Development of a simple serological method for diagnosing leptospirosis: a microcapsule agglutination test

0095-1137/82/050835-07$02.00/0

Development of

a

Simple

Serological

Method for

Diagnosing

Leptospirosis:

a

Microcapsule Agglutination

Test

YOSHIKOARIMITSU,* SHINZOKOBAYASHI, KIYOTO AKAMA,ANDTYOKUMATUHASI

TheSecond Departmentof Bacteriology, NationalInstituteofHealth,Kamiosaki2-10-35, Shinagawa-ku,

Tokyo

141,

Japan

Received3March1981/Accepted 29 December 1981

A

passive

microcapsule agglutination

testfor the

diagnosis

of

leptospirosis

was

developed

by utilizing

chemically stable

microcapsules

instead

of

sheep

erythro-cytes. In the test,

sonically

disrupted antigens

of

leptospira

were sensitized to

microcapsules treated

with

glutaraldehyde. Compared

with the

microscopic

agglutination

test, the

passive

microcapsule

agglutination

test

showed

a

relatively

genus-specific tendency and

a 4- to

32-fold-higher sensitivity.

The sensitized

microcapsule

antigens

were

stable

for

atleast 1 year. The

microcapsules coupled

with mixed

antigens

can be

used

as a

serodiagnostic

screening

test

for diseases

caused

by various

types

of

leptospira.

The

test,

which

is very

simple

and

reproducible and

requiring

no

specific training,

can

be

employed

easily

as a

routine

test in

diagnostic

laboratories.

For the

serological

diagnosis of leptospirosis,

the

microscopic

agglutination (MA)

test

(15) is

most

widely

employed

as

the

standard reference

test

because

of its

high specificity and

sensitiv-ity. However, it

requires multiple

serovars

of

live

leptospira,

involving the risk of infection

and maintenance of

a

large number

of stock

cultures

to

provide

antigens.

These

factors

limit

its

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 a

latex

agglutination

test

(12),

performing

serovar-specific

reactions, have been reported in

screen-ing human

serum

specimens.

Also,

highly

genus-specific reactions have been

investigated

as

screening

tests,

such

as

the

complement

fixation

test

(13,

14, 17), sensitized erythrocyte

lysis

test

(5, 6),

passive hemagglutination

(HA)

test

(4, 9,

19), and the

immunofluorescence

test (21). In

particular,

the HA test is

reported

as

highly

sensitive

and

satisfactory

as a

screening

test(18,

19), but

the

erythrocytes derived from animals

used

as

carrier

themselves show

antigenicity,

time-dependent change,

and an

animal-to-animal

fluctuation

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 routine

diagnostic

test, and

stability of

reagents, none has

been

qualified

ascompletely

satisfactory for

thepurpose.

We have

developed microcapsules (MC)

of

synthetic polymer

as

carriers

for

antigen

in the

passive MC agglutination

test

(MCA-LS test).

These MC

have

following

features: (i) absence of

antigenic substance

on the particle surface

avoiding nonspecific

reactions, (ii)

chemical

sta-bility,

(iii) possibility of

mass

production

with a

uniform

quality, and (iv)

possibility of

modifica-tion

on

particle characteristics such

as

particle

size,

specific gravity,

and

particle surface

prop-erties

according

to

the

testpurpose.

The MC used in our

study

are similar to the

conventionally

known

plastic spheres

in the sense

that

leptospira

antigen

is

coupled

to the external

surface of the

spheres and

that the

reaction

takes

place

on the

surface of spheres.

However, although it

is

generally

not easy to

modify the

particle size

or

chemical

composition

in

the

case

of plastic spheres, the MC provide

the

possibility of

selecting

an

optimum wall

material

for the

antigen

or

antibody

to

be

cou-pled and

an

optimum particle size for testing.

Also, the

MC

structure

is

positively utilized in

improving the performance

as

the

carrier for the

reaction in

twoaspects.

