Vol. 15, No. 5JOURNAL OF CLINICAL MICROBIOLOGY, May 1982, p. 835-8410095-1137/82/050835-07$02.00/0

Development of a Simple Serological Method for DiagnosingLeptospirosis: a Microcapsule Agglutination Test

YOSHIKO ARIMITSU,* SHINZO KOBAYASHI, KIYOTO AKAMA, AND TYOKU MATUHASIThe Second Department ofBacteriology, National Institute of Health, Kamiosaki 2-10-35, Shinagawa-ku,

Tokyo 141, JapanReceived 3 March 1981/Accepted 29 December 1981

A passive microcapsule agglutination test for the diagnosis of leptospirosis wasdeveloped by utilizing chemically stable microcapsules instead of sheep erythro-cytes. In the test, sonically disrupted antigens of leptospira were sensitized tomicrocapsules treated with glutaraldehyde. Compared with the microscopicagglutination test, the passive microcapsule agglutination test showed a relativelygenus-specific tendency and a 4- to 32-fold-higher sensitivity. The sensitizedmicrocapsule antigens were stable for at least 1 year. The microcapsules coupledwith mixed antigens can be used as a serodiagnostic screening test for diseasescaused by various types of leptospira. The test, which is very simple andreproducible and requiring no specific training, can be employed easily as aroutine test in diagnostic laboratories.

For the serological diagnosis of leptospirosis,the microscopic agglutination (MA) test (15) ismost widely employed as the standard referencetest because of its high specificity and sensitiv-ity. However, it requires multiple serovars oflive leptospira, involving the risk of infectionand maintenance of a large number of stockcultures to provide antigens. These factors limitits usefulness for routine application in diagnos-tic laboratories.To circumvent these limitations, various sim-

ple methods have been investigated. For exam-ple, a macroscopic agglutination test (7) and alatex agglutination test (12), performing serovar-specific reactions, have been reported in screen-ing human serum specimens. Also, highly genus-specific reactions have been investigated asscreening tests, such as the complement fixationtest (13, 14, 17), sensitized erythrocyte lysis test(5, 6), passive hemagglutination (HA) test (4, 9,19), and the immunofluorescence test (21). Inparticular, the HA test is reported as highlysensitive and satisfactory as a screening test (18,19), but the erythrocytes derived from animalsused as carrier themselves show antigenicity,time-dependent change, and an animal-to-animalfluctuation in the antigen-adsorbing activity. Al-though the above-mentioned methods have re-spective advantages with regard to reliability,reproducibility, ease of practice as a routinediagnostic test, and stability of reagents, nonehas been qualified as completely satisfactory forthe purpose.We have developed microcapsules (MC) of

synthetic polymer as carriers for antigen in thepassive MC agglutination test (MCA-LS test).

These MC have following features: (i) absenceof antigenic substance on the particle surfaceavoiding nonspecific reactions, (ii) chemical sta-bility, (iii) possibility of mass production with auniform quality, and (iv) possibility of modifica-tion on particle characteristics such as particlesize, specific gravity, and particle surface prop-erties according to the test purpose.The MC used in our study are similar to the

conventionally known plastic spheres in thesense that leptospira antigen is coupled to theexternal surface of the spheres and that thereaction takes place on the surface of spheres.However, although it is generally not easy tomodify the particle size or chemical compositionin the case of plastic spheres, the MC providethe possibility of selecting an optimum wallmaterial for the antigen or antibody to be cou-pled and an optimum particle size for testing.

Also, the MC structure is positively utilized inimproving the performance as the carrier for thereaction in two aspects. One is the possibility ofentrapping a coloring dye in the interior of theMC, thus arbitrarily adjusting the contrast ofagglutination pattern and improving the accura-cy of judgement, without affecting the antigen-antibody reaction taking place on the externalsurface of the MC. The other is the possibility ofobtaining a suitable specific gravity for the parti-cles. In the case of MC this can be achieved bychanging the mixing ratio of internal materials(diisopropylnaphthalene and chlorinated paraf-fin), thus reducing the time required for theformation of agglutination pattern and improvingthe sensitivity.The purposes of this study were to try the MC

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836 ARIMITSU ET AL.

as a carrier instead of erythrocytes in the passiveagglutination test for detecting leptospira anti-bodies and to evaluate the applicability of thisnew method as a simple serological screeningtest in comparison with the standard MA test.

