzyme (13), was synthesized andkindly supplied by Lars Svendsen of Pentapharm AG,Basel, Switzerland. It was dissolved freshly at 2 mMin pyrogen-free water (PFW).

Another substrate which is susceptible to the LALenzyme is available commercially from Kabi Diagnos-tica, Stockholm, Sweden.Limulus amoebocyte lysate. Limulus amoebo-

cyte lysate was purchased in a lyophilized state fromeither Mallinckrodt Inc., St. Louis, Mo. (Pyrogent) orfrom Millipore Corp., Bedford, Mass. (Pyrostat). TheMillipore product was found to be more economical inuse and was employed in all later tests. The contentsof one vial of Pyrostat 10 were routinely dissolved in10 ml of PFW or in 5 ml when increased sensitivitywas required.

Endotoxins. Endotoxins from the following speciesof organisms were used. (i) Escherichia coli endotoxinwas prepared from E. coli 0111; B4; K58; H- (obtainedfrom the National Collection of Type Cultures, Lon-don) in this laboratory by a phenol extraction methodbased on that of Westphal et al. (30). The endotoxinwas purified by ultracentrifugation, lyophiized, andstored at 5°C. (i) Serratia marcescens endotoxin wasprepared by the method of Webster et al. (29) andobtained from Difco Laboratories, Detroit, Mich. (iii)Shigella dysenteriae endotoxin was from samples ofthe First International Pyrogen Reference Prepara-tion, prepared from this organism (12), and was ob-

ENDOTOX IN

LA L pro-enzyme-- LA L enzyme(inactive) Ca (active)

COAGULOGEN GEL(Gel-able protein peptidefragmentss

in LAL)

FIG. 1. Schematicpathway ofLAL gelation. Open-headed arrows indicate activation; closed-headedarrows indicate progress ofa reaction.

tained from the National Institute for BiologicalStandards and Control, London.

Endotoxins were freshly diluted in PFW in 10-foldsteps between 1 ig/ml and 0.1 pg/ml.Sample collection. Blood was taken from healthy

volunteers by using the standard venipuncture tech-nique, with a sterile disposable plastic syringe andneedle (Gillette Industries Ltd., Isleworth, UnitedKingdom). The site (healthy skin) was thoroughlyswabbed with 70% isopropanol (British Pharmaco-poeia) beforehand, and hemolysis was avoided. Forplasma, the blood was anticoagulated with pyrogen-free heparin (Weddel Pharmaceuticals Ltd., London)at 25 U/ml of blood and immediately centrifuged at1,500 x g at room temperature. For serum, the bloodwas allowed to clot at 37°C for 1 h before centrifuga-tion.

Antibodies to endotoxin. Antibodies to E. coli0111;B4 were raised in New Zealand White rabbits byintravenous injection of heat-kiiled organisms (fivedoubling doses spread over 25 days). Four days afterthe last injection, blood was collected with sterileprecautions into pyrogen-free containers. The bloodwas allowed to clot at 37°C for 1 h, the sera werecollected and heated at 60°C for 15 min (see below),and part was absorbed with group A human erythro-cytes. Normal rabbit blood was collected and treatedin a similar fashion. Antibody titers were measured bypassive hemagglutination of human group A erythro-cytes coated with alkali-treated endotoxins (7) or withcrude lipid A (5). The anti-E. coli serum had a recip-rocal titer of 1.024 x 105 against E. coli endotoxin, 8against S. marcescens endotoxin, and 1,280 againstlipid A. Control serum titers were 32 against E. coliendotoxin, 4 against S. marcescens, and nil againstlipid A. Lipid A-coated erythrocytes were not agglu-tinated by antiserum to E. coli O antigen.

This E. coli antiserum was not characterized furtherto preserve its freedom from pyrogens. However, whensimilar antisera were prepared by the same method inthe past, they were found by gel filtration chromatog-raphy to contain both 2-mercaptoethanol-sensitivemacroglobulin and 7S antibody, the latter being pre-cipitating for the endotoxin.