One is the

possibility of

entrapping

a

coloring dye in the interior of the

MC, thus

arbitrarily adjusting the

contrast

of

agglutination pattern and

improving

the

accura-cy

of

judgement,

without

affecting

the

antigen-antibody reaction taking place

on the external

surface of

the MC. The other

is

the

possibility of

obtaining

a

suitable

specific gravity for

the

parti-cles. In the case

of MC this

canbe

achieved by

changing

the

mixing ratio

of

internal materials

(diisopropylnaphthalene

and

chlorinated

paraf-fin),

thus

reducing

the

time

required for

the

formation of agglutination

pattern

and

improving

the

sensitivity.

Thepurposes

of

thisstudy were to try the MC

835

on February 7, 2020 by guest

http://jcm.asm.org/

asacarrier instead of

erythrocytes

in the passive

agglutination

test for detecting leptospira

anti-bodies

and to evaluate the

applicability of

this new

method

as a

simple

serological

screening

test

in

comparison with the standard MA

test.

MATERIALS AND METHODS

Bacterial strains.Serovars and strains of leptospirae

used in this study were: icterohaemorrhagiae RGA,

copenhageni Shibaura, autumnalisAkiyami A,

hebdo-madisHebdomadis,australis Akiyami C, canicola H.

Utrecht IV, and pyrogenes Salinem. The virulent

Shibaura strain of serovar copenhageni maintained in

guineapigs was used (1, 2). Other strains were

culti-vated inKorthof medium containing 10% rabbit serum

at32°C.

Sera. Hyperimmunized rabbit antisera were

pre-pared by the method described previously (2).

Anti-sera wereobtained from two rabbitsintraperitoneally

immunized with living Shibaura strain of serovar

co-penhageni. Animals were bled firstdailyup today 9

and later at 2-week intervals. Thirteen paired sera

were collected from Weil's disease patients in our

laboratory, including some paired sera frompatients

with pyrexia and during convalescence. Also, two

series of sera collected from Weil's disease patients

alongthe course of illnesswereobtained from

hospi-tals. Ten paired serafrom Kawasaki disease patients

were madeavailable byKawasaki of the Japanese Red

Cross Medical Center. Five sera from infectious

mononucleosis patients, pooled anti-Borrelia duttonii

mouseserum, and anti-Brucella abortus rabbit serum

were used for the experiment. Twenty sera from

syphilis patients employed in the study were those

positive for Ogata (complement fixation test with

cardiolipin antigen) (3), Treponema pallidum antigen

(20), and fluorescent treponemal antibody (8) tests.

One hundred twenty sera from healthy individuals

negativefor theserologicaltestforsyphilis werealso

employed.

Preparation ofleptospira antigens.Samplesofa4-to

7-day-oldleptospira culture grown in Korthof medium

werecentrifuged at 9,000 rpm for 30 min. The

precipi-tatewaswashed once with saline (0.85% NaCl) and

then suspended in 1/10 theoriginal volume of

phos-phate-buffered saline (PBS), pH 7.2. The materials

weredisrupted withasonicator (Ohtake WorksCo.,

Ltd.; model 5202)at20kHz for10min and storedat

4°C aftertheaddition of0.1% sodium azide.

In thecase of mixedantigens, allstrains used inthe

test were sonicated in the same manner as for the

single antigenand then adjusted tothe same optical density at 280 nm and mixed together for use as

sensitizingantigens.

PreparationofMCparticles. Forpreparationof MC

particles (10), 2.5 g of urea, 0.25 g ofresorcin,and0.3

g of ammonium chloride weredissolved under

agita-tion in 25 g of a

10%o

aqueous solution of maleic

anhydridemethylvinylether copolymer (Gantrez-AN

139; molecularweight, ca. 25,000; GAF Corp.), and

the mixture was adjusted to pH 4.0 with a 20%

aqueoussolution of sodiumhydroxide.Thenamixture

of11.8gofdiisopropylnaphthalene(KurehaChemical

Industry Co., Ltd), 13.2 g of chlorinated paraffin

(chlorinecontent,ca. 50%; ToyoSodaManufacturing

Co., Ltd), and 0.1 g ofaoil-soluble red dye

(Aizen

Spiron Red; Hodogaya Chemical Co., Ltd.) was emul-sified in the resulting solution to obtain an oil-in-water