MATERIALS AND METHODSBacterial strains. Serovars and strains of leptospirae

used in this study were: icterohaemorrhagiae RGA,copenhageni Shibaura, autumnalis Akiyami A, hebdo-madis Hebdomadis, australis Akiyami C, canicola H.Utrecht IV, and pyrogenes Salinem. The virulentShibaura strain of serovar copenhageni maintained inguinea pigs was used (1, 2). Other strains were culti-vated in Korthof medium containing 10% rabbit serumat 32°C.

Sera. Hyperimmunized rabbit antisera were pre-pared by the method described previously (2). Anti-sera were obtained from two rabbits intraperitoneallyimmunized with living Shibaura strain of serovar co-penhageni. Animals were bled first daily up to day 9and later at 2-week intervals. Thirteen paired serawere collected from Weil's disease patients in ourlaboratory, including some paired sera from patientswith pyrexia and during convalescence. Also, twoseries of sera collected from Weil's disease patientsalong the course of illness were obtained from hospi-tals. Ten paired sera from Kawasaki disease patientswere made available by Kawasaki of the Japanese RedCross Medical Center. Five sera from infectiousmononucleosis patients, pooled anti-Borrelia duttoniimouse serum, and anti-Brucella abortus rabbit serumwere used for the experiment. Twenty sera fromsyphilis patients employed in the study were thosepositive for Ogata (complement fixation test withcardiolipin antigen) (3), Treponema pallidum antigen(20), and fluorescent treponemal antibody (8) tests.One hundred twenty sera from healthy individualsnegative for the serological test for syphilis were alsoemployed.

Preparation of leptospira antigens. Samples of a 4- to7-day-old leptospira culture grown in Korthof mediumwere centrifuged at 9,000 rpm for 30 min. The precipi-tate was washed once with saline (0.85% NaCl) andthen suspended in 1/10 the original volume of phos-phate-buffered saline (PBS), pH 7.2. The materialswere disrupted with a sonicator (Ohtake Works Co.,Ltd.; model 5202) at 20 kHz for 10 min and stored at4°C after the addition of 0.1% sodium azide.

In the case of mixed antigens, all strains used in thetest were sonicated in the same manner as for thesingle antigen and then adjusted to the same opticaldensity at 280 nm and mixed together for use assensitizing antigens.

Preparation ofMC particles. For preparation of MCparticles (10), 2.5 g of urea, 0.25 g of resorcin, and 0.3g of ammonium chloride were dissolved under agita-tion in 25 g of a 10%o aqueous solution of maleicanhydridemethylvinylether copolymer (Gantrez-AN139; molecular weight, ca. 25,000; GAF Corp.), andthe mixture was adjusted to pH 4.0 with a 20%aqueous solution of sodium hydroxide. Then a mixtureof 11.8 g of diisopropylnaphthalene (Kureha ChemicalIndustry Co., Ltd), 13.2 g of chlorinated paraffin(chlorine content, ca. 50%; Toyo Soda ManufacturingCo., Ltd), and 0.1 g of a oil-soluble red dye (Aizen

Spiron Red; Hodogaya Chemical Co., Ltd.) was emul-sified in the resulting solution to obtain an oil-in-wateremulsion; the agitation was terminated at an averagedrop size of ca. 7 ,um. After the addition of 25 g ofwater and 6.7 g of 37% formaldehyde solution, theemulsion was heated at 65°C, at which temperature thereaction was conducted for 2 h. The MC (averageparticle size, ca. 7 ,um; specific gravity, ca. 1.10) wereused in the study after washing three times with PBSto eliminate Formalin and protective colloids remain-ing on the MC.

Before the experiment for sensitization, we con-ducted a test to investigate the performance of MCused as a carrier for passive agglutination tests. Weinvestigated polyurea and polyurethane as the wallmembrane; we selected polyurea because of its superi-or stability, although the ability to couple antigen wasapproximately same for both materials. The MC,ranging 1.00 to 1.25 in specific gravity and 3 to 30 ,umin average particle size, were prepared by suitablyselecting the composition of core material (mixtureratio of diisopropylnapthalene and chlorinated paraf-fin) and drop size in the emulsification. An averagesize of 7 ,um proved to be most appropriate for theMC, in view of sensitivity in the passive agglutinationtest by the microtiter method.Also it was shown that MC containing a dye in the

core would allow adjustment of the pattern contrast inthis passive agglutination test without affecting thesurface property of the wall, thus improving the accu-racy of the test. Comparison of blue, red, and colorlessMC showed that MC containing red dye in the corewere easiest for pattern judgement. The presence ofdye in the MC did not show any adverse effect on theantigen-antibody reaction at the surface of the MC.Considering the above results, we used polyurea mem-brane MC of specific gravity 1.10, an average particlesize of 7 p.m, and containing red oil.

