APPLIED MICROBIOLOGY, Apr. 1974, p. 756-762Copyright 0 1974 American Society for Microbiology

Vol. 27, No. 4Printed in U.S.A.

Interactions of Standard Antibody with Australia Antigens inAu Ag-Ab Radioimmunoassay

HARRY DAUGHARTY, RUTH G. YOUNG, MILFORD H. HATCH, AND DONALD W. ZIEGLERCenter for Disease Control, Public Health Service, U.S. Department of Health, Education, and Welfare,

Atlanta, Georgia 30333

Received for publication 31 December 1973

Human antisera against Australia (Au) antigen have been characterized byliquid-phase radioimmunoassay (RIA) for their precipitation of '25I-labeled Auantigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of1251-Au antigen by RIA correlated well with complement fixation titers but had amuch wider range, indicating a greater precision and perhaps a better sensitivityof assay. Anti-Au serum diluted to precipitate 50% of 125I-labeled Au antigen wasused as standard antibody in RIA tests to detect either inhibition or enhance-ment of the reaction by preincubated mixtures of Au antigen and antibodyspecimens. Without free Au antigen or antibody in the resultant mixtures therewas no inhibition or enhancement; the mixtures presumably contained im-munoreactively equivalent proportions of Au antigen and antibody. RIA data fordiagnostic specimens indicated an end-point sensitivity which was proportionalto the dilution of the standard anti-Au sera used in the test. High concentrationsof the standard antibody permitted detectable inhibition of 125I-Au antigenprecipitation at lower antigen specimen concentrations. Similarly, low concen-trations of the standard antibody permitted detectable enhancement of 125I-Auantigen precipitation at lower antibody specimen concentrations. Omitting thestandard antibody altogether resulted in a more sensitive RIA for Au antibody intest sera.

Radioimmunoassay (RIA) has become amethod of choice in detection of Australia (Au)antigen and antibody (anti-Au) because of itsextreme sensitivity. The procedures generallyinvolve either solid phase (12) or liquid phase(5, 12, 15) systems. In this report factors affect-ing the sensitivity of a liquid phase system areinvestigated.Three liquid phase assay methods have re-

cently been developed (1, 5, 12, 15). In each ofthese methods a known antiserum (standardanti-Au) and a known amount of 125I-labeled Auantigen are the basic components of the ana-lytic system. When these components are mixedwith test specimens, precipitation of the 125I_labeled Au antigen by the standard anti-Au iseither enhanced (if antibody is present in thespecimen) or inhibited (if antigen is present inthe specimen).RIA previously demonstrated in blind studies

(14) had a high degree of sensitivity and re-quires the use of 125I-labeled Au antigen andstandard anti-Au of high specificity to avoidfalse positive results. The important role of thestandard anti-Au in the test was recognized by

several investigators who have sought to pro-duce anti-Au of high affinity and specificity.Purcell et al. (21) have noted variable responsesin specificity and potency of antisera to Aurelative to immunizing dose, time after inocula-tion, and animal species. Dreesman and col-leagues (7) have carried out controlled studiesto determine the most effective means for pro-ducing anti-Au of high specificity and sensitiv-ity.We have evaluated various Au antisera as

standard anti-Au in our procedure, and havedetermined the sensitivity of RIA relative to thedilution of standard anti-Au. These determina-tions of sensitivity have permitted us to quanti-tate antigen-antibody ratios in serum speci-mens containing both Au and anti-Au.

MATERIALS AND METHODSAntigen source and iodination. The Au antigen

used in these studies was a purified, concentratedform of hepatitis B associated antigen. It was ob-tained from Electro-Nucleonics Laboratories, Inc.Bethesda, Md., lot no. 86-24-1. The immunodiffusiontiter prior to iodination was 1:8, and the proteinconcentration was 633 Ag/ml.

