Monoclonal antibodies against hepatitis A virus

0095-1137/83/111237-07$02.00/0

Copyright © 1983, AmericanSocietyforMicrobiology

Monoclonal Antibodies

Against

Hepatitis

A

Virus

ANDREW

MAcGREGOR,l*

MYKOLA

KORNITSCHUK,'

JOHNG.R. HURRELL,1 NOREEN 1.

LEHMANN,2

ANTHONY G.

COULEPIS,2

STEPHEN A. LOCARNINI, AND IAN D.

GUST2

ImmunochemistryDepartment, Research andDev,elopment Division, Commonwealth Serum Laboratories,

Parkville, Victoria, 3052,Australia1; andVirology Department, Fairfield Hospital, Fairfield, Victoria,3078,

Australia2

Received3May1983/Accepted25July 1983

Three monoclonal antibodies

(K2-4F2, K3-2F2, and K3-4C8) of the

immuno-globulin G2a class were raised against hepatitis A virus. The specificity of these

antibodies was

confirmed

by

immune

electron

microscopy, solid-phase

radio-immunoassay, and in vitro neutralization in cell culture. Binding studies suggested

that

they

all

recognize

closely related

antigenic

determinants. These monoclonal

antibodies should prove to be

of

great value as

diagnostic

and research

reagents.

Hepatitis A virus (HAV) is a 27 to 32-nm

virus

(8)

whose

biophysical

and

biochemical

charac-teristics (5) have resulted in its classification as a

member

of the genus enterovirus (lOa) within

the

family

Picornaviridae

(2). HAV has proved

difficult to isolate in

vitro (22), and relatively

small

quantities

of virus have been recovered

from

the feces

of hospitalized

patients (4) and

experimentally

infected animals (21).

The

difficulty of

obtaining large quantities of

virus has

hampered the

production of

antisera

for

diagnostic

and research purposes. In this

paper we report

the

development and

character-ization of three monoclonal antibodies which are

specific

for

HAV, and which may be of value as

diagnostic reagents

and

research

tools.

MATERIALS ANDMETHODS

Viruspreparation. (i) Mouseinoculations. HAV for

mouseinoculationwasobtained fromafecal specimen

(HM-790) collected from a patient with naturally

ac-quired hepatitis in whom the clinical diagnosis of

hepatitisA was supported byliverfunction testsand

confirmedby thepresenceofspecific immunoglobulin

M (IgM) in the acute-phase sera (17). Virus was

identified by solid-phase radioimmunoassay (SPRIA)

andimmuneelectronmicroscopy (IEM) (19) andthen

purified by a process of differential centrifugation,

chloroform extraction, column chromatography with

agarosegelfiltration,andisopycnic ultracentrifugation

in cesium chloride as describedpreviously (3). During

purification, the presence of HAV was monitored by

SPRIA. The final identity of purified virus was as-sessedbydirect and IEM with human pre- and postin-fection hepatitis A sera (16), and no adventitious agents were isolated after routine viral culture (3).

HAV particle counts in this preparation exceeded

1,000per electronmicroscope400-meshgridsquare.

(ii) Reagent antigen. HAV as reagent antigen

com-prised four fecal preparations and three cell culture

isolates. The four fecal preparations were derived from differentpatientswithhepatitisAand werethree

individualspecimens (HM-947, HM-838,andHM-952)

andapool (pool A) of six fecal specimensknown to

contain HAV. The three cell culture isolates

(HM-790/7P, HM-165/1OP, and HM-172/5P) had been

pas-sagedinmonkeyembryonic kidney (MEK) (12),

Buf-falogreenmonkeykidney(BGM) (6),and fetal rhesus kidney (FRhK-4) (22) and wereatthe seventh, tenth,

and fifth passage, respectively. HAV extracted from

samples HM-952, pool A, HM-165/10P, and

HM-790/7Pwereusedfor IEM.