Radiolabeled endotoxin. Endotoxin from E. coliwas labeled with "'CrC13 (Radiochemical Centre,Amersham, United Kingom) by the method of Braudeet al. (3) modified by dissolving the endotoxin in 0.2 MTris-hydrochloride, pH 8.0. Free "Cr was removed byextensive dialysis against running water so that 98% ofthe retained radioactivity was precipitable by 95%ethanol saturated with sodium acetate. The labeledendotoxin had a specific activity of about 0.003 gCi/ig and was as active as the native material in the LALassay.

Experimental procedure. The general form ofeach experiment was to incubate together at 370C 50,ul of fluid containing endotoxin (or its control substi-tute), 100 1l of LAL (or substitute; i.e., PFW) and 50pl of substrate solution for various times. The reactionwas stopped by the addition of 2 ml of 6.25% aceticacid, and the optical density at 405 nm (1-cm lightpath) (OD"m'm) of the solution was measured. Appro-priate controls were used as follows: (i) the experimen-

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re without any added endotoxin, as a control tubes for estimation of radioactivity.oxin contamination, and (ii) the experimental (ii) Depletion of AT-II from plasma. Monospe-without LAL, as a blank for spectrophoto- cific rabbit anti-human anti-thrombin II (AT-II) an-easurement. tibody was purchased (Sigma Chemical Co., St. Louis,vation of endogenous amidases. Since Mo.) and was coupled to beads of Sepharose 4B-CLstains several enzymes (e.g., plasmin, throm- (Pharmacia Fine Chemicals AB, Uppsala, Sweden) byurokinase) which cleave some substrates hav- the method of Axen et al. (1) modified by the use of-Gly-Arg-p-nitroanilide terminal sequence, less cyanogen bromide. The coupled Sepharose wasidases must be removed or inactivated before used in an affinity column to absorb out the AT-Ilof substrate or LAL. present in normal human plasma (heparinized) tound that heating serum is a satisfactory which a trace of '25I-labeled bovine serum albumin hadfor inactivation of endogenous amidases. been added as a marker for dilution. (Molecularntaining 250,ul of fresh serum were incubated weights of bovine serum albumin and AT-II are 68,000for various lengths of time and then were and 64,000, respectively.) The absorbed plasma wastely cooled in ice. Subsequently, 50 ,il of sub- collected, and the column was washed well with phos-lution was added to each tube, and the tubes phate-buffered saline, pH 7.2. The bound AT-II wasicubated at 37°C for 1.5 h. The reaction was then eluted at room temperature with 3 M potassiumwith acetic acid and the OD4Js",mn of the solu- thiocyanate, pH 6.8. The presence or absence of AT-s read against a blank of serum to which Il in the eluate and from the absorbed plasma, respec-] had been added in the presence of acetic tively, were confirmed by Ouchterlony gel diffusion.en the fal in ODlS,05m was plotted against The absorbed plasma inevitably had been dilutedation time (at 60°C), the curve (Fig. 2) during chromatography, so it was necessary to recon-;hat inactivation of the endogenous amidases centrate it before testing in the LAL assay. Howeverwithin 15 min, andthiswas taken asastand- since the major anticoagulant action of heparin is-temperature combination. The experiment mediated by AT-II (15), when the absorbed plasmaormed with sera from several subjects, and was concentrated it would dot unless an alternativevere consistent. Since endotoxins are stable anticoagulant was added first. The calcium concentra-oiing, they are not inactivated by this treat- tion of the original plasma was assumed to be 2.5 mM,hen sera were preincubated at 55°C, 50toa 60 and 1 volume of 20 mM disodium ethylenediaminetet-required for complete amidase inactivation. raacetate (EDTA) was added, which was calculated to

tigation of LAL inhibition by blood. (i) Is be just sufficient to chelate the calcium present in the,in trapped when blood clots? I investi- dilute plasma. The plasma was concentrated by mem-e possibility that endotoxin might be physi- brane filtration over a Diaflo PM10 membrane (Ami-zoved from serum by clotting as follows. con Corp., Lexington, Mass.) until the concentration*h of three pairs of siliconized tubes containing of 125I-labeled bovine serum albumin was equai to thatr 0.2 mg of 5Cr-labeled endotoxin in 0.1 ml of of the starting material. Since it was not possible toîs added 4 ml of fresh human blood or saline. perform these maneuvers under pyrogen-free condi-ixing, half of each blood sample was trans- tions, the absorbed plasma was put up in the LAL>a plain glass tube to permit clotting, and to assaywoth the followmg three controls: (i) normal

fder was added 25 U of hepare.Acter 1i at plasma which had been kept in a clean, but not pyro-nperaturebanddduplicate 100-Mlcaliquotsewere gen-free, container; (ii) a portion of (i) to which S.

