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Copyright01974 American Society for Microbiology Printed inU.S.A.

Hepatitis B Core Antigen:

Immunology

and

Electron

Microscopy

LEWELLYSF.BARKER, JUNE D.ALMEIDA,JAY H. HOOFNAGLE,ROBERT J. GERETY,DANIEL R. JACKSON, AND PHILIP P. McGRATH

Divisionof Blood andBloodProducts, BureauofBiologics,Food andDrugAdministration, Bethesda,

Maryland,

and the WellcomeResearchLaboratories,

Beckenham,

Kent,

England

Received forpublication 20August 1974

Two distinct viral antigens are associated with the hepatitis B virus: the

hepatitis B surface antigen (HB8Ag, Australia antigen) and the hepatitis B core

antigen

(HB,Ag). HB.Ag,

purified from the serum of asymptomatic human

HB8Ag carriers, and

HBcAg,

purified from the liver of a chimpanzee acutely

infected with hepatitis B virus, were examined by serological and immune electron microscopic methods. Antisera raised against HB8Ag reacted with the outer, surfacecomponentof the Daneparticleandwith the 20-nmspherical and tubular particles present in HB8Ag-positive serum, but not with the internal componentof theDaneparticleorwithpurified

HBcAg

particles.Antiseraraised

against purified

HB,Ag

particles reacted with the internal component of the Dane particle and with

HBcAg,

butnotwiththesurfaceof the Daneparticleor

with the 20-nmspherical and tubular particlesassociatedwith

HB"Ag.

Purified

HBcAg

particles, 27 nm in diameter, demonstrated distinct subunits. The

infectious form of hepatitis B virus appears to be represented by the 42-nmDaneparticle composedofa27-nmnucleocapsidcorecomponent

(HBcAg)

surrounded byanantigenically andmorphologically distinct lipoprotein surface

component

(HB,Ag).

Evidence

from

several laboratories

has

dem-onstrated that the

hepatitis

B virus

(HBV)

possesses at least two separate

and distinct

antigens: the

hepatitis

B surface

antigen

(HB.Ag,

Australia antigen) and the

hepatitis

B core

antigen

(HB,Ag)

(1, 3, 6,

10).

HB8Ag,

the

lipoprotein

surface component of

HBV,

can

be

readily detected

by

a variety of

immunological

techniques

in

the

sera of

patients

acutely

or

chronically

infected with this virus

(4, 17).

HB,Ag,

the

nucleocapsid

component of

HBV,

is not

found

free in serum

but

is

found

in the nuclei of

hepatocytes

frompatients with type B

hepatitis

and can

also be extracted

from

HB8Ag-reactive

seracontaining Dane particles

by detergent

treatment (1, 11-13, 16, 18; R. H.

Purcell,

J. L.

Gerin,

J. D.

Almeida,

and P. V.

Holland,

Intervirology, in press). Both antigens are associated with distinctvirus-like structures visible on electronmicroscopy (1, 2, 11, 12, 18).

HB.Ag

inserum is associatedwith 20-nm

spher-ical

particles,

elongated

tubular

particles

(20-nm inwidth and several hundred nanometers in

length),

and with the outer, surface component of the

complex,

42-nm Dane particles (1, 7).

HBcAg

is associated with the inner, 27-nm,

electron-dense

core component of the Dane

particle

seen in HBsAg-positive serum and with

the

27-nm nucleocapsid found in the nuclei of

hepatocytes

of patientsinfected with HBV (1-3, 11, 12, 18).

This

report

describes

the purification and

characterization

ofnucleocapsid core particles from

the

liver of an experimentally infected

chimpanzee

(3; J. A.

Markenson,

R. J. Gerety,

J. H. Hoofnagle, and

L. F. Barker, J. Infect.

Dis.,

in press), and the demonstration by im-mune

electron

microscopy that these particles are

antigenically

identical to the inner, core component

(HB,Ag)

but

antigenically distinct from

the

outer, surface component

(HB8Ag)

of the Daneparticle.