The reagentantigen frombothfecesand cell culture

waspurified bydifferential centrifugation and

chloro-formextraction (16).

Immunization. Fourfemale BALB/cmice,6weeks

of age, were obtained from and maintained at the

breeding colonyattheCommonwealthSerum

Labora-tories, Melbourne. The limited quantities of antigen

availablepermittedtheimmunization oftwomicewith

each preparation of HAV (Table 1). Retroorbital

bleedingswereobtainedfromthemicebyusing

tribro-moethanol administeredintraperitoneallyas an

anaes-thetic (25). Bleedings were done the day before the

primary inoculation (day

0)

andatdays15and 90after

the immunization schedule wasbegun. The

develop-mentofspecificanti-HAVinthemicewasmonitored

by SPRIAasdescribedbelow. Furtherintraperitoneal

doses ofHAV weregiven toanimalsthat responded

poorlytotheinitial immunization. Whenrequired for

fusion, the animal with the highest serum antibody

level at theprevious bleedingwasselected and boost-ed.

Cellhybridization. Spleencells werefused with

NS-1 cells(13) by methods similartothoseof Galfreet al. (10). Briefly, spleen cells were dispersed through a stainless steel screen (0.3-mm mesh), mixed with 107

NS-1 cells per spleen, and washed three times by

centrifugationwithDulbecco modifiedEagle

medium-high glucose (MA Bioproducts) containing 2.4 g of

NaHCO3per liter(DME).The cellpelletwas

suspend-ed in 30% (vol/vol) polyethylene glycol 1000 (Sigma

ChemicalCo.)in DME(pH7.0to7.2)at37°C, pelleted

after 3 min by low-speed centrifugation, and diluted with alargevolumeofDMEat7.5min.The fused cells

werecentrifugedagainand diluted ingrowth medium

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1238 ET AL.

TABLE 1. Immunization schedule

Mouse Primary dose(day1),intraperitoneal Secondary dose, intraperitoneal Boost given3 days before fusion, intravenous(via tailvein)

Kl 500-p inoculum consisting of None Day 34,

500-pul

inoculum

con-250 pul of HAV inPBS,pH sisting of 250 pAlof HAV in

7.2(5 p.g ofprotein), mixed PBS, pH7.2 (5pugof

pro-with 250

pR1

of Freund com- tein), mixed with 250

pI

of

pleteadjuvantab PBSab

K2 Inoculumas formouse Kl b None Day 70,inoculumasfor

mouse K1'

K3 500

pL.

inoculum

consisting

of None

Day

119,

400-p.l

inoculum

con-250

RI1

ofHAVin PBS,pH sistingof 200

p.l

of HAV in

7.2(5p.g ofprotein), mixed PBS, pH 7.2 (4

p.g

of

pro-with250 p.l of Freundcom- tein), mixedwith200 p.1 of

pleteadjuvantb' PBS"

K4 400-p.l inoculumconsisting of Day 179,600-p.l inoculumcon- Day201,

500-p.l

inoculum

con-200p.l ofHAVinPBS, pH sistingof 300

pL.

ofHAVin sisting of300

p.l

of HAV in

7.2(4 p.g of protein), mixed PBS,pH 7.2(6

p.g

ofprotein), PBS,pH 7.2 (6

p.g

of

pro-with 200p.l ofFreund com- mixed with 300

p.l

of Freund tein), mixed with200

pL.

of

plete adjuvantb' complete

adjuvantb.'

PBSa

a Micewereimmunized(primary doseorbooster)with apreparation ofHM-790containing approximately

1012

HAVparticlesperml.TheHAVhad beenpurified by differentialcentrifugation, chloroform extraction,agarose

gelfiltration, and isopycnic ultracentrifugation.

bInocula also contained 0.05%mouseserum.

' Mice receivedprimaryorsecondary doses(orboth) ofapreparation of HM-790containing approximately

1010

HAVparticles per ml.TheHAVhad beenpurified by the first three purificationstepsonly.

