New, Sensitive Rocket Immunoelectrophoretic Assay forMeasurement of the Reaction Between Endotoxin and Limulus
Amoebocyte LysateLEIF BAEKt
Statens Seruminstitute, Department of Clinical Microbiology, Rigshospitalet and the University Clinic forInfectious Diseases, Rigshospitalet, Copenhagen, Denmark
Received 19 January 1983/Accepted 17 March 1983
To study the active proteins which participate in the reaction of Limulusamoebocyte lysate (LAL) with lipopolysaccharide, antibody was raised in rabbitsagainst LAL. When LAL was run against rabbit antiserum in crossed immuno-electrophoresis, a complex precipitin pattern appeared. Profound changes tookplace after reaction of LAL with lipopolysaccharide. The most distinct changewas the complete disappearance of the cathodic migrating protein coagulogen,because the antigenicity of coagulogen was lost. Based on this observation, a newrocket immunoelectrophoretic method was developed to detect the disappearanceof coagulogen after reaction of LAL with lipopolysaccharide. This assay methodwas used on clinical specimens (cerebrospinal fluid, plasma, ascites, and urine). Itwas used as a qualitative test, when a single sample is tested, or as a quantitativeassay, when a number of sample dilutions were tested. The new method showed a
higher degree of accuracy and sensitivity in comparison with the tube test and itcan be used for both research and diagnostic purposes.
The Limulus amoebocyte lysate (LAL) testintroduced by Levin and Bang (13) in 1964 is themost sensitive assay for detection of lipopoly-saccharide (LPS) from gram-negative bacteria.The original form of the LAL test, performed intubes, was based on the formation of a gel or clotwhen LAL was incubated with LPS.The active material in LAL consists of a pro-
clotting enzyme which is activated in the pres-ence of LPS and Ca2+. The clotting enzyme actson the substrate, coagulogen, which splits intocoagulin and a C-peptide. The result is clotformation (22, 24, 26, 27, 32).
Although the number of different applicationsof the LAL test has increased in the years sinceits first description, there are, however, someproblems related to its sensitivity and reproduc-ibility. This is because of the subjective methodof visually reading the gelation endpoint, evenwhen done by an experienced investigator. Theproblems are especially pronounced when solu-tions with variable concentrations of proteins,lipoproteins, lipid, sugar, or other highly viscousfluids are to be tested.
Several modifications of the basic LAL testhave been made to increase the sensitivity and
t Present address: Department of Microbiology, Rigshospi-talet, Afsnit 7806, Tagensvej 18, DK-2200 Copenhagen N,Denmark
to develop a more objective method of readingthe test.
Levin and Bang (14) demonstrated that theLPS-activated LAL reaction can be followed bymeasuring a change in optical density or anincrease in light scattering. This observation hasresulted in the development of spectrophotomet-ric and nephelometric methods (2, 7, 25).Based on studies of the reaction mechanism in
LAL (17, 18, 26, 27), other modifications of theLAL test have been published. These include aprotein assay (19), a radioisotopic labeling ofcoagulogen (16), and enzymatic methods mea-suring cleavage of a synthetic chromogenic sub-strate by the clottable enzyme (5).
In the present study, a new immunoelectro-phoretic method which is able to detect thereaction of LAL with LPS is described. Themethod is a combination of the tube test and arocket immunoelectrophoretic method that mea-sures the loss of the antigenicity of coagulogenwhen it is split by the LPS-activated enzyme.This is the last step in the reaction mechanism inthe LAL system.The method can be used as a qualitative and
as a quantitative assay. Examples of its clinicalapplications are presented.
MATERIALS AND METHODSPyrogen-free materials. Pyrogen-free materials em-
test tubes (10 by 80 mm), rubber stoppers, and Carls-berg pipettes (Bie & Berntsen, Copenhagen, Den-mark). These were depyrogenated by washing in de-tergent solution and by heating as previouslydescribed (M. Tvede and L. Baek, Acta Pathol. Micro-biol. Immunol. Scand. Sect. B, in press). Pyrogen-freewater (from the dispensary of Rigshospitalet, Copen-hagen, Denmark) was used in all experiments.LAL test. LAL was prepared by a method previous-
ly described (Tvede and Baek, in press), and a com-mercial LAL product (Pyrogent) was purchased fromMallinckrodt Inc. (St. Louis, Mo.). LPS of Escherich-ia coli 026:B6 (W) from Difco Laboratories (Detroit,Mich.) was diluted 10-fold in pyrogen-free water withCarlsberg pipettes. The concentration of LPS in testsolutions ranged from 1 mg/ml to 0.1 ng/ml. Twofolddilutions were made from 1 ng/ml to 0.1 pg/ml. Usual-ly, 100 u1l of LAL and 100 ,ul of test solution weremixed in test tubes and incubated for 1 to 4 h in a 37°Cwater bath. To test the reproducibility of the rocketmethod, experiments with 10, 25, and 50 ,u1 of LALwere performed. Only LAL which could detect at least0.1 ng of LPS per ml was accepted in all experiments.LAL (100 pul) mixed with pyrogen-free water (100 ,ul)was used as a control.