emulsion; the agitation was terminated at an average

drop size ofca. 7 ,um. After the addition of 25 g of

water and 6.7 g of 37% formaldehyde solution, the

emulsion was heated at 65°C, at which temperature the reaction was conducted for 2 h. The MC (average

particle size, ca. 7 ,um;specificgravity, ca. 1.10) were

used in the study after washing three times with PBS

toeliminate Formalin and protective colloids

remain-ingontheMC.

Before the experiment for sensitization, we

con-ducted a test to investigate the performance of MC

used as acarrier for passive agglutination tests. We

investigated polyurea and polyurethane as the wall

membrane; we selected polyureabecause of its

superi-orstability,although the ability to couple antigen was

approximately same for both materials. The MC, ranging 1.00 to 1.25 in specific gravity and 3 to 30 ,um

in average particle size, were prepared by suitably

selecting the composition of core material (mixture

ratioofdiisopropylnapthalene and chlorinated

paraf-fin) and drop size in the emulsification. An average

size of 7 ,um proved to be most appropriate for the

MC, in viewof sensitivity in thepassiveagglutination

testby the microtiter method.

Also it was shown that MC containing a dye in the

corewould allowadjustment of the pattern contrast in

this passive agglutination test without affecting the

surface property of thewall, thusimproving the

accu-racyof the test. Comparison of blue, red, and colorless

MC showed that MC containing reddye in the core

were easiest forpattern judgement. The presence of

dye in the MC didnotshow any adverse effect on the

antigen-antibody reaction atthe surface of the MC.

Considering the aboveresults,weusedpolyurea

mem-brane MC of specificgravity1.10,anaverageparticle

size of 7 p.m, andcontainingred oil.

Preparation of sensitized MC antigen. MC were

washed twice with saline, suspended in PBS to a

concentration of1.5%,mixed withthesamevolumeof

0.25% glutaraldehyde at37°C for 1 h, washed twice

with saline, and suspended in twice the volume of

PBS. Thesuspensionwasmixed withanequalvolume

of single antigen or mixed antigens of the optimal

concentration, incubated for1 h ina37°Cwaterbath

underagitation, and allowed to standovernightin a

refrigerator. This sensitized MC suspension was

washed twice with0.2%glycine-salinebycentrifuging

at 3,000 rpm for10 min and then suspended to the

originalvolume in3% bovineserumalbumin-PBS.

MCA-LStest.TheMCA-LStest wasconductedby

the microtitration method with disposable V-type

plates. Asolution of 1% bovine serumalbumin-PBS

was usedfor allserumdilutions. Theserumspecimens

wereserially twofold diluted witha0.025-ml

microdi-luter, and thesuspensionofsensitized MCantigenwas

delivered to each serum dilution with a 0.025-ml

dropper. The mixtures were shaken well and kept overnightat5°C,and theagglutination patternsof the

bottomwereobservedonthenextday.The titerof the

specimen was expressed as the reciprocal of the

highest serum dilution showing a definite positive

MCA-LS pattern. Normal rabbit serum and human

healthyserum wereusedascontrol.

MAtest. Theagglutinin titers wereestimatedbya

modification of the Schuffner-Mochtar MA method

J.CLIN.MICROBIOL.

on February 7, 2020 by guest

http://jcm.asm.org/

LEPTOSPIROSIS DIAGNOSIS BY MCA-LS TEST 837

TABLE 1. CFactivityof theantigens separated byultracentrifugation

CFtitera

Antigens Untreated Ultracentrifugationat:

20,000xg 40,000xg 80,000x g 160,000xg

Untreated 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)

aExpressedasthereciprocalof thehighestantigendilutionshowingnohemolysis.The

optical

density

at280

nmof eachantigenused is shown withinparentheses.