(DME containing 1 mM sodium pyruvate, 100 U of penicillinGperml, 100 p.gofstreptomycin sulfateper ml, 20 mM

N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acidbuffer, [pH 7.5], 1% 100x Eagle nones-sential amino acids solution[CSL,Australia] and 15% [vol/vol] fetal calfserum[CSL]). For initial plating, the growth mediumwassupplementedwith0.1mM hypo-xanthine(Calbiochem)and0.4 mMthymidine (Calbio-chem) (15).

Thecellsuspensionwasplatedoutin96-well tissue cultureplates(Costar). Every 24 h, 50% of the medium was replaced with growth medium containing hypo-xanthine and thymidine. From 48hafterfusion, 0.04 mM aminopterin (Sigma) wasalsoincluded. After14 days, aminopterinwasomittedfrom the freshgrowth medium; 7 days later, growthmediumalonewasused forsubsequent cell cultivation.

Hybridomas were visible from day 8 after fusion, andsupernatants wereassayed forantibody from day 11. Cells from wells positive for specific antibody production were cloned twice by limiting dilutionon 3T3 BALB/c feederlayers in 96-well plates (1).

Detection ofanti-HAVby SPRIA.Antibody activity in cellculture supernatants was detected by usinga modified SPRIA (14). Wells of polyvinyl microtiter plates (Cooke Engineering Inc.) were coated with a 1:1,000 dilution of humanconvalescent-phase hepati-tis Aserumcontaining anti-HAV (18) in 0.85% NaCl (saline) and 0.1% mouse serum for 4 hat 20°C. The wells were then washed with phosphate-buffered sa-line(PBS), pH 7.4, and incubated with HAV (pool A) overnightat4°C. Wellswerethenwashedthreetimes with PBS(pH 7.4) and drained. Test samples (50 p.1) wereinoculated into the wells andincubated for 4 hat

20°C.Fifty microliters of

1251I-labeled

human anti-HAV

serum wasthen addedtoeachwell,and theincubation

was continued overnightat4°C. The wells were then

washed threetimeswithPBS,cut outwith a hot wire,

and counted.Twopositive andtwonegative controls

wereincluded ineachassay.Theresult wasexpressed

aspercent countsbound([countsperminuteinsample

well/mean counts perminute in negative controls] x

100). A figure of 60% reduction in the binding of

labeled antibodies per well or less was taken as

positive forantibody activity.

Isotyping. The three monoclonal antibodies were

isotyped by immunodiffusion with isotype-specific

antisera(LittonBionetics) (20).

Monoclonal antibody production. Monoclonal

anti-bodies wereproduced both in ascites fluid andincell

culture. For ascites fluid

production,

BALB/c mice

older than 6 weeks wereinoculated intraperitoneally

with 0.5 mlof Pristane (AldrichChemicalCo.). After

10 to 14days, 5 x 105 hybridoma cells in 0.5 ml of

Dulbecco PBSwere inoculated

intraperitoneally

into each mouse.When abdominalswelling

occurred,

asci-tes fluid washarvesteddaily

using

a20-gauge needle.

EDTA(2.5mg/ml)and NaN3(0.2

mg/ml)

wereadded

to the pooled harvest. Cells and

precipitates

were

removed by centrifugation. Forcell culture

superna-tantproduction, hybridomacellsweregrown in 1-liter

volumes ofgrowth mediumfromaninitialdensity of2

x 104cells to a final density of2 x 10" cellsperml, and the supernatants were harvested

by

centrifuga-tion.