MATERIALS AND METHODS

Serological testing. HB8Agwasdetectedby coun-terelectrophoresis (9) and by solid-phase radioim-munoassay (Ausria; Abbott Laboratories, North Chi-cago,Ill.) using theroomtemperature procedure (15). AntibodytoHB8Ag

(anti-HB.)

was assayed by coun-terelectrophoresis, passive hemagglutination (19), and solid-phase radioimmunoassay (Ausab; Abbott Laboratories [Reagents kindly provided by Abbott Laboratories.]). Selectedsampleswerealsotested for

anti-HB.

by radioimmunoprecipitation (14). HBcAg andantibodyto

HB,Ag

(anti-HB,)

weredetectedbya 1552

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further processing. Electron microscopyof liver tions revealed numerous 27-nm intranuclear core

particles. Because of this, attempts were made to purify core particles from this liver by differential centrifugation. Liver (80g) wassuspendedin320ml of hypotonic (0.45%) saline and homogenized in a Waring blender. The 20% (wt/vol) homogenate was clarified by centrifugationat2,500 rpm for30min in an International PR-2 centrifuge (1,100 x g). The supernatant fluidwascentrifugedat25,000 rpmfor 2 hina no.30 rotorina Beckman L-2ultracentrifuge (75,000 x g). Theresultingpelletwasresuspendedin 35 ml ofdistilled water, and thehigh-speed centrifu-gationwasrepeated. Thepelletwasthenresuspended

in 35 ml of distilled water and clarified again by centrifugation at 2,500 rpm for30min(1,100 x g).

Theresultingsupernatantwasusedastheantigen

in serological testing for both complement fixation and counterelectrophoresis to detect

anti-HB,.

To furtherpurify thissupernatant, itwaslayeredonto a continuousCsClgradient,density1.2to1.5g/ml.The gradient mixturewascentrifugedat22,000 rpm for16 h in an SW25.2rotor in a Beckman L-2 ultracentri-fuge (75,000 x g). Fractionswerecollecteddropwise from the bottom ofthetube in approximately 1-ml amounts. Thespecific gravity ofthe resultant frac-tionswas determined byusing anAmericanOptical refractometer. Each fractionwas assayedfor HB.Ag byradioimmunoassayandfor

HBcAg

by complement fixation and examined forvirus-like particles under the electronmicroscope. Fractionscontainingtypical

27-nm core particles were dialyzed for 1 to 3 days againstphosphate-buffered saline and usedto immu-nize animals and for immune electron microscopic studies.

HB.Ag

was purified from the plasma ofhuman

chronic

HB.Ag

carriersbyaseries ofisopycnic band-ings in CsCl and rate zonal ultracentrifugations in sucrose (5). Samples were

dialyzed

against

phos-phate-buffered salinepriorto use.

Immuneelectronmicroscopy.

Specimens

for elec-tron microscopy were prepared by dialyzing either purified

HB.Ag

or

HBcAg

againstphosphate-buffered saline. Specimens were then diluted with an equal quantity of phosphate-buffered saline, and 0.5-ml portions ofeither antigen

(HB.Ag

or

HB,Ag)

were

mixed with 0.02 ml of the antisera to be tested

(anti-HB.,

anti-HB,,

or normal control serum). For

studies of the Dane particles, purified

HB.Ag

was treated with Tween 80priortomixingwith antisera (1). Antisera were tested under code, identified by species only. The antigen-antibody mixtures were

allowedtoreactovernightat4C and thencentrifuged for1h at18,000 xg.Thesupernatantwas

discarded,

HB,Ag

(anti-HBc, anti-core) were prepared in guinea

pigs (10; R. J. Gerety, J. H. Hoofnagle, and L. F. Barker, J. Immunol., in press). Hartley guinea pigs weighing 200 to 400 g were inoculated subcutaneously with 0.2 ml of HB8Ag or

HB,Ag

(diluted 1:3 to 1:10in normal saline), emulsified in an equal volume of complete Freund adjuvant, and were boosted with a similar amount of antigen emulsified in incomplete Freund adjuvant at 14 days. The animals were bled by cardiac puncture at 21 days and at weekly intervals thereafter. In addition, antisera to both HB.Agand HBcAgwere prepared in rabbits andrhesusmonkeys using similar immunization schedules. Naturally oc-curring antibodies to

HB.Ag

and HBcAg were ob-tained from chimpanzees experimentally infected with HBV and from humans at varying stages of convalescence from type B hepatitis.

Anti-HB.

was obtained from achimpanzee (chimp 959) who devel-oped this antibody 5 weeks after exposure to infec-tious serum and inwhom

HB.Ag

was neverdetected (Markenson et al., J. Infect. Dis., in press).

Chimpan-zee

anti-HB,

was obtained from three animals

(chimps 23, 920, and 921) all in the immediate convalescent period of typical, experimentally in-duced type B hepatitis (after the disappearance of

HB.Ag

and prior to the appearance of

anti-HB.).