(15), inwhich the sera were serially twofold diluted

with a microtitrating diluter. The reciprocal of the

dilution of the serashowing about50%agglutination

wastaken as the MAantibodytiter.

Complement fixation test. The

activity

of

comple-ment was determined by a modification of Mayer's

microtitrationmethod(11).Theantigenwastreatedby

sonic oscillationat20 kHzfor 40 min andseparated by

ultracentrifugation at 20,000, 40,000, 80,000 and

160,000 x g. The optical densities at280nm of the

supernatants and pellets are shown in Table 1. The

antigenpreparationswereseriallytwofolddiluted with

a0.025-mldiluter. A 0.025-mlsampleof 550%

hemo-lytic complement units ofcomplementand 0.025 ml of

a10,000-folddilution of anti-RGAserumwithatiterof

12,800 in the MAtestweredeliveredtoeachsample

dilution. After the mixturewaskeptovernightat4°C,

0.025ml of the sensitized sheep erythrocyte

suspen-sion(5x 108cells perml)wasadded,and the mixture

wasincubated at 37°C for 30 min. Titers were recorded

after standing at4°C for 2 to 3 h. The complement

fixation titer was expressed as the reciprocal of the

highestdilution of theantigenpreparationsshowingno

hemolysis.

RESULTS

AND

DISCUSSION

RGA

strain,

serovar

icterohaemorragiae,

was

treated

by

sonic

oscillation

at

20

kHz for 5,

10,

20, and 40 min. The

antigenicity

of sensitized

MC

was

little affected

by

the

sonicating

time

(Fig.

1), and

a

10-min

sonication

seemed

to

provide

practically

sufficient destruction,

al-though the optical density of antigenic solution

at

280

nm

decreased with

a

longer sonic

treat-ment.

The

optimum antigen density for MC

sensitization

giving

a

stable

and

reproducible

agglutination

pattern

was

found

to

be

in

the range

of

0.2 to 0.4

unit

of

optical density

at 280 nm. An

antigen concentration higher

than 0.5

unit of

optical

density

resulted in a

nonspecific

reaction.

The

antigen

(10

ml), treated

by sonic

oscilla-tion

at 20 kHz

for

40 min, was

separated

by

ultracentrifugation

at

20,000, 40,000, 80,000,

and

160,000

x g

for

60min

(fixed

angle rotor 65, L-4

centrifuge, Spinco; Beckman Instruments,

Inc.),

and the

resulting

supernatant

and pellet

fractions

were

respectively used for

MC

sensitization

and

the

complement

fixation

test. The

pellets

were

suspended

in 10 ml of PBS

by

sonic

oscillation.

However,

thesumof the

optical density

values ofthe

supernatant

andthe

pellet obtained

from

each

centrifugation

was

found

to

be

slightly

lower

than the

optical density

value of the untreated control

antigen;

this

difference

maybe considered as the result ofthe

aggregation

of

antigenic

substances

in thecourseof

centrifuga-tion. The

optical density

at280nmof the

super-natants and

pellets

are

shown

in Table 1. For

MC

sensitization,

each

fraction

was

diluted

toan

optical density

of 0.30 at 280 nm. The

pellet

fractions showed

no

significant difference

in

activity

for MC

sensitization,

but the

superna-tant

fractions showed

adecrease in the

sensitiz-ing activity

with the

increase

in

centrifugation

speed,

and theMC sensitized with the 160,000x

g

fraction

did not respond even to the

antiserum

at a

200-fold dilution

(Fig.

2).

On the other hand, the

antigenicity

of each

fraction

was

checked

by

the CF test. The

pellet

fractions showed

no

difference

inthe

CF

antigen

activity.