Purification of monoclonalantibodyfrom ascites

flu-id.Ascitesfluidpoolswereclarified

by

centrifugation

at12,000 xg for 20min and filtered

through

a0.2-p.m

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membrane. The filtrate was adjusted to pH 8.1 and passed through an 8-ml protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) column which had beenequilibrated with 0.1 M phosphate buffer, pH 8.1 (washing buffer). Washing bufferwaspassed through the column untiloptical densityat280nmof theeluate

was less than 0.05. The monoclonal antibody was eluted with 0.1 M phosphate buffer in 0.9% [wt/vol] NaCl (pH 3.0), and 1-ml fractions (optical density at

280 nmofgreaterthan0.1)were pooledand immedi-atelyadjusted topH 7 (7). Proteinconcentrationwas estimated by the biuret method and adjusted to 1 mg/ml.

lodination of antibody. Antibodieswereiodinatedby adding 100 ,ug of antibodyin 0.1ml ofPBS(pH 7.4)to

1.2,ug of lodogen(Pierce Chemical Co.). 1251(1 mCi; AmershamCorp., Amersham, UnitedKingdom)in 10 ,ul of distilledwaterwasaddedand reacted for 10min

atroomtemperature;0.2 ml of0.1%NaN3inPBSwas addedtostopthereaction,and free125Iwasseparated from the iodinatedantibody bypassage over a Sepha-dexG-25(Pharmacia) columnthathad been preequili-brated with PBS (9). The proportion of specific anti-body in iodinated preparations was estimated by incubating serialdilutions onHAV attached towells andmeasuringthepercentageofcounts boundatthe asymptoteinaplot againstdilution.

IEM.HAVwascentrifuged at55,000 xgfor 4hat

4°C in a SW60 rotor (Beckman Instruments, Inc.). Pellets were incubated with 100 ,u1 of cell culture supernatant containing eachmonoclonal antibodyfor

1 h at 34°C. After overnight incubation at 4°C the

samples were centrifuged for 2 h at 34,000 x g,

suspendedinapproximately30,ul of PBS, andstained with 3%phosphotungstic acid (pH 7.4). The prepara-tions were examined for immune complexes in a PhilipsEM301 electron microscopeataplate magnifi-cationof44,000.

Virus neutralization. Cell lysates of FRhK-4 cells infected with HAV isolate HM-165/1OPwerediluted in 10-fold stepsfrom 10-1 to10-5, and 0.1-ml volumes were incubated with 0.1-ml volumes of monoclonal antibody(1 mg/ml)at37°Cfor 2 handthen inoculated

onto monolayers of FRhK-4 cells in 25-cm2 plastic disposable flasks (Costar). Control flasks were inocu-latedwith 0.1-ml volumesof eachvirus dilutionwhich had previously been incubated with 0.1 ml of PBS. Foursetsof flasksweresetupand incubatedat37°C. Atweekly intervals cellsfromone setofflasks were strippedoffbyabriefexposure at37°C toa trypsin-versene solutionconsisting of 0.12% (wt/vol) trypsin (Difco Laboratories)and 0.2%(wt/vol) versene-EDTA in calcium- andmagnesium-free Hanks balanced salt

solution. The cells weredisrupted by three cycles of freeze-thawing, and the presenceof HAV was

moni-tored bySPRIA (14).

Specificity testing. Each monoclonal antibody was

tested for its ability to capture both cell cultureand fecally derived HAV by SPRIA.Ascitesfluid contain-ing each of the monoclonal antibodies, purified by protein A and diluted 1:1,000 togive a final protein concentration of 25,ug/ml, wasusedto coatthewells of polyvinyl microtiter plates (Cooke Engineering Inc.)for 4 hat20°C. The wellswerethenwashed with PBS (pH 7.4), and 50-,ul volumes of the different HAV preparationwereadded and incubated for 4 hat20°C. The wells were washed onceagain with PBS before the addition of 50 ,ul of 1251I-labeled humananti-HAV convalescent serum. Wells were incubatedovernight

at4°Cand washed withPBS,andcountsboundwere calculated. The humancoating antibodywas purified asdescribed previously (17).