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For iodination the antigen was diluted in phosphatebuffer (pH 7.4) containing EDTA and 1.0 mM MgCl,at pH 7.4. Iodination followed the procedure ofGreenwood et al. (10) with modifications by Hollingeret al. (12). While 50 gsg of purified Au antigen wasbeing mixed in an ice bath, 1.0 mCi of 125I in 0.1 MNaOH and an equal volume of 0.1 M HCl were added.After the suspension was thoroughly mixed, 2.5 mg ofchloramine T were added dropwise. The oxidationreaction was allowed to proceed for 4 to 5 min whilestirring was continued. The reaction was stopped byadding 5.0 mg of potassium metabisulfite. After thesample was mixed for 4 to 5 min, it was exhaustivelydialyzed 'against borate saline buffer (BSB; 0.15 MNaCl, 0.002 M H3BO,, pH 7.4). Iodinated antigen wasfurther purified by filtration on a Sephadex G200column (2.2 by 32 cm) with BSB. The eluted fractionsassociated with the initial radioactive peak werepooled and used as the '29-Au antigen for the RIAprocedure. The radioactivity of this pool was 90 to95% precipitable with 10% trichloracetic acid andapproximately 80% precipitable with our standardantiserum against Au antigen. Assuming about 80%recovery of protein from the Sephadex column, thespecific activity of our 125I-Au preparations was about2.25 x 103 counts per min per ug.

Specimens and reagents. The standard anti-Aureagent used in these studies was purchased fromMedical Sciences International, Inc., Medical Serv-ices Division, Stoneham, Mass. The antiserum hadbeen obtained from a hemophilic individual who hadreceived multiple therapeutic transfusions. Comparedto several other anti-Au sera, the selected serumexhibited maximal precipitation of "'2I-Au antigen inour RIA so as to preclude any possible false positivereactions. Additional human sera tested for Au anti-body were also from hemophiliacs or they wereselected from diagnostic specimens supplied by theEpidemiology Program, Viral Diseases Branch, Cen-ter for Disease Control, Atlanta, Ga.The specimen of Au antigen positive plasma was

obtained from the Atlanta Chapter of the AmericanRed Cross. It was found to be positive for Au antigenby agar-gel diffusion, by crossover electrophoresis(titer 1: 2), and by complement fixation (titer 1: 128).RIA procedure. Evaluation of standard anti-Au

sera and quantitation of both Au antigen and Auantibody in specimens were performed by RIA. TheRIA procedure was essentially the double antibodymethod of Hollinger et al. (12) and Purcell et al. (15).The double antibody procedure was modified by usingColler and his colleagues' (5) method of ammoniumsulfate precipitation for separating bound "25I-Auantigen from unbound "25I-Au antigen.The titration of standard anti-Au and the assays of

specimens for Au antigen or antibody are essentiallysimilar procedures. In these procedures 0.5 ml oftwofold diluted standard anti-Au is used for thetitration. For antigen or antibody assay, preincubated(2 h at 37 C and 18 h at 4 C) mixtures of 0.5 ml ofstandard anti-Au and 0.05 ml of 1:10-diluted testspecimens are reacted in glass tubes (10 by 75 mm)with 0.5 ml of a dilution of "25I-Au antigen havingapproximately 3,000 counts/min. The final volume is

adjusted to 1.1 ml by the addition of BSBSA diluent(0.1 M borate, 0.85%; NaCl buffer containing 0.04%bovine serum albumin, pH 8.4). After incubation ofthis final mixture for 2 h at 37 C and 18 h at 4 C, thespecimen either synergistically enhances the precipi-tation of "2'I-Au antigen if it contains Au antibody orcompetitively inhibits precipitation if it contains Auantigen.

In both the standard anti-Au titration and thespecimen assays, double antibody complexes areinduced by incubating the final mixtures for 2 h at37 C and 18 h at 4 C with 0.05 ml of a 1: 2 dilution ofgoat antiserum to human IgG. Double antibodycomplexes which form are precipitated by adding 0.82ml of 60.9% ammonium sulfate (final concentration of25.0%) and incubating for 1 h at room temperature.The precipitate is then sedimented for 1 h at 20 C in aSorvall RC 3 centrifuge at 7,000 x g. Supernates areaspirated into separate test tubes. The distribution of"25I-Au antigen between the precipitate (containingbound antigen) and its corresponding supernate (con-taining unbound antigen) is determined by countingthe respective fractions in a gamma-scintillation spec-trometer. The end point for a standard anti-Autitration is defined as the dilution of the antiserumwhich precipitates 50% of the "2'I-Au antigen (the 50%"25I-Au precipitating level). With each group of speci-mens assayed, a standard set of 10 randomly selectednegative control sera is tested to establish a meanbaseline value and standard deviation of "25I-Auantigen precipitated by negative sera. Minimal pre-cipitation of Au-antigen by these control sera gave aGaussian (bell-shaped) distribution (14). A positivespecimen is inferred when the percentage of "25I-Auantigen precipitated by the specimen deviates morethan 2 standard deviations from the mean value of"5I-Au antigen precipitated by the negative controls.Other methods of Au antibody titrations. The

passive hemagglutination (PHA) (23) tests on theserum specimens were performed at the AmericanNational Red Cross, Washington, D.C., to determinethe Au antibody PHA titers.Complement fixation (CF) titers were determined

by the Hepatitis and Enteric Virology Section, Centerfor Disease Control. The micro method for CF (4) wasused for the block titration of antigen and antibody.