The supernatant

fractions

showed a

tendency

ofdecrease in the CF

activity

at

higher

centrifugation speed, but retained

considerable

activity even at

160,000

x g

(Table

1). The

results

suggestthat the antigen of a certain

size

5

1,200-'

25,600-2 12,800

D

6,400-a,

3,200

*Z

1,600--n

800-8 400

.2

200

z 100

0

E C: 0

0.500

0.4 .,

0-8

-0.2 X .1 0.1 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-RGA

serum tested by MCA-LS with MC sensitized with

antigen of RGA strain sonicated under different condi-tions; 0, optical density (280 nm) after the treatment.

VOL. 15,1982

on February 7, 2020 by guest

http://jcm.asm.org/

C,)

e'

51,200

E25,600

>12,800

-0 6,400

a) 3,200

*- 1,600

X 800

o 400

c- 200

a) 200>

A *- * *

0--0

Untreated 20 40 80 160

control

UItracentrif ugation (1 03X °) FIG. 2. MC sensitizing activityof theantigens sep-aratedbyultracentrifugation. Antibody titers ofrabbit anti-RGA serum were tested by MCA-LS with MC sensitized with the supernatants and pellets ofRGA strain samples separated under different conditions. Symbols: 0, supernatant; 0,pellet;andA,untreated

control. Each fractionwasdilutedtoanoptical density (280 nm) of 0.30atMC sensitization.

ismoreeasily linkedtothe membrane surfaceof MCmadefrompolyureausedin thisexperiment thanis thesoluble antigen.

The sonicated antigens of typical strains for five serovars

(icterohaemorrhagiae,

autumna-lis, hebdomadis, austraautumna-lis, and canicola) were

usedtosensitize MC, andcross-reactivity in the MCA-LS test was studied in comparison with thatof the MAtest(Table 2). Although there isa

slightdifference in cross-reactivity dependingon the serovar used, the MCA-LS test is more

cross-reactive

and genus specific than the MA

test,butit is consideredtobemoretypespecific thanthe HAtestreportedpreviously by several workers(4, 9). Such datasuggestthatthemixed antigen method canbe usedas a

serodiagnostic

screening test for diseases caused by various

types ofleptospira.

In an experiment with rabbit antiserum, the MCA-LS testwith MC sensitized with the pool of threeantigens (autumnalis, hebdomadis, and pyrogenes) showed the same titer of 12,800 to

each

corresponding

homologous antiserum, which is the same level of sensitivity experi-enced with thesingle antigen. This MCA-LStest

also gave 1,600- to 3,200-fold antibody titer to

heterologous antisera. These results showed thatanMCA-LSreagentwith three mixed

anti-gens could detect antibodies induced by three

strains inthesame manner asthe single antigen and alsowas sensitive to heterologous antisera (Table 3).

This findingwasalso confirmed inatestwith

patients' sera in which the MCA-LS test with

three mixedantigensgave satisfactoryantibody titers to heterologous Weil's disease sera in

TABLE 2. Antibody titers of rabbitantileptospira serainMCA-LS and MAtests Antibodytiter'

MCA-LStest MA test

Antiserum(immunized

with serovar) ictero- ictero- altum- hebdo- austra-

can-haemor- nalis madis lis cola hemor- nalis madis lis cola

rhagiae rhagiae

icterohaemorrhagiae 12,800 3,200 1,600 3,200 6,400 6,400 200 <200 <200 1,600

autumnalis 800 12,800 1,600 800 800 <200 6,400 <200 <200 <200

hebdomadis 1,600 1,600 12,800 1,600 1,600 <200 <200 12,800 <200 <200

australis 800 1,600 3,200 12,800 1,600 <200 <200 <200 12,800 <200

canicola 3,200 1,600 800 3,200 12,800 1,600 <200 <200 <200 6,400

a Expressedasthereciprocal of the endpoint titer.