Preparation of sensitized MC antigen. MC werewashed twice with saline, suspended in PBS to aconcentration of 1.5%, mixed with the same volume of0.25% glutaraldehyde at 37°C for 1 h, washed twicewith saline, and suspended in twice the volume ofPBS. The suspension was mixed with an equal volumeof single antigen or mixed antigens of the optimalconcentration, incubated for 1 h in a 37°C water bathunder agitation, and allowed to stand overnight in arefrigerator. This sensitized MC suspension waswashed twice with 0.2% glycine-saline by centrifugingat 3,000 rpm for 10 min and then suspended to theoriginal volume in 3% bovine serum albumin-PBS.MCA-LS test. The MCA-LS test was conducted by

the microtitration method with disposable V-typeplates. A solution of 1% bovine serum albumin-PBSwas used for all serum dilutions. The serum specimenswere serially twofold diluted with a 0.025-ml microdi-luter, and the suspension of sensitized MC antigen wasdelivered to each serum dilution with a 0.025-mldropper. The mixtures were shaken well and keptovernight at 5°C, and the agglutination patterns of thebottom were observed on the next day. The titer of thespecimen was expressed as the reciprocal of thehighest serum dilution showing a definite positiveMCA-LS pattern. Normal rabbit serum and humanhealthy serum were used as control.MA test. The agglutinin titers were estimated by a

modification of the Schuffner-Mochtar MA method

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LEPTOSPIROSIS DIAGNOSIS BY MCA-LS TEST 837

TABLE 1. CF activity of the antigens separated by ultracentrifugationCF titera

Antigens Untreated Ultracentrifugation at:

20,000 x g 40,000 x g 80,000 x g 160,000 x gUntreated control 768 (4.43)Supernatant 256 (1.37) 128 (1.30) 64 (1.31) 32 (1.18)Pellet 512 (2.52) 512 (2.46) 512 (2.50) 512 (2.40)

a Expressed as the reciprocal of the highest antigen dilution showing no hemolysis. The optical density at 280nm of each antigen used is shown within parentheses.

(15), in which the sera were serially twofold dilutedwith a microtitrating diluter. The reciprocal of thedilution of the sera showing about 50% agglutinationwas taken as the MA antibody titer.Complement fixation test. The activity of comple-

ment was determined by a modification of Mayer'smicrotitration method (11). The antigen was treated bysonic oscillation at 20 kHz for 40 min and separated byultracentrifugation at 20,000, 40,000, 80,000 and160,000 x g. The optical densities at 280 nm of thesupernatants and pellets are shown in Table 1. Theantigen preparations were serially twofold diluted witha 0.025-ml diluter. A 0.025-ml sample of 5 50% hemo-lytic complement units of complement and 0.025 ml ofa 10,000-fold dilution of anti-RGA serum with a titer of12,800 in the MA test were delivered to each sampledilution. After the mixture was kept overnight at 4°C,0.025 ml of the sensitized sheep erythrocyte suspen-sion (5 x 108 cells per ml) was added, and the mixturewas incubated at 37°C for 30 min. Titers were recordedafter standing at 4°C for 2 to 3 h. The complementfixation titer was expressed as the reciprocal of thehighest dilution of the antigen preparations showing nohemolysis.

RESULTS AND DISCUSSIONRGA strain, serovar icterohaemorragiae, was

treated by sonic oscillation at 20 kHz for 5, 10,20, and 40 min. The antigenicity of sensitizedMC was little affected by the sonicating time(Fig. 1), and a 10-min sonication seemed toprovide practically sufficient destruction, al-though the optical density of antigenic solutionat 280 nm decreased with a longer sonic treat-ment. The optimum antigen density for MCsensitization giving a stable and reproducibleagglutination pattern was found to be in therange of 0.2 to 0.4 unit of optical density at 280nm. An antigen concentration higher than 0.5unit of optical density resulted in a nonspecificreaction.The antigen (10 ml), treated by sonic oscilla-

tion at 20 kHz for 40 min, was separated byultracentrifugation at 20,000, 40,000, 80,000, and160,000 x g for 60 min (fixed angle rotor 65, L-4centrifuge, Spinco; Beckman Instruments, Inc.),and the resulting supernatant and pellet fractionswere respectively used for MC sensitization andthe complement fixation test. The pellets were