'perature,and duplicate 00-tLI aliuomarcescens endotoxin was added at 2 gg/ml (this wasm each of the serum, plasma, or saine control assumed to be considerably more than the concentra-

tion of endotoxin which would be acquired off of cleanglassware); and (iii) serum from the same donor, col-

-i lected and stored pyrogen-free, to which S. marcescensendotoxin was added at 2 ,ug/mi.The serum and plasma samples were heated at 60°C

for 15 min before testing. Approximately 200 tig ofcalcium chloride was added to the absorbed plasma toensure that sufficient free Ca2+ was present in theassay. Clotting did not occur because the plasma wasdiluted by LAL and substrate.

* \ The eluate from the column, containing purifiedAT-Il, was concentrated over a PM10 membrane, andthe potassium thiocyanate was removed by dialysisagainst saline at 5C.

_ I Investigation ofthe effect of antibody to endo-

0 2 4 6 8 10 12 toxin. Equal volumes of similar dilutions of endo-toxins from E. colt O111;B4 and S. marcescens were

Minutes added to E. coli antiserum and control serum (both

2. Inactivation of endogenous amidases in diluted 1:4 with pyrogen-free saline), or to pyrogen-ncubation time at 60°C is plotted against fail free saline alone, and incubated at 37°C for 30 min.""" for tu'o typical sera. LAL and substrate were added, and the assay was

tal mixtu:for endotmixture smetric m

Inactiblood coIbin, and u

ing thethese amiaddition i

We fomethodTubes coiat 600Cimmediatestrate solwere reinstopped stions wasubstrateacid. Whpreincubshowed toccurredard timewas perfresults w

even to bment. W]min were

Invesiendotoxgated thcally remrTo eac

0.8, 0.4, osaline waAfter miferred tothe rema37°C, al]room tentaken frc

0.2

EC6 0.1

o

FIG. 2serum. Iiin OD

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COLORIMETRIC ASSAY OF ENDOTOXINS IN BLOOD

performed in the standard manner.Investigation of endotoxin inactivation by se-

rum factors. Endotoxin (S. marcescens) was dilutedto 10 ug/ml in human serum which had been heatedat 560C for 1 h. Portions (25p1) (0.25 pg) were added to100-pl samples of fresh or heated (600C, 15 min) serumwhich were incubated at 37°C for various periods oftime. Additional samples of each serum were kept at5°C for 24 h. After incubation, the fresh serum sampleswere heated at 60°C for 15 min. Ail tubes then received250 pl of pyrogen-free saline, 50 »d of LAL, and 50 pl ofsubstrate, and the OD«0*' was measured after incu-bation at 37°C for 1 h.

RESULTS

Investigation ofLAL inhibition by blood.(i) Io endotoxin trapped when blood clots?After allowing for the volume occupied by eryth-rocytes, from which endotoxin is excluded (4),recovery of radioactivity was almost 100% in theserum samples and consistently 4.4 to 4.8% lessin the corresponding plasma. The finding wasconfirmed by using blood taken from rats in-jected with 51Cr-labeled endotoxin 1 h beforesampling, where the final endotoxin concentra-tion was approximately 100 ng/ml of blood.Thus, significant trapping of endotoxin in theclot was not demonstrable.

(ii) Inhibition of LAL by AT-fl-depletedplasma and by purified AT-Il. The results ofthe assay, when AT-II-depleted plasma was in-cubated with LAL for 2 h, showed that thisplasma displayed scarcely diminished inhibitionof the LAL enzyme. However, when the columneluate which contained the recoverable, purifiedAT-Il from 5 ml of plasma in about 0.4 ml ofphosphate-buffered saline was tested in the LALassay, very substantial inhibitory activity waspresent. Quantitation of this AT-Il activity, interns of a plasma standard, was not practicable.These results imply that although AT-II is apotent inhibitor of the LAL enzyme, it is notsolely responsible for the inhibition shown byundiluted plasma. There are known to be atleast two other enzymes in plasma which pro-gressively and irreversibly inhibit thrombin (15)and which possibly could affect the LAL en-zyme.Most of the residual inhibition present in se-

rum could be abrogated by dilution (19). Figure3 shows the results of an experiment where 25,ul of S. marcescens endotoxin, at 10 jug/ml inpyrogen-free human serum, was added to 0.5-mlportions of serum from each of three normalvolunteers. The heated sera were diluted in py-rogen-free saline as shown, and 50 gl of eachdilution was tested in the LAL assay. This ex-periment was performed three times with similarresults.