Human

anti-HB.

was obtained from a hemophiliac patient, and human anti-HBc wasobtained from a patient recently convalescent from type B hepatitis (1). Control sera consisted of prebleeds from uninocu-lated guinea pigs, rabbits, rhesus monkeys, and one chimpanzee (chimp 23). Human control serum con-sisted of plasma negative forHB.Ag,

anti-HB.,

and anti-HBc from a person with no known history or exposure to type Bhepatitis.

RESULTS

Electron

microscopic

examination of intact liver sections from the

chimpanzee

revealed many intranuclear core particles. Both the crude homogenate and pellet from

the

second

high-speed

ultracentrifugation

revealed 27-nm

HBcAg

particles and typical

HB.Ag

particles

(20-nm spheres and

tubules)

(Fig. 1).

Examina-tion of fractions from CsCl gradients revealed typical 27-nm

nucleocapsid

core particles in fractions at a

density

from1.30 to1.33

g/ml

and the typical

HB.Ag

particles in fractions at a

density

from 1.20 to 1.23 g/ml.

Figure

2 illus-trates the patterns of

reactivity

of

HB.Ag

(by

radioimmunoassay)

and

HB,Ag

(by comple-ment fixation) found in each fraction from a

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--FIG. 1. Pellet of second high-speed ultracentrifugation ofthe

chimpanzee

liver

homogenate.

Both

HB,Ag

particles (20-nm spheres andtubules) andHBAgparticles (27-nm

cores)

are seen. x140,000.

2 4 6 8 10 12 14 16

FRACTION NUMBER

FIG. 2. Patternof serological reactivity inCsCIgradientfractions ofthe materialshown inFig.1.HB,Ag,as

detectedbyradioimmunoassay (0),was seenatdensities(A) of1.20 to1.23g/ml,whereasHBeAg,asdetected

by complement fixation (0), was seenat densitiesof1.30to1.33g/ml.ElectronmicroscopyrevealedHB8Ag particles infractions12and 13andHBAg particlesinfractions 6 and 7ofthisgradient.

CsCl gradient.

HB,Ag

reactivity was seen in fractionswithdensities of from1.30to1.33g/ml andHB8Ag reactivity at densities of from 1.20 to 1.23g/ml. Thispattern wasfound repeatedly onsubsequent isopycnic bandings in CsCl.

Because of the high degree of purity ofthe

HBcAg

particles, itwaspossibletoobtain better

morphological characterization than has been

possible with previous serum-derived

prepara-tions. The great majority of particles were 27 nm in diameter. From the viewpoint of

sub-structure, the core particles displayed distinct

subunits (Fig. 3). Although subunits could be resolved, it was not possible to determine the exactnumber ofcapsomeresforming the capsid.

Insomeareas,particles showed

breakdown,

and

1554

E 1.30

E

z

0

1.20.

RIACPM

(HBSAg)

3000

2500

2000

1500

1000

500

0

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(4)

FIG. 3. PurifiedHBAg particlesseen at adensityof1.32 inaCsCIgradientshown inFig.2. Thepurity ofthe preparation and morphologyofthecoreparticles is shown. The average diameter

of

the

particles

was27nm, withoccasionalparticles only20 nmin diameter. x230,000. The insert showsasinglecoreparticleat a

higher

magnification (x350,000). This demonstrates the distinctive characteristicoftheHB¢Ag particle, the subunits whichcomprisetheparticle.Itwas notpossibleto establish theexactnumber

of

subunitspresent.

in

these

it

appeared

that

partially disrupted

core

particles coalesced

toform

highly aberrant

forms

(Fig.

4).

Results

of

serological testing

and

immune

electron microscopy on antisera from

guinea

pigs,

rabbits, rhesus monkeys, chimpanzees,

and humans

are

summarized

in

Table

1.