suspended in 10 ml of PBS by sonic oscillation.However, the sum of the optical density valuesof the supernatant and the pellet obtained fromeach centrifugation was found to be slightlylower than the optical density value of theuntreated control antigen; this difference may beconsidered as the result of the aggregation ofantigenic substances in the course of centrifuga-tion. The optical density at 280 nm of the super-natants and pellets are shown in Table 1. ForMC sensitization, each fraction was diluted to anoptical density of 0.30 at 280 nm. The pelletfractions showed no significant difference inactivity for MC sensitization, but the superna-tant fractions showed a decrease in the sensitiz-ing activity with the increase in centrifugationspeed, and the MC sensitized with the 160,000 xg fraction did not respond even to the antiserumat a 200-fold dilution (Fig. 2).On the other hand, the antigenicity of each

fraction was checked by the CF test. The pelletfractions showed no difference in the CF antigenactivity. The supernatant fractions showed atendency of decrease in the CF activity at highercentrifugation speed, but retained considerableactivity even at 160,000 x g (Table 1). Theresults suggest that the antigen of a certain size

5 1,200-' 25,600-2 12,800D 6,400-a, 3,200*Z 1,600--n 800-8 400.2 200z 100

0

EC:00.5 00

0.4 .,

0-8-0.2 X.10.1 a

5 10 20 40 (min)Sonication time 20KHz

FIG. 1. MC-sensitizing activity of sonicated anti-gens. Symbols: 0, antibody titers of rabbit anti-RGAserum tested by MCA-LS with MC sensitized withantigen ofRGA strain sonicated under different condi-tions; 0, optical density (280 nm) after the treatment.

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838 ARIMITSU ET AL.

C,)

e' 51,200E 25,600> 12,800-0 6,400a) 3,200*- 1,600X 800o 400c- 200a) 200>

A *- * *

0--

0

Untreated 20 40 80 160controlU Itracentrifugation ( 1 03X °)

FIG. 2. MC sensitizing activity of the antigens sep-arated by ultracentrifugation. Antibody titers of rabbitanti-RGA serum were tested by MCA-LS with MCsensitized with the supernatants and pellets of RGAstrain samples separated under different conditions.Symbols: 0, supernatant; 0, pellet; and A, untreatedcontrol. Each fraction was diluted to an optical density(280 nm) of 0.30 at MC sensitization.

is more easily linked to the membrane surface ofMC made from polyurea used in this experimentthan is the soluble antigen.The sonicated antigens of typical strains for

five serovars (icterohaemorrhagiae, autumna-lis, hebdomadis, australis, and canicola) were

used to sensitize MC, and cross-reactivity in theMCA-LS test was studied in comparison withthat of the MA test (Table 2). Although there is aslight difference in cross-reactivity depending on

the serovar used, the MCA-LS test is more

cross-reactive and genus specific than the MAtest, but it is considered to be more type specificthan the HA test reported previously by severalworkers (4, 9). Such data suggest that the mixedantigen method can be used as a serodiagnosticscreening test for diseases caused by varioustypes of leptospira.

In an experiment with rabbit antiserum, theMCA-LS test with MC sensitized with the poolof three antigens (autumnalis, hebdomadis, andpyrogenes) showed the same titer of 12,800 toeach corresponding homologous antiserum,which is the same level of sensitivity experi-enced with the single antigen. This MCA-LS testalso gave 1,600- to 3,200-fold antibody titer toheterologous antisera. These results showedthat an MCA-LS reagent with three mixed anti-gens could detect antibodies induced by threestrains in the same manner as the single antigenand also was sensitive to heterologous antisera(Table 3).This finding was also confirmed in a test with

patients' sera in which the MCA-LS test withthree mixed antigens gave satisfactory antibodytiters to heterologous Weil's disease sera in

TABLE 2. Antibody titers of rabbit antileptospira sera in MCA-LS and MA tests

Antibody titer'

MCA-LS test MA testAntiserum (immunized

with serovar) ictero- ictero- altum- hebdo- austra- can-haemor- nalis madis lis cola hemor- nalis madis lis colarhagiae rhagiae

icterohaemorrhagiae 12,800 3,200 1,600 3,200 6,400 6,400 200 <200 <200 1,600autumnalis 800 12,800 1,600 800 800 <200 6,400 <200 <200 <200hebdomadis 1,600 1,600 12,800 1,600 1,600 <200 <200 12,800 <200 <200australis 800 1,600 3,200 12,800 1,600 <200 <200 <200 12,800 <200canicola 3,200 1,600 800 3,200 12,800 1,600 <200 <200 <200 6,400

a Expressed as the reciprocal of the endpoint titer.