0.8 -

0.7 -

0.6 -

0.5 -E

E C

* 0.4 -

0.3 n

0.2-

0.1-

u -_0

I~~~~~~~~~~~~~~~C

0i/s

O/dV4A

Parts PFS per part serumFIG. 3. Effect of dilution on residual inhibition of

LAL in three human sera. Change in OD'rniplotted against the number ofparts ofpyrogen-freesaline (PFS) addedperpart of serum.

Effect of antibody to endotoxin upon thesensitivity ofthe LAL assay. The results (Fig.4) show that although S. marcescens endotoxinwas approximately 10 times more active than E.coli endotoxin, the activities of each endotoxinwere similar in both sera and also bear the samerelationship to each other as was obtained insaline. This signifies that endotoxin activity inthe LAL assay is unaffected by specific antibodyagainst the O antigen or by some anti-lipid Aantibodies. This experiment was performedtwice, with similar results.Inactivation of endotoxin by serum fac-

tors. Results are summarized in Fig. 5. It can beseen that inactivation of endotoxin proceedsvery much more slowly in heated serum than infresh serum, although the former is not devoidof inactivating ability. Inactivation by fresh se-rum would appear to be biphasic, and the rateof the first phase (up to 3 h of incubation)approximates to 600 ng/ml per h in this experi-ment. At 5°C, inactivation was slower, but ac-counted for a substantial fraction of the addedendotoxin in 24 h. This experiment was repeatedtwice, with similar results.

DISCUSSIONThe objective of this work was to investigate

further the factors which might bear upon the

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method, variations of which have been usedquite widely in clinical studies (8, 14, 18, 20).

,------ The fact that Levin et al. (19) found that"'-~ much of the inhibition of theLALenzymeby. serum could be diluted out made very unlikely

,V,,</ the trapping of endotoxin during clotting as an,8,' ,I explanation of this inhibition, and I have shown

that there is no less endotoxin in serum than inplasma. Nevertheless, the facts remained thatsome endotoxin activity in the LAL assay ap-

,O peared to be lost during clotting, and yet, thatendotoxin added to serum had more activitythan a similar amount added to plasma. Thissuggested that the inhibitor of the LAL enzyme

,present in plasmamightbe inactivated by clot--~ / . /. t ting, but in thepresence of endotoxin is partially

spared from such inactivation. The inhibitor isnot heparin (19), but is possibly AT-II, which isknown to inhibit serine esterase enzymes, to beactivated by heparin, and of which about 20 to

1.0 10 102 103 30% is consumed during clotting (15). AT-IIEndotnxin concentration (ng/ml) activity is also susceptible to those procedures

ifect ofantibody to E. coli 0111 endotoxin which have been found to remove plasma inhib-ion with LAL. Symbols: *, activation of itors of LAL (2).coli 0111 endotoxin; O, activation by S. My results show that the inhibition of theendotoxin. Assay was performed in the LAL enzyme is due to more than a single com-saline (-----), E. coli 0111 rabbit antise- ponent of plasma. Other serine esterase inhibi-or normal rabbit serum tors (e.g., al-antitrypsin and a2-macroglobulin)

could be expected to play a part, and some lossof sensitivity may be due to substrate competi-tion between coagulogen and the chromogenic

_* . . substrate. However, I fmid that dilution of serumremoves much of the residual inhibition presentand renders the assay of equal quantities ofendotoxin in different sera reproducible, al-though I have evidence that the degree of dilu-tion necessary to achieve this varies between

* species. Others have reported the total recoveryof endotoxin activity, as estimated by the gela-tion assay, when human sera were diluted 10-fold (19), but by using a chromogenic substrate,

_ . I find no improvement in sensitivity when sera. * are diluted beyond fivefold.