Competitive bindingassay.The SPRIAfor the detec-tion of anti-HAV was carried out on serial 10-fold dilutions of eachmonoclonal antibody. However, in-steadof

125I-labeled

humananti-HAV,labeled mono-clonal antibodyK3-4C8wasaddedtothe wells asthe competingprobe. Wells were incubated overnightat

4°Candwashed withPBS,and thecountsboundwere calculated.Thecompetitive bindingassay was repeat-edwith 125I-labeled K2-4F2and

125I-labeled

K3-2F2.

RESULTS

Hybridomas. Spleens from four mice

(Kl

through K4)

were

used

in this

study (Table 2).

After fusion, of the 1,250

wells

seeded,

842

(67.4%) contained

hybridomas,

and of

these

only

4

(0.5%) prodticed

anti-HAV. One of the

four

hybridomas

lost its

ability

to

produce

anti-HAV

on

subsequent passaging.

The three

re-maining

parent

hybridoma

lines

(K2-4F2,

K3-2F2, and K3-4C8)

were

each

cloned twice

(K2-4F2-3G2-1D9, K3-2F2-2C7-1C8,

and

K3-4C8-1E8-3F6,

respectively)

to ensure

their

monoclonality.

Isotyping of

the three

antibodies

showed that

they

were all

of the

IgG2a class.

IEM.

Anti-HAV-containing

supernatants

from the

three

positive hybridoma cell lines

were

tested by

IEM. Supernatants from

hybri-domas that

were

negative by

SPRIAwere

used

as

negative controls. All three monoclonal

anti-bodies

produced

immune

complexes

with

HAV

TABLE 2. Hybridoma production

Developmentof anti-HAV No. of No. of No. ofhybridomas

Mouse onday: Day of wells wells with producing

anti-0 15 fusion seeded hybridomas HAV

Kl - + + 37 350 212 0

K2 - + 73 300 300 2a

K3 - - + + 122 300 300 2

K4 - - + 204 300 30 0

aOne

hybridoma

failedtomaintainantibody production.

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A

lOOnrrt-.

:.

j100nm

FIG. 1. (A) Immune electron

micrograph

ofHAV strain HM-925 with acell culture

supernatant

from a

hybridomalinenot

producing

anti-HAV.The

specimen

wasstainedwith3%

(wt/vol)

phosphotungstic

acid,

pH

7.4.Immunecomplexes and individual virus

particles

coatedwith

antibody

couldnotbe

visualized. (B)

Immune electron

micrograph

of HAV strain HM-925 with a cell culture supernatant from

hybridoma

K2-4F2.

The

specimenwasstainedwith3%

(wt/vol)

phosphotungstic

acid,

pH

7.4. Immune

complexes

were

commonly

seen

inthis

preparation.

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TABLE 3. Neutralization of HAV in cell culture

withmonoclonal antibodies

HAV- P/Nratio at weeka: Antibody 165/1OP

dilutions 1 2 3 4

Control 10- 0.9 2.4(+) 3.5(+) 4.4(+)

10-2 1.0 4.1(+) 4.3(+) 4.9(+)

10-3

1.3 1.8 4.0(+ 5.0(+

10-4

0.8 1.6 4.2()5.5(+

10-5

1.3 1.2 3.3(+) 4.3 (+)

K2-4F2 10- 1.1 1.2 2.0 2.9(+)

10-2 1.0 1.1 1.1 2.7 (+)

10-3 0.9 1.0 1.0 1.5

10-4

1.0 1.3 1.3 1.2

10-5

1.2 1.0 1.2 1.3

K3-2F2 10- 0.9 1.1 1.3 2.3

10-2 1.0 1.2 0.9 0.9

10-3 1.2 1.0 1.2 1.0

10-4

0.8 1.3 1.0 0.9

lo-5

1.0 0.9 0.9 0.9

K3-4C8 10- 1.0 1.0 1.1 2.7(+)

10-2

1.2 1.2 1.0 2.6(+)

10-3

0.9 0.9 1.2 2.3(+)

10-4 1.0 1.3 1.1 1.8

10-5

0.8 1.2 1.0 1.4

aValues are expressed as

positive/negative

(P/N)

ratios where P represents the counts per minute of

bound

125I-anti-HAV

human convalescent serum in

test wells and N represents the counts per minute

bound in the control wells. Ratiosof2.1 orgreaterare

regardedas

positive

for HAV.

strain

HM-952 which were not visualized on

reaction of HAV with control supernatants.