RESULTSStandard anti-Au titration. The potency of

an antiserum intended for use as a standardreagent in the RIA procedure was evaluated. Inthe titration of the standard Au antiserumtwofold dilutions were reacted with constantamounts of "2'I-Au antigen. An RIA titrationcurve for a standard anti-Au preparation with25I-Au antigen is presented in Fig. 1. At dilu-tions between 1: 1,280 and 1: 81,920, a decreas-ing proportion of the "2'I-Au antigen was precip-itated. A maximum of 80% of the 25I-Auantigen was precipitated at standard anti-Audilutions of 1: 1,280 or less. At dilutions greaterthan 1: 81,920 a negligible (less than 10%)

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precipitation of "25I-Au antigen occurred. Byinterpolation, 50% of the '25I-Au antigen wasestimated to be precipitated at a dilution be-tween 1: 5,120 and 1: 10,240.

Titration of several sera by both CF and RIAshowed a good correlation between CF titersand RIA titers (the dilution factor associatedwith precipitation of 50% of the "25I-Au anti-gen). Sera with the highest CF titers alsoexhibited highest RIA titers. These results,presented in Table 1, indicate that whereas CFresults exhibited a fourfold range (8 to 32) intiters, the RIA titers varied over a 32-fold range(160 to 5,120).Equivalence proportions (1: 1) of Au anti-

gen and antibody determined by standardanti-Au. The potential of the RIA method fordetecting either reactive Au antigen or antibodyin specimens was investigated by preincubatingfixed concentrations of Au antigen positiveplasma (0.025 ml) with variable dilutions of Auantiserum (0.5 ml). The reaction mixtures as

80 *_-._7025

60-

I-Au 50

Precipitated 4030

20

10 .

It WW N q (O (N q (O (N

-. N q -a

Standard antiserum dilution factors

FIG. 1. RIA titration of serial twofold dilutions of astandard anti-Au serum with 125I-Au to determine theend-point dilution of anti-Au at which one-half of the125I-Au is precipitated.

TABLE 1. CF titers of standard anti-Au comparedwith RIA end points for precipitating 50% "2'I-Au

antigen

RIA titersa. (50% 1251-Au antigenSerum specimen CF titer precipitating

end point)

F.Z. <8 160D.G. 8 1,000No. 1407 16 1,300No. 1402 16 1,800No. 1397 16 3,500No. 1369 32 3,500No. 1393 32 4,000G.K. 32 5,120

a End-point dilution factors.

specimens were incubated for 2 h at 37 C andthen for 18 h at 4 C. These reaction mixtureswere added to the test system consisting of1251-Au antigen and standard anti-Au at a 50%precipitating concentration. Excess Au antigenin the reaction mixtures was indicated by inhi-bition of precipitation of 125I-Au antigen. Con-trawise, excess anti-Au was indicated by en-hancement of 1251-Au antigen precipitation. InFig. 2 each precipitation curve represents aconstant amount of Au antigen plasma reactedwith increasing dilutions of anti-Au in thereaction mixture.

Greatest inhibition of '251-Au antigen precipi-tation was observed when anti-Au was prein-cubated with the highest concentration of Auantigen positive plasma; also, inhibition wasgreatest when Au antigen positive plasma waspreincubated with the lower concentrations ofanti-Au. Thus, precipitation below the 50%level was not attained when the reaction mix-ture consisted of anti-Au and the higher dilu-tions (1:1,000, 1:10,000) of Au antigen positiveplasma. However, precipitation below the 50%level was readily attained with mixtures ofhigher dilutions of anti-Au and antigen positiveplasma dilutions at 1:100 and 1:10. The brokenline parallel to the abscissa indicates the 50%precipitation of 1251-Au antigen observed whenthe negative control instead of the reactionmixtures was added to the standard test sys-tem. When there is a 50% precipitation of the125I-Au antigen in the final mixture with thestandard test system, one may assume thatthere is neither free Au antigen nor antibody in

70-

125I-Au 50-

precipitated(%) 40-

30-

0 0 0 0 0 0 0 0 0IT ( D ID T 0to (0

to CO C O O uXanti-Au serum dilution factors

FIG. 2. RIA for either excess, unbound Au antigenor antibody in specimen mixtures of the two atvarying ratios. Horizontal dashed line indicates pre-cipitating level observed for the dilution of standardantibody of the standard test system with negativecontrol specimens. Dilutions of low concentrations ofanti-Au and high concentrations of Au positiveplasma inhibit 125I-Au precipitation.