My finding that the presence of antibody to_ | | either the O antigen or lipid A of an endotoxin

o 1 2 3 4 5 does not affect its performance in the LAL assayHours is reminiscent of the findigs of others for differ-

nactivation of endotoxin by enzymes in ent properties of endotoxins. Thus, Perry re-and unheated (-) human serum. Endo- ported that the administration to rabbits of anty is plotted against incubation time with excess of precipitating antitoxin had no effect7°C. upon the endotoxin's pyrogenicity (22a), and

antibody has been found not to affect an endo-and reproducibility of the LAL assay toxin's tumor-necrotizing abiity (27).Oxin in the blood. The advent of chro- However, it has been reported that antibodyubstrates susceptible to the LAL en- to lipid A, as opposed to the anti-O antigenws significant improvements in sensi- antibody used by Perry et al., does suppress theobjectivity to be made, but some of endotoxin's pyrogenicity in vivo, provided thatgs are also applicable to the gelation the animal is prepared beforehand with lipid A

0. 5-

0.4 -

E 0.3 -E

0.2

0.1 -

0

'O-J

FIG. 4. Eon its reactLAL by E.marcescens

presence ofrum (.....

0.6

0.5

E

E= 0.4

° 0.3

0.2

0.1

O

FIG. 5. I]heated (A)toxin activiserum at 37

sensitivityfor endotomogenic siazyme allovtivity andmy finding

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COLORIMETRIC ASSAY OF ENDOTOXINS IN BLOOD 649

(24), which suggests that either the determinantsin lipid A which are responsible for pyrogenicityare not identical with those which cause activa-tion ofLAL or that the physical configuration ofthe endotoxin in our experiments rendered thesedeterminants inaccessible. Inaccessibility oflipidA could prevent the induction of neutralizingantibodies during immunization or could pre-vent the subsequent combination of antibodywith the active sites. Thus, I do not suggest thatsome antibodies to lipid A might not affect thereactivity of endotoxins with LAL, but simplythat this reactivity appears to be unaffected byantibody to the O antigen, which is immunodom-inant in all gram-negative bacteria.Endotoxin inactivation in unheated serum has

been shown to be relatively rapid, and, therefore,it becomes of some importance to prepare theserum or plasma to be assayed without delay. Itis likely that several methods for the removal ofLAL inhibitors from plasma remove also theenzymes responsible for endotoxin inactivation.Alternatively, heating of neat serum at 60°C for30 min is practicable. The most expeditiousmethod I have used for the preparation ofserumhas been to defibrinate blood by gentle mixingwith pyrogen-free glass beads.

In conclusion, the inactivation of serine ester-ases in blood by heating allows extremely lowendotoxin activities to be assayed objectively bythe use of a chromogenic substrate for the LALenzyme. This technique could have importantapplications as a research tool and as a clinicalaid. Endotoxin activities could be estimated fora single incubation period, but preferably wouldbe determined kinetically, and the results wouldbe reported in terms of a specified referenceendotoxin (e.g., the First International PyrogenReference Preparation from S. dysenteriae).

ACKNOWLEDGMENTS

I am very grateful to J. H. Humphrey for helpful discussionand suggestions, P. Hilgard, for advice concerning plasmainhibitors, and R. Frearson for typing the manuscript.

C.J.W. is a Medical Research Council Research Student.

LITERATURE CMD

1. Axen, R., J. Porath, and S. Ernback, 1967. Chemicalcoupling of peptides and proteins to polysaccharides bymeans of cyanogen halides. Nature (London) 214:1302-1304.

2. Biggs, R., and K. W. E. Denson. 1972. Natural andpathological inhibitors of blood coagulation, p. 133-158.In R. Biggs (ed.), Human blood coagulation, haemosta-sis and thrombosis. Blackwell Scientific Publications,Oxford.

3. Braude, A. I., F. J. Carey, D. Sutherland, and M.Zalesky. 1955. Studies with radioactive endotoxin. I.The use of 5"Cr to label endotoxin of Escherichia coli.J. Clin. Invest. 34:850-857.