Fig-ure 1

shows

representative

results

of these

IEM

experiments

with monoclonal

antibody

K2-4F2.

Similar

findings were obtained with a number of

other HAV

preparations, including

HM-165/1OP, HM-790/7P,

and pool A.

Virus

neutralization. The

results of HAV

neu-tralization by

the

monoclonal antibodies are

shown

in Table 3.

HAV

was

not

detected in any

of the

dilutions of control

and test

flasks

after

the

first

week. At

week 2, HAV was only

detected

at

10-1

and 10-2

in the control flasks.

At

week 3, all the

control flasks were positive,

but

virus

was

still

undetectable in the test flasks.

However, at week 4 all three test flasks showed

signs

of HAV

activity. The monoclonal

antibod-ies appeared to

reduce infectivity rather than

completely

neutralize

the virus.

Specificity

testing. The

ability of

each of the

three

monoclonal antibodies

to recognize HAV

purified from

both

feces and cell culture is

shown

in

Table 4. In most cases the use of

monoclonal antibodies

resulted in

higher

posi-tive/negative ratios than those

obtained with

polyclonal

human

convalescent

sera.

The

reac-tivity of the

monoclonal antibodies K2-4F2 and

K3-4C8 appeared to be similar and generally

higher

than that of K3-2F2 for both the cell

culture and the fecally derived virus, except that

K3-4C8

failed

to

recognize

cell culture isolate

HM-175/5P.

Competitive binding assay. All three

monoclo-nal antibodies inhibited the

binding

of

125I-la-beled

K3-4C8. Similar results were obtained

with

125I-labeled K3-2F2

and K2-4F2.

The

kinet-ics of

binding

inhibition of K3-4C8 for all three

monoclonal

antibodies

(Fig.

2) suggest that

they

bind to

antigenically

similar or

sterically

related

determinants.

DISCUSSION

The

percentage

of hybridomas

recognized

as

producing

HAV-specific antibody

was

very

low,

being

less than

1%. This

was

probably due

to

the

screening method

used in

this

study, namely,

a

"blocking"-type

SPRIA with human

polyclonal

convalescent-phase

anti-HAV. This system

would miss

any

specifically reacting monoclonal

antibodies

that were not

effective

in

blocking

labeled

polyclonal

anti-HAV and

would select

for antibodies

that react

with the

major

surface

antigens of

HAV.

There can

be

little

doubt

regarding the

speci-ficity of

the three

monoclonal

antibodies for

TABLE 4. Ability of each of the three monoclonal

antibodiestobindHAVin anSPRIA

P/N ratio withthefollowingcoating

HAV antibodya:

Human K24F2 K3-2F2 K3-4C8

Fecal strains

PoolA 17.1 47.0 27.6 40.0

HM-947 7.5 25.4 20.0 23.8

HM-972 2.2 3.1 2.2 3.5

Cell culture iso-lates HM-790

(7thpassage)b 23.1 32.2 12.0 23.1

HM-165

(10th

passage)c

11.0 13.9 7.8 13.3

HM-172

(Sth

passage)d

3.7 5.3 2.4 1.2

aValues are expressed as positive/negative

(P/N)

ratios where P represents the counts per minute of

bound

125I-anti-HAV

human convalescent serum in

thetestwellsandNrepresents thecountsper minute

bound inthecontrolwells.Ratiosof 2.1 orgreaterare

regarded as positive for HAV. Negative controls

(PBS, completemedium, hybridoma

supernatant,

and

ascites) all had P/N values of less than2.1.

b Passagedinmonkey kidneycells(MEK) (6).