TABLE 3. Results ofMCA-LStestwith singleand three mixedantigensonrabbitantileptospira sera

Antibody titer' fromMCA-LStestwith:

Antiserum(immunized Single antigen

with serovar) Mixed

antigensS

autumnalis hebdomadis pyrogenes

icterohaemorrhagiae 3,200 3,200 1,600 3,200

autumnalis 12,800 12,800 1,600 1,600

hebdomadis 12,800 1,600 12,800 800

australis 3,200 1,600 3,200 800

canicola 1,600 1,600 800 1,600

pyrogenes 12,800 3,200 800 12,800

Normal rabbitserum <40 <40 <40 <40

aExpressedasthereciprocalof theendpointtiter.

bThe threeantigenswerefromserovarsautumnalis, hebdomadis,andpyrogenes.

on February 7, 2020 by guest

http://jcm.asm.org/

839

TABLE 4. ResultsofMCA-LStestwith three mixedantigenson serafrom humanleptospirosispatients

Antibodytitersa

Patient Specimen MCA-LS test with MAtest

mixedantigensb autumnalis hebdomadis pyrogenes icterohaemorrhagiae

1 1 20 <20 <20 <20 <20

2 640 <20 <20 80 <20

2 1 20 <20 <20 <20 <20

2 160 <20 <20 80 <20

3 1 40 <20 <20 <20 <20

2 320 <20 <20 80 <20

4 1 20 <20 <20 <20 <20

2 160 <20 <20 80 <20

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

2 2,560 320 <20 <20 <20

6 1 2,560 320 <20 <20 <20

7 1 160 <20 40 <20 <20

2 1,280 <20 160 <20 <20

8 1 1,280 <20 160 <20 <20

9 1 640 <20 160 <20 <20

10 1 20 <20 <20 <20 <20

2 320 <20 <20 <20 40

11 1 320 <20 <20 <20 40

2 640 <20 <20 <20 80

12 i 320 <20 <20 <20 80

2 320 <20 <20 <20 80

13 1 160 <20 <20 <20 40

2 320 <20 <20 <20 80

a

Expressed

asthereciprocal of the endpoint titer.

bThe threeantigenswerefromserovarsautumnalis,

addition

to

homologous

patients

sera

(Table

4).

The

immunological

response

of

two

rabbits

to

live virulent Shibaura strain

was tested by the MCA-LS and MA tests

(Fig. 3).

The

results

showed

good

correlation

between the MCA-LS

and 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

testgave a

higher sensitivity

by

8- to

16-fold

in

antibody

titer

than

the MA test, but

showed

good correlation of rise and fall of

antibody titer.

Sulzer

et

al.

(18,

19)

stated that the HA

test

appeared

to

be much

more

sensitive than the

MA test to

the

serum

samples in the early

stage

of

illness

and

pointed

outthe

probable dominant

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

Injection Days after infection

Injection

FIG. 3. Immunological responseoftworabbit seracollectedat different times after infection in MCA-LS

(rabbitsA[0]andB[-])andMA(rabbitsA[0]and B

[EU)

tests.

VOL.15,1982

on February 7, 2020 by guest

http://jcm.asm.org/

110 3 50

Daysof illness

70 90 110

FIG. 4. Immunologicalresponseoftwo Weil's diseasepatient seracollected alongthecourseof illness in

MCA-LS(patientsA[@] and B [U]) andMA(patientsA [0] and B[El])tests.

role of immunoglobulin M antibody in the HA reaction based on a fact that HA-positive sera

were nonreactive when treated with

2-mercap-toethanol.Also inourresults(Fig. 4), the

MCA-LS test appeared to have a somewhat higher

sensitivity to early serum antibodies, of which

the titersweredistinctly decreased bytreatment

with

2-mercaptoethanol

(data not shown). It therefore seems that the MCA-LS test shows results similarto thoseof Sulzeretal.

The paired seraof 13 Weil's disease patients werealso tested, and the MCA-LSgave a4-to

32-foldhigher sensitivity in antibody titer than the MAtest(Table 5). The correlation between theMCA-LS and the MAtests wasreasonably

satisfactory.