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the reaction mixture (ratios of Au antigen andantibody are equivalent, i.e., 1:1). Thus addi-tion of disproportionate amounts of either Auantigen or antibody in the reaction mixture willcause precipitation of 125I-Au antigen inamounts greater or less than 50%. Precipitationgreater than 50% indicates enhancement byexcess Au antibody (Au antigen: antibody ratioless than one), and precipitation less than 50%indicates inhibition by excess Au antigen (Auantigen: antibody ratio greater than one).RIA sensitivity relative to standard

anti-Au concentration. It was observed that thesensitivity of the RIA method for assaying Auantigen and antibody was related to the concen-tration of standard anti-Au in the test system.Variable concentrations of standard anti-Auwere used in the test system to determine thesensitivity for the assay of Au antigen (Fig. 3)and anti-Au (Fig. 4) in specimens. These dilu-tions of standard anti-Au antibody representeda full range for minimum (10%) to maximum(80%) precipitation of 1251-Au antigen. The endpoint for a test specimen may be considered asthe highest dilution of the specimen at whichthere is detectable inhibition (antigen) or en-hancement (antibody) of 1251-Au antigen precip-itated. These specimen end points increase ordecrease relative to the dilution of standardanti-Au antibody used in the test (Fig. 3).Although less obvious in Au antigen determina-tions, higher concentrations of standard anti-Augenerally resulted in higher end points for theantigen positive plasma specimen tested. Simi-larly (Fig. 4), lower concentrations of standard

70

60

251-Au 50

precipitated40-

30-

20-

10-

A A -&---A-- A A-

-

go--o- o0

00 0 0 0 0 0 0

x * x x x

6 6 6 6 6 6As0 0 0 0 0 0 0 0 0

_ In ID N

Au Ag positive plasma dilution factor

FIG. 3. RIA of Au antigen in serial dilutions of anantigen positive plasma. Higher concentrations ofstandard anti-Au antibody resulted in higher endpoints for the Au antigen positive specimen as indi-cated by later occurring plateau regions. Standardanti-Au dilutions: A, 1:320; A, 1:3,200; 0, 1:5,120;0, 1:20,000; x, 1:80,000.

anti-Au usually resulted in higher end points forthe antibody positive specimen tested.The increased sensitivity attained in these

experiments suggested that even greater sensi-tivity in detecting Au antibody could be at-tained by the complete omission of standardanti-Au in the test system. Therefore, RIA wascarried out for a group of serum specimens, withand without standard anti-Au. In Table 2 theseresults are compared with titers determined bythe conventional PHA method. When a dilutionof standard anti-Au antibody precipitating 50%of the 125I-Au antigen was included in the test,

802

70.

60

125I U

Preo~putoted(%) 40

30

20

10

^ _^ _ _A ^ xi ^

A- -\ O O\; * °-O

\ *X\ * ^ ^ _ ^ ^

XX

XX X

X-X X

8 8 @ 8 TO 88 8N 8- 0 0

Au Ab positive plosrno dilution factor

FIG. 4. RIA ofAu antibody in serial dilutions of anantibody positive plasma. Lower concentrations ofstandard Au antibody resulted in greater end pointsfor the Au antibody positive specimen as indicated bylater occurring plateau regions. Standard anti-Audilutions: A, 1:320; A, 1:3,200; 0, 1:5,120; 0,

1: 20,000; x, 1: 80,000.

TABLE 2. Au antibody detected at lower levels ofconcentration when standard antibody was absent

1576, 18431664, 18641574, 15661086, 14941987, 21571568, 26771026, 18301619, 16441495, 164311001800, 2036

1:81:161:321:641:1281:2561:5121:1,0241:2,0481:5,120

I 1:10,240

RIA results withstandard Ab

Presenta Absentb

+

++

+++++++

aStandard anti-Au present at a dilution whichprecipitated approximately 50% of the 125I-Au.bBSBSA diluent (0.5 ml) substituted in place of

standard antibody.