4. Braude, A. I., F. J. Carey, and M. Zalesky. 1955.Studies with radioactive endotoxin. II. Correlation of

physiologic effects with distribution of radioactivity inrabbits injected with lethal doses of E. coli endotoxinlabeled with radioactive sodium chromate. J. Clin. In-vest. 34:858-866.

5. Burton, A. J., and H. E. Carter. 1964. Purification andcharacterisation of the lipid A component of the lipo-polysaccharides from Escherichia coli. Biochemistry 3:411-418.

6. Cooper, J. F. 1975. Principles and applications of theLimulus test for pyrogen in parenteral drugs. Bull.Parenter. Drug. Assoc. 29:122-130.

7. Davies, D. A. L, M. J. Crumpton, L. A. Macpherson,and A. M. Hutchison. 1958. The adsorption of bacte-rial polysaccharides by erythrocytes. Immunology 1:157-171.

8. Fossard, D. P., V. V. Kakkar, and P. A. Elsey. 1974.Assessment ofLimulus test for detecting endotoxaemia.Br. Med. J. 2:465-468.

9. Goto, H., and S. Nakamura. 1979. Dry-up method as arevised Limulus test with a new technique for gelationinhibitor removing. Jpn. J. Exp. Med. 49:19-25.

10. Harris, N. S., and R. Feinstein. 1977. A new Limulusassay for the detection of endotoxin. J. Trauma 17:714-718.

11. Holme, R., and N. O. Solum. 1973. Electron microscopyof the gel protein formed by clotting of Limulus poly-phemus hemocyte extracts. J. Ultrastruct. Res. 44:329-338.

12. Humphrey, J. H., and D. R. Bangham. 1959. Theinternational pyrogen reference preparation. Bull. W.H. O. 20:1241-1244.

13. Iwanaga, S., T. Morita, T. Harada, S. Nakamura, M.Niwa, K. Takada, T. Kimura, and S. Sakakibara.1978. Chromogenic substrates for horseshoe crab clot-ting enzyme-its applications for the assay of bacterialendotoxins. Haemostasis 7:183-188.

14. Jones, R. J., and E. A. Roe. 1979. Measurement ofendotoxins with the Limulus test in burned patients. J.Hyg. 83:151-156.

15. Lane, J. L., and R. Biggs. 1977. The natural inhibitorsof coagulation: antithrombin III, heparin cofactor andantifactor Xa, p. 123-139. In L. Poller (ed.), Recentadvances in blood coagulation (2). Churchill Living-stone, London.

16. Levin, J., and F. B. Bang. 1964. The role of endotoxinin the extracellular coagulation of Limulus blood. Bull.Johns Hopkins Hosp. 115:265-274.

17. Levin, J., and F. B. Bang. 1968. Clottable protein inLimulus: its localisation and kinetics of its coagulationby endotoxin. Thromb. Diath. Haemorrh. 19:186-197.

18. Levin, J., T. E. Poore, N. P. Zauber, and R. S. Oser.1970. Detection of endotoxin in the blood of patientswith sepsis due to gram-negative bacteria. N. Engl. J.Med. 283:1313-1316.

19. Levin, J., P. A. Tomasulo, and R. S. Oser. 1970. De-tection of endotoxin in human blood and demonstrationof an inhibitor. J. Lab. Clin. Med. 75:903-911.

20. Nachum, R., A. Lipsey, and S. E. Siegel. 1973. Rapiddetection of gram-negative bacterial meningitis by theLimulus lysate test. N. Eng. J. Med. 289:931-934.

21. Nakamura, S., T. Morita, S. Iwanaga, M. Niwa, andK. Takahashi. 1977. A sensitive substrate for the clot-ting enzyme in horseshoe crab hemocytes. J. Biochem.(Tokyo) 81:1567-1569.

22. Nakamura, S., T. Takagi, S. Iwanaga, M. Niwa, andK. Takahashi. 1976. Amino acid sequence studies onthe fragments produced from horseshoe crab coagulo-gen during gel formation: homologies with primate fi-brinopeptide B. Biochem. Biophys. Res. Commun. 72:902-908.

22a.Perry, W. L. M. 1954. Standards of pyrogenic activity. J.Pharm. Pharmacol. 6:332-338.

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650 WEBSTER

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