Immu-nization of guinea pigs with

HB,Ag

yielded

antisera

that contained

anti-HB,

by

comple-ment fixation

and

counterelectrophoresis

in

the

absence

of

detectable

anti-HB8

by passive

he-magglutination, radioimmunoassay, and

radi-oimmunoprecipitation. These antisera failed to

agglutinate

HB.Ag

particles

or attach to the surface component of

the

Dane particle

(HB.Ag),

but distinct

antibody attachment was

seen to

the

internal core component of

the

Dane

particle previously released by

Tween 80 treat-ment of

the

serum (1). Antisera raised in guinea

pigs against purified

HB8Ag

gave the opposite pattern of

reactivities. Although

a high titer of

anti-HB8

was found in these antisera, no

anti-HB,

was

detectable

by complement

fixation or

counterelectrophoresis. Furthermore, when

guinea pig

anti-HB8

was

reacted with purified,

disrupted

Dane

particles,

a

large

amount of

antibody

was seen

attached

tothe outersurface component of the Dane

particle

aswellas to

the

20-nm

spherical

and

tubular

particles,

butnone was seen

attached

to

the

inner, core component ofthe Dane

particle (Fig.

5).

Immune

electron

microscopy using

purified

core

particles

and

purified HBSAg

particles

confirmed these

findings.

Guinea

pig,

rabbit,

chimpanzee, and human

sera

positive

for

anti-HB,

by serological

tests

demonstrated both

agglutination

and

antibody

attachment to

puri-fied core

particles (Fig. 6). However, when these

anti-HB,

reactiveserawere

reacted

with

HB.Ag

purified

from serum, no

agglutination

or

anti-body attachment

was seen.

Conversely,

guinea

pig,

chimpanzee, and human

anti-HB.

did

not reactwith the

purified

HB,Ag

preparations,

but

demonstrated

agglutination

and

antibody

at-tachment

to

HBSAg

particles (Fig. 5). Control

sera

(prebleeds and human

serum

negative

for

antibodies

toHBV

antigens) showed

no reactiv-ity with either

HB8Ag

or

HBcAg

particles.

DISCUSSION

The

immune electron

microscopic and

sero-logical data presented

here reinforces

the

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[image:4.499.48.443.72.334.2]
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[image:5.499.67.449.80.286.2]

FIG. 4. Malformed HB^Agparticles. Occasionallygroupsof malformedandseemingly coalesced

particles

were seen. Theseparticles mayhave been altered anddisruptedbythepurification procedure. Alternatively, such particlesmay represent incompletely formedvirus. x238,000.

TABLE 1. Reactivityand titersofantiserawithHBAgand

HB8Ag

Antisera Antigensa

HBCAg HB,Ag

Specificity Species

CF CEP IEM PHA RIA IEM RIP

Anti-HBc GuineaPig 1:64 1:16 + - - _

Rabbit 1:32 1:8 + - - _

Rhesus 1:512 1:32 NT - - NT

Chimpanzee 23 1:256 1:16 + - -

-920 1:1,024 1:128 + - - - NT

921 1:1,024 1:128 + - - - NT

Human(Pugh) 1:512 1:64 + - - - NT

Anti-HB.

GuineaPig - - - 1:128,000 + +

Rabbit - - NT 1:64,000 + NT

Ihesus - - NT 1:8,192 + NT

Chimpanzee959 - - _ 1:64,000 + +

Human - - NT 1:256,000 + NT

Control GuineaPig _- -

-Rabbit - - NT _ - NT

Rhesus - - NT _ _ NT

Chimpanzee23(pre) - - _ _ _

Human - - NT NT

aAbbreviations:CF,complementfixation;CEP,counterelectrophoresis; IEM,immuneelectronmicroscopy; PHA, passivehemagglutination; RIA,radioimmunoassay; RIP, radioimmunoprecipitation; NT,nottested.

mulating

evidence

regarding

the existence of

two

independent

antigen-antibody

systems

as-sociated

with type B

hepatitis

(1, 3, 6, 10,

12).

HBcAg

particles

have been

purified

from the liver of a

chimpanzee

that died

during

acute viral

hepatitis,

type B. Immune electron

mi-croscopy using this purified

HBcAg

and deter-gent-treated, Dane-rich

HB.Ag-positive

prepa-rations

have

revealed the antigenic identity of the 27-nm intranuclear core particle with the internal component of the Dane particle. Immu-nization of a variety of laboratory animals with 1556

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[image:6.499.51.441.46.304.2]

4sikS: * : S ; Aj;w;

FIG. 5. Reactionseenwhenguinea pig antiserum against HBAg

(anti-HB,)

was mixed with a preparation of Dane particles disrupted by detergent treatment. The antibody agglutinated and attached to the surface component of the Dane particle and to the 20-nm spherical and tubular particles. Antibody was not seen attachedtothe intemal component of the disrupted Dane particles, and when this antiserum was mixed with purified HBAg no agglutination was seen, but the particles appeared dispersed and uncoated as shown in Fig. 3. x230,000.