Preparation of polyspecific antibody against LAL.Ten rabbits were injected intradermally with equalvolumes of LAL and Freund incomplete adjuvant andbled according to the schedule of Harboe and Ingild(6). Each animal received 100 .1A of LAL per injectionand was reimmunized with four injections at intervalsof 14 days. The initial five injections were followed bymonthly booster vaccinations for 6 months before thefirst bleeding. Thereafter, the immunization contin-ued, and 1 week after each monthly vaccination, 40 mlof blood was obtained from each rabbit. The serumsamples from the 10 rabbits were pooled and immuno-globulin G (IgG) and IgA were purified and concentrat-ed (6).
Preparation of monospecific antibody against coagu-logen. Coagulogen was purified according to Tai et al.(27). To test the immunochemical purity of coagulo-gen, crossed immunoelectrophoresis (CIE, see below)with polyvalent antibody against LAL, as described
A
earlier, was performed. The large cathodic precipitatewas cut out of the gel and incubated overnight at 4°Cwith an aliquot of phosphate-buffered saline. The gels(containing the coagulogen precipitate) were then ho-mogenized at low speed by a Micro-Dismembrator(type 854 480; B. Braun Melsunger AB, W. Germany).The suspension was mixed with Freund incompleteadjuvant, and five rabbits were immunized and bled asdescribed earlier (6). IgG and IgA from the pooledantisera were isolated and concentrated (6).CIE of LAL. CIE was performed according to the
method of Weeke (29) on glass plates (10 by 10 cm) byusing 1% Litex-type HSA agarose gel, -mr = 0.13(Litex, Copenhagen, Denmark), and Tris-barbital buff-er containing 10 mM calcium lactate (ionic strength,0.02; pH 8.6). A 10-,ul volume of LAL was run in thefirst dimension at 10 V/cm, guided by a bromphenolblue albumin marker which migrated 5 cm. The first-dimension gel slab was placed on the middle of theglass plate with antibody-containing gel on both sidesto detect the anodically and cathodically migratingproteins (Fig. 1). The second dimension electrophore-sis was run at 2 to 3 V/cm overnight. The temperatureof the cooling water was 12°C. Identification of coagu-logen was carried out by means of crossed-line immu-noelectrophoresis (11) and tandem CIE (10), usingpurified coagulogen and the LAL reference CIE sys-tem (Fig. 1A).RIE. Rocket immunoelectrophoresis (RIE) was per-
formed according to Weeke (30). The electrophoresiswas run with antibody in the whole gel and with thewells at the anodic part of the gel, on 10 by 10 or 11 by20.5 cm glass plates with high voltage (10 V/cm) for 4to 5 h. The mixtures of LAL and test solutions werediluted with 3,000 pI of pyrogen-free water aftercompletion of the reaction, except when the commer-cial LAL was used; the commercial LAL was dilutedwith 1,500 pI because of the lower content of coagulo-gen. Five microliters of the dilutions was used in thewells in RIE.To avoid further reactions due to contamination in
the mixtures of LAL and test solutions after incuba-tion, another dilution procedure was performed. Amodification of the acidification procedure described
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FIG. 1. CIE ofLAL before (A) and after (B) reaction with LPS. Antigen, (A) 10 p.l of LAL, (B) 20 p.l of LALplus LPS mixture (1:1); LPS concentration, 10 jig/ml; antibody, polyspecific antibody against LAL (10 p.l/cm2 ofgel); staining, Coomassie brilliant blue.