To determine the existence of nonspecific reactions in MCA-LS test, various infectious disease sera, consisting of 10 paired sera of

Kawasaki disease, 5seraof infectious

mononu-cleosis patients, anti-Borrelia duttonii mouse

serum, and anti-Brucella abortus rabbit serum,

were checked. Twenty sera from syphilis pa-tients and120serafromhealthy individualswere

also tested. The greater partof such leptospiro-sis-negative seraresulted in 20-to40-foldtiters

in theMCA-LStest,whereasafewseragave

80-foldtiters, andtherewere noabnormalreactions

such as the nonspecific reaction with the MC. The borderline between positive and negative

appearstolieatabouta40-to80-foldtiter;ifso,

the results from the 13 Weil's disease patients (Table 5)suggestthat theMCA-LStestrevealed apositive reaction earlierthan the MAtest.This may lead toobtainingwith the MCA-LS testa meaningful diagnosisfromasingleserumsample

from

patients suspected of having leptospirosis,

thus

dispensing with the necessity of always

using paired

sera.

The

determination of

the

TABLE 5. Antibody titers of 13pairedserafrom

Weil's disease patients inMCA-LS and MAtests

Antibody titer Patient Dayafter onset

MCA-LStesta MA test

1 5 2,560 80

13 5,120 320

2 12 2,560 320

19 2,560 320

3 7 80 20

61 2,560 1,280

4 16 640 40

20 640 160

5 15 1,280 80

29 1,280 160

6 30 1,280 1,280

31 1,280 640

7 17 1,280 160

32 1,280 320

8 8 1,280 320

18 1,280 320

9 8 1,280 80

22 2,560 160

10 9 5,120 160

25 5,120 160

11 11 1,280 640

28 1,280 640

12 6 640 40

60 1,280 80

13 7 1,280 80

17 5,120 640

aThe MCA-LS test was done with antigen from

serovaricterohaemorrhagiae.

10,240-

5,120- 2,560-

1,280-._

640-

320-8

a

160-a)

80-

40-

20-

10-I

on February 7, 2020 by guest

http://jcm.asm.org/

borderline, however, has

to

be based

on

the

results

of

more

samples.

The same

antibody titer

was

obtained

with two

lots of

antigen in

tests

done

over a

12-month

period.

Therefore, the sensitized MC antigens

are

considered

to

be

stable for

at

least

1 year whenstored at 4 to

8°C.

A

factor

which

may

affect the results

in

the

practice

of the MCA-LS

test

is the

stability of

the

sensitized MC

antigen

to

pH, but the

pH of

the

diluent within the studied

range

of pH

4 to10

did

not

affect the

test.These

results

suggest

that

MC

are

chemically

stable

for

a

prolonged

period

and

firmly sensitized by antigen and

that the

sensitized

MC

antigen is stable

to a

wide

range

of specimen

conditions.

Taking advantage of

the

features

of MC

made

of

synthetic polymer, the MCA-LS

test

with

sonically

disrupted antigen of

leptospira

has

been

developed for the

diagnosis of

leptospiro-sis.

A

good

correlation between

the

MCA-LS

and the

MA tests was

obtained with

sera

from

the

Weil's disease

patients. The

test

has

the

advantage of

simplicity, rapidity, and stability

and appears to

be applicable

as a

routine

serodi-agnostic

test

sensitive enough

to

detect

antibod-ies

even

in

an

early

stage

of leptospirosis

where

the

conventional

MA test

is unable

to

show

a

meaningful

result.

Such high

sensitivity in

an

early

stage

of disease

seemsvery

important from

the

diagnostic

point of view, since this

may

eliminate the

use

of

paired

sera as

required in

diagnosis with

the

conventional

MA test.

ACKNOWLEDGMENTS

Wearegrateful to MitsutakaSofue for his helpful advice duringpreparation of themanuscript.

This work was supported by grants-in-aid for Scientific Research, theMinistry ofEducation, Science and Culture.

LITERATURE CITED

1. Akama,K., S. Otani, M. Mori, and Y. Arinmitsu. 1975. Studiesonthe potency testof antiserum for Weil's disease by theintracutaneous method. Jpn. J. Med. Biol. Sci. 28:1-9.