FIG. 6. Reaction seen when chimpanzee antiserum against HBAg

(anti-HBc)

was mixed withpurified HB,Agparticles. Thepreparation shown inFig. 3wasreacted withchimpanzeeanti-HB8naturally

acquired

fromanHBVinfection. The particlesareagglutinated andareobscuredbyahaloofattachedantibody.Similar resultswereobtainedusing antiserum from animals immunized withHBAg. Thesameantisera hadnoeffect

on

HB,Ag particles.

x

158,000.

1557

[image:6.499.47.443.378.621.2]
(7)

BARKERET AL.

this

purified

HB,Ag

preparation

yielded

anti-sera

which

had

high

titers of

anti-HBc,

but

which

were negative for

anti-HB.

by

the

most

sensitive

techniques (14). Also

demonstrated

wasthe

ability

of

purified

HB.Ag

to

induce

high titers

of

anti-HB.

in the absence of

anti-HB,

detectable by complement

fixation,

coun-terelectrophoresis,

or immune

electron

micros-copy.

Finally,

the

antigenic duality shown

by

serological techniques

was

confirmed visually

by immune electron microscopy using

both

purified

HBcAg

and

purified, disrupted

serum-derived

Dane

particles.

The

purity

of

the

core

preparation

described

here has

allowed

for a better

morphological

description

of the core

particle.

Distinct

sub-units were

visualized, although

theexact

num-ber of capsomeres

forming

the

nucleocapsid

couldnot

be

determined.

The presenceof

sub-units

differentiates

this agent from

en-teroviruses and

rhinoviruses

which also

fall

in the 27-nmrangein

diameter.

Indeed,

the struc-ture, size, and

characteristics

of

HBV

appearto

place it in aviruscategoryof itsown.Failure of anti-HB8 and

anti-HBc

to react with the

re-centlydiscovered

hepatitis

Aantigen instools of patients acutely ill with viral

hepatitis,

typeA, makes it unlikely that the hepatitis A and B viruses are

immunologically

related (8). The recent

demonstration

of a specific DNA-dependent, DNA polymeraseintheheavycore

fractions from

detergent-disrupted

Dane parti-cles isstrongevidencethat HBV isaDNA virus

(13).

Immunological

and electron microscope data

now allows the description ofthe

physical

and

antigenic madeupofthehepatitisBvirus. The 42-nm Dane

particle

is most likely the intact, infectious form of the virus andismadeupofa

27-nm

nucleocapsid

core

(HBcAg)

surrounded

by a

lipoprotein

surface component

(HB.Ag).

The morenumerous20-nm

spherical

and tubu-lar structures seen in

HB8Ag-positive

serum

probably

representthe

lipoprotein

surface

anti-gen

(HB.Ag)

produced

by infected hepatocytes

far inexcessof what isnecessarytoencapsulate the number ofnucleocapsid forms. The exact role of

HBcAg

andanti-HBcinthecourseoftype B

hepatitis

isnotknown.

Hopefully,

the

discov-eryand

characterization

ofthissecond

antigen-antibodysystemin typeBhepatitis will

permit

aclearerunderstanding of this still uncontrolled

andsometimes fatal viral disease.

ACKNOWLEDGMENTS

Wethank Audrey J. Shawver and DavidE. Gilbertfor technical assistance and Robert Purcellfortestingselected

samples byradioimmunoprecipitation. LITERATURE CITED

1. Almeida, J. D.,D.Rubenstein,and E. J. Stott. 1971. New antigen-antibodysysteminAustralia-antigen-positive hepatitis. Lancet 2:1225-1227.

2. Almeida, J. D.,A. P. Waterson, J.M.Trowell, and G. Neale. 1970. The findingofvirus-likeparticlesintwo

Australia-antigen positive human livers. Microbios 2:145-153.

3. Barker, L. F., F. V. Chisari, P. P. McGrath, D. W. Dalgard, R. L. Kirschstein, J. D. Almeida, T. S. Edgington, D. G. Sharp, and M. R. Peterson. 1973. Transmission ofviral hepatitis, typeB, to

chimpan-zees.J.Infect. Dis.127:648-662.