TABLE 3. Results of MCA-LS test with single and three mixed antigens on rabbit antileptospira sera

Antibody titer' from MCA-LS test with:Antiserum (immunized Single antigen

with serovar) MixedantigensSautumnalis hebdomadis pyrogenes

icterohaemorrhagiae 3,200 3,200 1,600 3,200autumnalis 12,800 12,800 1,600 1,600hebdomadis 12,800 1,600 12,800 800australis 3,200 1,600 3,200 800canicola 1,600 1,600 800 1,600pyrogenes 12,800 3,200 800 12,800Normal rabbit serum <40 <40 <40 <40

a Expressed as the reciprocal of the endpoint titer.b The three antigens were from serovars autumnalis, hebdomadis, and pyrogenes.

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LEPTOSPIROSIS DIAGNOSIS BY MCA-LS TEST 839

TABLE 4. Results of MCA-LS test with three mixed antigens on sera from human leptospirosis patientsAntibody titersa

Patient Specimen MCA-LS test with MA testmixed antigensb autumnalis hebdomadis pyrogenes icterohaemorrhagiae

1 1 20 <20 <20 <20 <202 640 <20 <20 80 <20

2 1 20 <20 <20 <20 <202 160 <20 <20 80 <20

3 1 40 <20 <20 <20 <202 320 <20 <20 80 <20

4 1 20 <20 <20 <20 <202 160 <20 <20 80 <20

5 1 2,560 160 <20 <20 <202 2,560 320 <20 <20 <20

6 1 2,560 320 <20 <20 <207 1 160 <20 40 <20 <20

2 1,280 <20 160 <20 <208 1 1,280 <20 160 <20 <209 1 640 <20 160 <20 <2010 1 20 <20 <20 <20 <20

2 320 <20 <20 <20 4011 1 320 <20 <20 <20 40

2 640 <20 <20 <20 8012 i 320 <20 <20 <20 80

2 320 <20 <20 <20 8013 1 160 <20 <20 <20 40

2 320 <20 <20 <20 80

a Expressed as the reciprocal of the endpoint titer.b The three antigens were from serovars autumnalis,

addition to homologous patients sera (Table 4).The immunological response of two rabbits to

live virulent Shibaura strain was tested by theMCA-LS and MA tests (Fig. 3). The resultsshowed good correlation between the MCA-LSand MA tests.The immunological response of two sera from

Weil's disease patients collected along the

hebdomadis, and pyrogenes.

course of illness was tested (Fig. 4). The MCA-LS test gave a higher sensitivity by 8- to 16-foldin antibody titer than the MA test, but showedgood correlation of rise and fall of antibody titer.Sulzer et al. (18, 19) stated that the HA testappeared to be much more sensitive than theMA test to the serum samples in the early stageof illness and pointed out the probable dominant

0 1 2 3 4 5 6 7 8 9 14 28 42 56 70

Injection Days after infectionInjection

FIG. 3. Immunological response of two rabbit sera collected at different times after infection in MCA-LS(rabbits A [0] and B [-]) and MA (rabbits A [0] and B [EU) tests.

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840 ARIMITSU ET AL.

110 3 50

Days of illness70 90 110

FIG. 4. Immunological response of two Weil's disease patient sera collected along the course of illness inMCA-LS (patients A [@] and B [U]) and MA (patients A [0] and B [El]) tests.

role of immunoglobulin M antibody in the HAreaction based on a fact that HA-positive serawere nonreactive when treated with 2-mercap-toethanol. Also in our results (Fig. 4), the MCA-LS test appeared to have a somewhat highersensitivity to early serum antibodies, of whichthe titers were distinctly decreased by treatmentwith 2-mercaptoethanol (data not shown). Ittherefore seems that the MCA-LS test showsresults similar to those of Sulzer et al.The paired sera of 13 Weil's disease patients

were also tested, and the MCA-LS gave a 4- to32-fold higher sensitivity in antibody titer thanthe MA test (Table 5). The correlation betweenthe MCA-LS and the MA tests was reasonablysatisfactory.To determine the existence of nonspecific