wate,gr wasY ade to th tues Anie, l ofthdilutedmixture~in s4Z1tep 2lwas applieinth wells in theimmuoplte (ste 3) cotrl 10 Rl of LA an 100B%
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FIG. 2. New method illustrated as a four-step pro-cedure. The tube test (step 1 and step 2) and the RIEmethod (step 3 and 4). In step 1, 100 p.1l of LAL wasadded to 100 p.1 of LPS diluted in pyrogen-free water.After incubation (step 2), 3,000 p.1 of pyrogen-freewater was added to the tubes. Antigen, 5 p.1 of thediluted mixture in step 2 was applied in the wells in theimmunoplate (step 3); control, 100 p.1 of LAL and 100p.1 of pyrogen-free water; antibody, polyspecific anti-body against LAL in the whole gel (7.5 p.11cm2);staining, Coomassie brilliant blue.
by Solum (23) was simultaneously used to stop thereaction in LAL and to dilute the reaction mixturebefore RIE was run. The method was performed asfollows. After incubation and preliminary reading ofthe tubes, 1,500 ,ul of 0.2 N HCI was added to the testtubes. After shaking on a Vortex mixer and incubationat 20°C for 10 min, 1,500 ,ul of 0.2 N NaOH was added,and the tubes were then centrifuged for 10 min at 5,000x g (Sorvall RC2-B, rotor SS-34); 5 p.1 of the superna-tant was then added to the wells for RIE (Fig. 7).Treatment of plasma specimens. Venous blood was
obtained from patients with infections and from nor-mal controls. Blood was drawn into sterile pyrogen-free plastic syringes and transferred into pyrogen-freeglass tubes containing heparin (10 U/ml of blood). Theplasma was added to 2 ml of pyrogen-free water andboiled for 10 min. After centrifugation, the supernatantwas tested with LAL.
RESULTSCIE of LAL. When LAL was analyzed by
CIE, most proteins migrated to the anode, butseveral proteins migrated to the cathode (Fig.1A). Altogether, approximately 50 precipitatescould be counted. Coagulogen moved to thecathode, and the corresponding precipitate wasidentified by means of tandem CIE and crossed-line immunoelectrophoresis with purified coagu-logen (L. Baek, manuscript in preparation). Af-ter the reaction of LAL with LPS, coagulogendisappeared totally (Fig. 1B), whereas the con-trol plate without LPS showed no change (Fig.1A).RIE of LAL. The new method as a four-step
procedure is illustrated in Fig. 2. Figure 3 showsRIE of a twofold standard dilution of LPS incu-bated with LAL. When LPS was in excess, allcoagulogen was split and the rocket disap-peared. When the concentration of LPS was 63pg/ml, the rocket reappeared and the heightincreased to the endpoint of 1 pg of LPS per ml.Further incubation of LAL and LPS did notchange the results as measured by the RIE. Theendpoint measured as a solid clot by the clot
FIG. 3. RIE of a twofold dilution of a standard LPS (E. coli 026:B6). Antigens, 5 p.1 of the diluted LAL plusLPS mixtures were applied to each well; antibody, polyspecific antibody against LAL in the whole gel (7.5p./cm2); control, LAL plus pyrogen-free water; staining, Coomassie brilliant blue.
FIG. 4. RIE of twofold dilution of a standard LPS (E. coli 026:B6). Antigens, 5 jil of the same LAL plus LPSmixtures used in Fig. 3; antibody, monospecific antibody against coagulogen in the whole gel (15 Vtl/cm2);staining, Coomassie brilliant blue.
method was 63 pg of LPS per ml. In Fig. 3,polyvalent antibody to LAL was used in the gel.The same standard dilution of LPS incubatedwith LAL and detected with monospecific anti-body against coagulogen is shown in Fig. 4.
Reproducibility of the RIE method. The repro-ducibility of the RIE method was estimated byrepeated determinations of the same sample onthe same immunoplate, on different immuno-plates on the same day, and on different days.The results are expressed as variation coeffi-cients.The variation coefficient was 1.02% when the
same pipette was used on four different mea-surements on the same sample on the sameimmunoplate and the experiment was repeatedfour times. The variation coefficient was 3.1%
when different pipettes were used on six differ-ent measurements on the same sample on thesame immunoplate and the experiment was re-peated four times. The 95% confidence limits ofreported results are therefore ± 6.2% and the99% confidence limits are ± 8%. The variationcoefficient was 5.5% when different pipetteswere used on six different measurements on thesame sample on six different days (28).