4. Blumberg,B.S., A.I.Sutnick, and W. T. London. 1968.

Hepatitis and leukemia. Their relation to Australia antigen. Bull.N.Y.Acad.Med.44:1566-1586. 5. Bond, H. E., and W. T. Hall. 1972. Separation and

purification ofHepatitis-associated antigen into

mor-phologic typesby zonalultracentrifugation.J. Infect. Dis. 125:263-268.

6. Brzosko,W.J.,K. Madalinski,K.Krawczynski, and A. Nowoslawski. 1973. Dualityofhepatitis B antigen and itsantibody.I.Immunofluorescence studies.J. Infect. Dis.127:424-428.

7. Dane, D. S., C. H. Cameron, and M. Briggs. 1970. Virus-likeparticlesinserumofpatients with Australia-antigenassociated hepatitis. Lancet 1:695-698. 8. Feinstone, S. M., A. Z. Kapikian, and R. H. Purcell.

1973. Hepatitis A: detection byimmuneelectron

mi-croscopyofavirus-like antigen associated withacute

illness.Science 182:1026-1028.

9. Gocke, D. J., and C. Howe. 1970. Rapid detection of Australia antigen bycounterimmunoelectrophoresis.J. Immunol. 104:1031-1034.

10. Hoofnagle, J. H., R. J. Gerety, and L. F. Barker. 1973. Antibody to hepatitis-B-virus core in man. Lancet 2:869-873.

11. Huang,S.N. 1972.Hepatitis-associatedantigen hepati-tis. An electronmicroscopic study of virus-like particles inlivercells. Amer. J. Pathol. 64:453-470.

12. Huang, S.N., andV.Groh.1973. Astudyonantibodies

produced with liver tissue containing Australiaantigen andvirus-like particles. Lab.Invest.29:743-750. 13. Kaplan, P. M., R. L. Greenman, J. L. Gerin, R. H.

Purcell, andW.S. Robinson. 1973. DNA polymerase associatedwith human hepatitis B antigen. J. Virol. 12:995-1005.

14. Lander, J. J., H. J. Alter, and R. H. Purcell. 1971. Frequency of antibodytohepatitis-associatedantigen

asmeasured bya newradioimmunoassay technique. J. Immunol. 106:1166-1171.

15. Ling, C. M., and L. R. Overby. 1972. Prevalence of hepatitisBvirusantigenasrevealed by direct radioim-mune assay with 1125 antibody. J. Immunol.

109:834-841.

16. Lipman,M.B., W. J.Hierholzer,Jr.,and A. Schlueder-berg. 1973. Isolation ofcoresfrom hepatitis B Dane particles.J. Infect.Dis. 128:664-667.

17. Prince, A. M. 1968. An antigen detected in the blood duringtheincubationperiodofserumhepatitis.Proc. Nat.Acad.Sci.U.S.A. 60:814-821.

18. Stannard, L. M.,A.Polson,A.Kipps, andJ.R.Parker. 1973. Twoantigen-antibodysystemsinserum

hepati-tis:preparationofimmunoglobulins andtheir reaction with Australia antigen and particles in liver cell homogenates.Microbios 8:87-100.

19. Vyas,G.N.,and N. R.Shulman.1970.Hemagglutination

assay for antigen and antibody associatedwith viral hepatitis.Science170:332-333.

1558

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Figure

FIG. 1.particles Pellet of second high-speed ultracentrifugation of the chimpanzee liver homogenate
FIG. 3.preparationwithmagnificationwhich Purified HBAg particles seen at a density of 1.32 in a CsCI gradient shown in Fig
FIG. 4.weresuch Malformed HB^Ag particles. Occasionally groups of malformed and seemingly coalesced particles seen
FIG. 5.Danepurified3.attachedcomponent Reaction seen when guinea pig antiserum against HBAg (anti-HB,) was mixed with a preparation of particles disrupted by detergent treatment

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In the UK study men- tioned previously, infants who had a high score on the “ infant guidelines ” dietary pattern (fruit, vegetables, and home-prepared foods) at 6 months gained

4 In this system of medicine, Parada (Mercury) a back- bone of Rasashastra has been blended with different potent Herbs, Minerals and drugs of animal origin by

and to the recflnt introduction of the sales of reduced price butter for dirBot consumption (Christmas butter) which has reduced the oppor·cuni ties. for selling

This is consistent with the result of a research conducted by PISA 2012 that openness to problem solving is related to financial literacy, while perseverance

The researcher has planned the entire process of research work in such a way that it can focus on the social, family, psychological and economic problems of the single mothers