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only the anti-Au specimens with PHA titers1:5,120 or greater were classified as positive.However, when only BSBSA diluent was substi-tuted for standard anti-Au in the test, almost asmany positive specimens were detected by RIAas by the PHA method.

DISCUSSIONSeveral characteristic reactions of Au anti-

body have been noted in the RIA procedures.For example, complete precipitation of 125I-Auantigen by anti-Au has not been attained. Whenwe tested standard anti-Au dilutions for thepercent of 125I-Au antigen which they were ableto precipitate, values greater than 80% wererarely obtained. This may be due to soluble,nonprecipitable Au antigen-antibody com-plexes or aggregates, or perhaps free 125-iodine.Our value of 80% for maximum precipitation isconsistent with results of others (5, 12, 15).Although all of our Au antisera have been fromhumans, antisera from other species have alsobeen compared. It was shown that specific andpotent immunoreactivity of Au antibody wasthe greatest for human, guinea pig, and rabbitantisera, in that order (21).There was good correlation between CF titers

and RIA end-point dilutions of Au antibodyprecipitating 50% of the 126I-Au antigen. Al-though sera with high CF titers also had highRIA end-point dilutions, there was a muchwider range of titers by the RIA method than bythe CF method. It is evident that Au antiserathat react quite similarly in the CF test mayvary widely in their capacity to precipitate125I-Au antigen (Table 1). The greater variabil-ity of the RIA method reflects a definite qualita-tive difference in Au antisera having equal CFactivity. This qualitative difference among Auantisera of the same CF activity is obviously afunction of antibody dilution since certain seracontain more reactive (higher degree of'affinity)antibodies at lower dilutions. This greater reac-tivity of certain sera is evident (Table 1) wherehigher dilutions are just as capable of precipi-tating 50% of the I25I-Au antigen as lowerdilutions of other sera which may have exactlythe same CF titers.Those sera which precipitate larger amounts

of 1251-Au antigen, presumably from theirhigher affinity constants, would serve moreeffectively as standard anti-Au in RIA than seraof lower precipitating capacity. At a fixeddilution the sera with high precipitating titersare more sensitive to inhibition by Au antigen inspecimens because 125I-Au antigen precipitationis proportional to the concentration of standard

anti-Au (Fig. 1). Antiserum of extremely highaffinity for Au antigen as determined by RIAwould also be very useful in simplifying Auantigen purification procedures. Such elaborateprocedures involving gradient and rate-zonalcentrifugation (7, 8, 17) may be eliminated byusing affinity chromatography and anti-Auserum of high potency. The feasibility of thismethod of affinity chromatography has previ-ously been demonstrated for the isolation ofAleutian disease virus (13).The selectivity of RIA for anti-Au of high

affinity would also have application for thera-peutic procedures. In the past, randomly pooledhuman globulin has been used in treatment ofpersons accidentally exposed or under high riskof infectivity with hepatitis virus. RIA testingand PHA assays (20) now offer a better basis forselection of antisera with higher titers for use inglobulin pools. These pools should be moreeffective in the elimination of hepatitis virus.This application is consistent with the demon-stration by Alpers et al. (2) that high affinityantibody is more effective than low affinityantibody in eliminating circulating proteinantigen.We have described a procedure whereby

equivalence proportions of Au antigen and anti-body can be determined. Mixtures of im-munoreactively equal amounts of the Au anti-gen and antibody result in nonreactivity ofeither unbound antigen or unbound antibody.This is a result of complex formation betweenthe added antigen and antibody so that neitheris able to react by inhibiting (excess antigen) orenhancing (excess antibody) the precipitationof the 125I-Au antigen used in the test. Thus, onecan quantitate combining ratios of Au antigenand antibody for varying ratios of antigen-anti-body complexes. Similar complexes of amylaseand specific antibody have been demonstratedto have greater immunogenicity than free anti-gen (19). The complexes were also shown to bephagocytosed and degraded more rapidly ineliciting a primary response than was the freeantigen. At the other extreme, antigen in four-fold excess was shown to have only approxi-mately 0.5% the immunogenicity of unboundantigen (11).With our proposed method of RIA one can

obtain Au antigen-antibody complexes in mix-tures of the antigen and antibody in im-munoreactively equivalent proportions. Dispro-portionate amounts of either Au antigen orantibody result in antigen-antibody complexeswhich may resemble human globulin as antigenin combination with excess globulin-specific