Detection of LPS in plasma. When LPS wasdiluted in plasma from a normal control andtested with LAL, a concentration of 10 ng ofLPS per ml of plasma was detected by visualreading of the tubes (increased opacity) and aconcentration of 1 ng of LPS per ml was deter-mined by RIE (Fig. 5). Figure 6 shows RIE ofthe same 10-fold dilution of LPS in plasma as
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FIG. 5. RIE of a 10-fold dilution of LPS in untreated fresh plasma from a control. LPS was diluted 10-fold inplasma from 10 ,ug of LPS per ml of plasma to 10 pg of LPS per ml plasma (only the LPS concentrations withpositive tests are given). Antigens, 5 ±1 of the diluted LAL plus LPS plasma mixtures was applied to the wells;antibody, polyspecific antibody against LAL in the whole gel (7.5 ,ul/cm2); control, LAL plus plasma; staining,Coomassie brilliant blue.
FIG. 6. RIE of a 10-fold dilution of LPS in fresh plasma from a control treated by heating. Antigens, 5 ,ul ofthe same LAL plus LPS plasma mixtures used in Fig. 5, but treated by heating before the reaction with LAL;antibody, control, and staining as described for Fig. 5.
that used in Fig. 5, but it was treated by dilutionand heating before incubation with LAL. Thisprocedure increased the sensitivity of the meth-od 1,000 times, as 1 ng of LPS per ml of plasmaby visual reading of the tubes and 1 pg of LPSper ml with the RIE was now recorded aspositive.Examples of clinical applications. Figure 7
shows the results of culture and LAL tests and afew other parameters from repeated cerebrospi-nal fluid (CSF) specimens obtained from a 3-week-old patient with E. coli meningitis. It is
seen that although the CSF was sterilized byantibiotics, the LAL tests remained positive for20 days (last measurement). The patient died 11days later due to hydrocephalus.
Figure 8 shows RIE of plasma specimens fromeight patients with suspected gram-negative bac-teremia or endotoxemia. Three patients hadpositive LAL tests, and all were treated formeningococcal septicemia. In one of these threepatients with a strong positive LAL test, menin-gococci were cultured from the blood. The re-maining five patients were LAL negative, and
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FIG. 7. RIE of CSF from a patient with E. coli meningitis. Other laboratory results are correlated with thoseof the RIE. Antigens, 5 IlI of the diluted LAL plus CSF mixtures was applied to the wells; antibody, polyspecificantibody against LAL in the whole gel (7.5 jll/cm2); control, from a patient suspected of lymphocytic meningitis,but with no cells, no bacteria, and normal protein concentration in CSF; staining, Coomassie brilliant blue.
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FIG. 8. RIE of plasma specimens from eight patients suspected of gram-negative bacterial infections. Theplasma are treated by heating before the reaction with LAL. Antigens, 5 ,ul of the diluted LAL plus plasmamixtures was applied to each well; antibody, as described in Fig. 7; control, LAL plus pyrogen-free water;staining as described in Fig. 7.
the suspected septicemia was not bacteriologi-cally confirmed.
DISCUSSIONIn the present study, a well-established sensi-
tive immunochemical method (RIE), using spe-cific antibody to coagulogen, was used to deter-mine the reaction between LPS and LAL. Themethod records the last step in the coagulationmechanism in LAL, namely the splitting ofcoagulogen into coagulin and C-peptide. By thisprocess, the antigenicity of cleaved coagulogenis lost.CIE ofLAL before and after the reaction with
LPS, using high avidity antibody to LAL,showed (Fig. 1A and B) that LAL contains atleast 50 different proteins of which 9 were in-volved in the reaction of LAL with LPS (L.Baek, manuscript in preparation). Several minorprecipitins were present at the cathodic part ofthe gel, but these did not disturb the reading ofthe rocket corresponding to coagulogen in RIEbecause of the high antigen dilution before theelectrophoresis was run. The coagulogen, repre-senting 40 to 50% of the total proteins in LAL,was a strong immunogen in rabbits, and highavidity antibody was obtained by the immuniza-tion schedule described by Harboe and Ingild(6). The antibody was purified according to themethod described by Harboe and Ingild (6) toavoid background staining and to enhance theresolution and visibility of the precipitins (rock-ets), especially in the wet gel shown in Fig. 2(step 3).There was no difference in the reproducibility
of the method by using polyspecific antibody toLAL or monospecific antibody to coagulogen.The best reproducibility of the RIE method was
achieved when the height of the rockets was 40to 50 mm (30). Therefore it was important to findthe antigen/antibody ratio, using as small anamount of antibody as possible, that would givethe most distinct precipitate in the immunoplate.The sensitivity of LAL was not exclusivelydependent on the amount of coagulogen, butalso on other substances in LAL. This wasshown when a commercial LAL (Pyrogent) wasused which contained only half as much coagu-logen, as measured by the height of the rocket inRIE; however, the sensitivity was the same.