2. Arimitsu, Y., M. Mori, and K. Akama. 1980. Cross antigenicities of leptospira interogans serovar copenha-geni Shibaurastrainforpreparing biological products in Japan.Jpn.J. Med. Biol. Sci. 33:223-229.

3. Beutner, E.H., T.P. Cherzelski, S. F. Bean, and R. E. Jordon. 1973. Immunopathology of the skin labeled anti-bodystudies, part 3. New contributions to syphilis serolo-gy andtreponemal antibody tests from Japan. Dowden, Hutchinson andLoss, Inc.,Stroudsburg, Pa.

4. Chang, R. S., and D. E. McComb. 1954. Erythrocyte sensitizing substance from five strains of leptospirae. Am. J.Trop. Med. Hyg.3:481-489.

5.Chang, R. S., D. J. W. Smith, D. E. McComb, C. F. Sharp, and J. I. Tonge. 1957. The use of erythrocyte sensitizing substance in thediagnosis of lysistest.Am. J. Trop. Med. Hyg. 6:101-107.

6.Cox, D. D. 1955. Hemolysis of sheeperythrocytes sensi-tized with leptospiral extracts. Proc. Soc. Exp. Biol. Med. 90:610-615.

7. Galton,M.M.,D. K.Powers,A.D.Hale,andR.Cornell. 1958. A rapid macroscopic-slide screening test for the serodiagnosis of leptospirosis. Am. J. Vet. Res. 19:505-512.

8. Hunter, E. F., W. E. Deacon, and P. E. Meyer.1964. An improved FTA test forsyphilis, theabsorption procedure (FTA-ABS). Public Health Rep. 79:410-412.

9. Imamura, S., H.Matsui, and Y. Ahizawa. 1972. Studies on indirect hemagglutination test for leptospirosis. Jpn. J. Exp. Med. 42:563-568.

10. Kondo, A.1979.Microcapsule processing and technology. MarcelDekker, Inc., New York.

11. Mayer, M. M. 1961. Complement and complement fixa-tion, p. 133. In E. A. Kabat and M. M. Mayer (ed.), Experimental immunochemistry, 2nd ed. Charles C. Thomas,Publisher, Springfield, Ill.

12. Muraschi, T. F. 1958. Latex-leptospiral agglutination test. Proc.Soc. Exp. Biol. Med. 99:235-238.

13. Randall, R., P. W. Wetmore, and A. R. Warner. 1949. Sonic vibratedleptospirae asantigens in the complement fixation test for the diagnosis ofleptospirosis. J. Lab. Clin. Med. 34:1411-1415.

14. Rothstein, N., and F. Wolman. 1959. Studies of the immunochemistry of leptospires. Serologic characteriza-tion of the complement-fixacharacteriza-tion antigen. J. Infect. Dis. 105:280-287.

15. Schikffner, W., and A. Mochtar. 1927. Versuche zur Aufteilung von Leptospiren-Stammen, mit einleitenden Bemerkungenuber VerlautvonAgglutinationundLysis. Zentralbl.Bakteriol. Parasitenkd.Infektionskr.Hyg. Abt.

1Orig.101:405-413.

16. Stoenner, H. G., and E.Davis.1967.Furtherobservations

on leptospiral plate antigens. Am. J. Vet. Res. 28:259-266.

17. Sturdza, N.,M.Elian, and G. Tulpan. 1960. Diagnosis of humanleptospirosisby the complement fixationtestwith

a single antigen. Arch. Roum. Pathol. Exp. Microbiol. 19:561-582.

18. Sulzer, C. R., J.W.Glosser, F. Rogers,W. L.Jones, and M. Frix.1975.Evaluation of anindirect hemagglutination test for the diagnosis of human leptospirosis. J. Clin. Microbiol. 2:218-221.

19.Sulzer, C. R., and W. L. Jones. 1973. Evaluation of a hemagglutinationtestfor humanleptospirosis.Appl. Mi-crobiol. 26:655-657.

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

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

VOL. 15,1982

on February 7, 2020 by guest

http://jcm.asm.org/