Passaged in buffalo green

monkey kidney

cells

(BGM) (12).

dPassaged in fetal Rhesuskidney cells (FRhK) (22).

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$100'

0

-C.)

2

W' 50-C)

"ft.

0 I..

10000

o?

K2-4F2

: K2.3F2

X K3-4C8

g,~~~~~

D~

1000 100 10 1

CONCENTRATION OF

ANTIBODY

(ng)/WELL

FIG. 2. Inhibition of

binding

of

1251I-labeled

K3-4C8 monoclonal

antibody by

each of the three monoclonal

antibodies.

HAV.

First, the virus used to produce the

antibodies was prepared by a method known to

produce material of high purity (3, 16), and mice

immunized with this material

produced

specific

anti-HAV.

Second,

all three

monoclonal

anti-bodies

produced immune complexes with HAV

by IEM.

Third,

the

antibodies recognized both

cell

culture-derived and fecally derived HAV

when used

in a

SPRIA test. Fourth, they

partial-ly neutralized

the

infectivity of

HAV

in

cell

culture.

The

inability of

the

monoclonal

antibod-ies

to

completely

neutralize

cell

culture-derived

HAV may

reflect

either the presence of virus

aggregates

before neutralization

(11) or

inade-quacies

of the neutralization

conditions used

in

this

study (23). The

suppression of

neutraliza-tion

by the presence

of

virus aggregates has

been

observed

with

other enteroviruses that were

grown in

cells of

monkey origin (12).

Monodis-persion

of viral

aggregates with an agent such as

deoxycholate before

neutralization may be

re-quired

before total

neutralization

can be

demon-strated (11). In

addition,

many enteroviruses

require

prolonged

incubations with antibody

(several

hours

at

room

temperature or

370C

followed

by

overnight

incubation at

4°C)

before

total

neutralization

can

be

achieved

(23).

The

competitive binding data of Fig. 2, in

which the

binding

of labeled monoclonal

anti-body

K3-4C8 to HAV in the SPRIA test was

blocked

by the other monoclonal antibodies,

suggest

that these antibodies recognize closely

related

(antigenically similar

or

sterically

associ-ated)

antigenic

determinants.

In

general, the monoclonal antibodies

yielded

higher

positive/negative ratios

when

reacted

with HAV than those observed

with

human

anti-HAV

used at

optimal

conditions. The

choice of

monoclonal

antibodies

and the

optimization

of

conditions

for their

use

could therefore

prove

valuable in

generating diagnostic

reagents that

would be

free of

the inherent

difficulties of

quality and supply

involved in the

use

of

human

convalescent

serum.

Other

possible

applications

for the

monoclo-nal

antibodies include

searching

for

antigenic

variants

of

HAV, differentiation of

wild-type

from

attenuated

strains, comparison

with future

monoclonal antibodies

against

cell culture

iso-lates

of

HAV,

purification

of cell

culture-derived

HAV

by

affinity

chromatography,

and

detection

of

HAV-specific antigen

producing

colonies of

DNA

recombinant

organisms

into

which

ele-ments

of the

HAV

genome have been cloned

(24).

ACKNOWLEDGMENTS

This work was supported by the Commonwealth Serum Laboratories, Melbourne, Australia, andbygrants from the National Health and Medical ResearchCouncilof Australia.

Weacknowledge theassistance ofLinda E. Smith,David A.Harrison, and ZhuangHui in cellhandling,JohnC.Cox for isotyping the monoclonal antibodies, Alan R. Coulter for assistance in iodination, Kathleen Gavin in preparation of materials,John Marshall for theIEM,and RobertPringle for helpful discussions.

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