Quantitation of LPS by the clot method isusually performed by testing twofold dilutions ofan unknown sample in comparison with a stan-dard LPS (20). The endpoint is a solid clot.Although sensitive, these clot procedures arehighly subjective and are referred to as semi-quantitative. Other more sensitive methods havebeen developed, utilizing different physico-chemical properties of LAL, i.e., measuring theprotein concentration of the clot (19), spectro-photometric assay measuring increases in opti-cal density or turbidity (7, 25), and nephelomet-ric methods measuring increases in lightscattering (2). All of these methods are more orless indirect, measuring the sum of events whenLAL is reacting with LPS.The RIE method directly measures the most
important protein in LAL, the coagulogen, andextremely low concentrations of LPS induce thecleavage of this protein, which results in dimin-ished rockets (Fig. 4). Furthermore, the repro-ducibility of the method was very high as reflect-ed by the low variation coefficients. Because ofthese properties, the RIE method may be usedas a reference method for other LAL methods.The clinical utilization of the LAL test demon-
strates an excellent correlation with the pres-ence of gram-negative bacteria and endotoxin inurine and CSF (4, 9). An example is shown inFig. 7, where different laboratory results werecorrelated with results of the LAL test in aclinical course of E. coli meningitis. The patientdeveloped hydrocephalus, which may be ex-plained by decreased clearance of endotoxin asdemonstrated by a positive LAL test even after20 days of illness.The most difficult challenge for the LAL test
has been the measurement of endotoxin in bloodor plasma. First, the concentration of circulatingendotoxin in blood is usually low because of itsrapid clearance in the liver (21). Second, plasmacontains inhibitors to LAL (15), endotoxin-inac-tivating substances (8), and endotoxin-bindingproteins (31). Another important variable is thatthe levels of substances inhibiting endotoxinprobably vary not only from person to personbut also in a patient during various stages ofillness associated with gram-negative infections(12).
Different methods have been used to extractendotoxin from plasma. Levin et al. (15) intro-duced chloroform extraction, but the methodrequires hours to complete. Dilution is some-times adequate, but may decrease the endotoxinconcentration below detectable level. A thirdmethod comprising dilution combined with heat-ing has been used successfully (1, 3). All threemethods have been tested with the RIE method,and the combination of dilution and heatingrevealed the best results. A paradox reactionwas seen when LPS was diluted 10-fold in freshuntreated plasma from a normal control. Asshown in Fig. 5, the highest concentration ofLPS (10 jig) revealed a weaker reaction withLAL than the two following concentrations (1and 0.1 R,g). This may be explained by increasedrelease of LAL-inhibiting substances in plasmawhen high concentrations ofLPS are present. Atlower concentrations of LPS, the reaction withLAL decreased, probably because of its bindingto plasma proteins that mask the active sites ofLPS (Fig. 5). Dilution and heating of plasmaincreased the sensitivity of the method 1,000times (Fig. 6).These experiments indicated that plasma has
the capacity to bind and mask the active sites ofabout 1,000 pg of LPS (E. coli 026:B6) and thatthe clot method was difficult to interpret whenused on plasma specimens.
In conclusion, the RIE method is a highlysensitive assay method for reading the reactionbetween LAL and LPS. The method measuresthe last step in the multienzyme system in LALwhen reacting with LPS, and the extremely lowconcentrations of LPS added to LAL are reflect-ed in the loss of the antigenicity of cleaved
coagulogen, expressed by a diminished rocket inRIE. The method has been applied on clinicalspecimens, including plasma specimens, and hasdemonstrated a good correlation with the clini-cal diagnosis in patients suspected of gram-negative bacterial infections.
ACKNOWLEDGMENTS
Aase Stricker-Nielsen is thanked for skillful technical assist-ance.
This work was supported by grants from the NationalDanish Medical Research Foundation (J. no. 512-8782, 12-0648, 12-2346).
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