reactions in MCA-LS test, various infectiousdisease sera, consisting of 10 paired sera ofKawasaki disease, 5 sera of infectious mononu-cleosis patients, anti-Borrelia duttonii mouseserum, and anti-Brucella abortus rabbit serum,were checked. Twenty sera from syphilis pa-tients and 120 sera from healthy individuals werealso tested. The greater part of such leptospiro-sis-negative sera resulted in 20- to 40-fold titersin the MCA-LS test, whereas a few sera gave 80-fold titers, and there were no abnormal reactionssuch as the nonspecific reaction with the MC.The borderline between positive and negativeappears to lie at about a 40- to 80-fold titer; if so,the results from the 13 Weil's disease patients(Table 5) suggest that the MCA-LS test revealeda positive reaction earlier than the MA test. Thismay lead to obtaining with the MCA-LS test a

meaningful diagnosis from a single serum sample

from patients suspected of having leptospirosis,thus dispensing with the necessity of alwaysusing paired sera. The determination of the

TABLE 5. Antibody titers of 13 paired sera fromWeil's disease patients in MCA-LS and MA tests

Antibody titerPatient Day after onset

MCA-LS testa MA test

1 5 2,560 8013 5,120 320

2 12 2,560 32019 2,560 320

3 7 80 2061 2,560 1,280

4 16 640 4020 640 160

5 15 1,280 8029 1,280 160

6 30 1,280 1,28031 1,280 640

7 17 1,280 16032 1,280 320

8 8 1,280 32018 1,280 320

9 8 1,280 8022 2,560 160

10 9 5,120 16025 5,120 160

11 11 1,280 64028 1,280 640

12 6 640 4060 1,280 80

13 7 1,280 8017 5,120 640

a The MCA-LS test was done with antigen fromserovar icterohaemorrhagiae.

10,240-

5,120-

2,560-

1,280-

._ 640-

320-8a 160-a)

80-

40-

20-

10-

I

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LEPTOSPIROSIS DIAGNOSIS BY MCA-LS TEST 841

borderline, however, has to be based on theresults of more samples.The same antibody titer was obtained with

two lots of antigen in tests done over a 12-monthperiod. Therefore, the sensitized MC antigensare considered to be stable for at least 1 yearwhen stored at 4 to 8°C.A factor which may affect the results in the

practice of the MCA-LS test is the stability ofthe sensitized MC antigen to pH, but the pH ofthe diluent within the studied range of pH 4 to 10did not affect the test. These results suggest thatMC are chemically stable for a prolonged periodand firmly sensitized by antigen and that thesensitized MC antigen is stable to a wide rangeof specimen conditions.Taking advantage of the features of MC made

of synthetic polymer, the MCA-LS test withsonically disrupted antigen of leptospira hasbeen developed for the diagnosis of leptospiro-sis. A good correlation between the MCA-LSand the MA tests was obtained with sera fromthe Weil's disease patients. The test has theadvantage of simplicity, rapidity, and stabilityand appears to be applicable as a routine serodi-agnostic test sensitive enough to detect antibod-ies even in an early stage of leptospirosis wherethe conventional MA test is unable to show ameaningful result. Such high sensitivity in anearly stage of disease seems very important fromthe diagnostic point of view, since this mayeliminate the use of paired sera as required indiagnosis with the conventional MA test.

ACKNOWLEDGMENTSWe are grateful to Mitsutaka Sofue for his helpful advice

during preparation of the manuscript.This work was supported by grants-in-aid for Scientific

Research, the Ministry of Education, Science and Culture.

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2. Arimitsu, Y., M. Mori, and K. Akama. 1980. Crossantigenicities of leptospira interogans serovar copenha-geni Shibaura strain for preparing biological products inJapan. Jpn. J. Med. Biol. Sci. 33:223-229.

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16. Stoenner, H. G., and E. Davis. 1967. Further observationson leptospiral plate antigens. Am. J. Vet. Res. 28:259-266.

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20. Tomizawa, T., S. Kasamatsu, and S. Yamaya. 1969. Use-fulness of the hemagglutination test using Treponemapallidum antigen (TPHA) for the serodiagnosis of syphilis.Jpn. J. Med. Biol. Sci. 22:341-350.

21. Torten, M., E. Shenberg, and J. Van der Hoeden. 1966.The use of immunofluorescence in the diagnosis of humanleptospirosis by a genus-specific antigen. J. Infect. Dis.116:537-543.

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