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VOL 27, 1974 STANDARD ANTIBODY WIT

antibody whereby immune precipitates aremetabolically processed differently by the host(11). These complexes can be used for determin-ing the role of Au antigen in the pathogenesis ofvirus-induced immune complex disease and forstudying their interactions with rheumatoidfactor and complement. Similar investigationswith herpes (18) and influenza (6) viruses haveindicated increased virus sedimentation charac-teristics and enhanced neutralization of thevirus-antibody complexes upon interaction withrheumatoid factor and complement. A moreprecise understanding of the roles of Au antigenin immune complex diseases will require furtherstudy of the factors which influence the associa-tion and dissociation of these complexes.There was an inverse relationship between

the concentration of standard anti-Au and sen-sitivity in Au antibody determinations (Fig. 4).However, the direct relationship between stan-dard anti-Au concentration and Au antigendeterminations was not as obvious (Fig. 3).Plateau regions indicating end points for Auantigen assays did occur later with higherconcentrations of standard anti-Au. However,the trend is less noticeable and has lessreproducibility than with Au antibody determi-nations. For this reason one should perhaps useas low a dilution of standard anti-Au as possiblein Au antigen assays for achieving maximum1251-Au precipitation (Fig. 1). On the otherhand, higher dilutions of standard anti-Au oromitting it entirely would afford a more sensi-tive Au antibody assay.The advantages of RIA for detecting Au

antigen and antibody have been demonstratedin recent epidemiological investigations. Nu-merous surveys for determining the prevalenceof Au antigen and antibody (3, 9, 15, 22) havedepended heavily upon the sensitivity of theRIA methodology. Without the sensitivity ofRIA for carrying out these surveys our presentstate of knowledge concerning the incidence,transmission, and persistence of an agent asso-ciated with hepatitis B would be far moreincomplete. Development of more refined RIAtechniques will be essential for future epidemio-logical endeavor in subtyping of Au antigen andantibody. Presently, characteristic dilutioncurves for the major Au antigen subtypes serveas a basis for RIA differentiation of "ad" and"ay" specimens (9). However, labeling a mono-specific typing serum of single serological speci-ficity has proven useful in determining type-specificities of hepatitis B viral antigen (16).These methods of RIA subtyping will requiremore selective and more specific standard Auantisera for subtyping purposes.

'H AUSTRALIA ANTIGENS 761

LITERATURE CITED

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2. Alpers, J. H., M. W. Steward, and J. F. Soothill. 1972.Differences in immune elimination in inbred mice. Therole of low affinity antibody. Clin. Exp. Immunol. 12:121-132.

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6. Daugharty, H. 1971. Immunoprecipitin reaction of influ-enza virus-antibody complex with anti-IgG. J. Immu-nol. 107:802-809.

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8. Gerin, J. L., P. V. Holland, and R. H. Purcell. 1971.Australia antigen: large-scale purification from humanserum and biochemical studies of its protein. J. Virol.7:569-576.

9. Ginsberg, A. L., W. H. Bancroft, and M. E. Conrad. 1972.Simplified and sensitive detection of subtypes of Aus-tralia antigen (HBAg) using a solid-phase radioim-munoassay. J. Lab. Clin. Med. 80:291-296.

10. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963.The preparation of "'I-labeled human growth hor-mone of high specific radioactivity. Biochem. J.89:114-123.

11. Henney, C. S., and K. Ishizaka. 1968. Studies on theimmunogenicity of antigen-antibody precipitates. 1.The suppressive effects of anti-L and anti-H chainantibodies on the immunogenicity of human y-Gglobulin. J. Immunol. 101:896-904.

12. Hollinger, F. B., V. Vorndam, and F. R. Dreesman. 1971.Assay of Australia antigen and antibody employingdouble-antibody and solid-phase radioimmunoassaytechniques and comparison with the passive hemag-glutination methods. J. Immunol. 107:1099-1111.

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15. Lander, J. L., H. J. Alter, and R. H. Purcell. 1971.Frequency of antibody to hepatitis-associated antigenas measured by a new radioimmunoassay technique. J.Immunol. 106:1166-1171.

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18. Notkins, A. L. 1971. Infectious virus-antibody complexes:interaction with anti-immunoglobulins, complement,and rheumatoid factor. J. Exp. Med. 124:41-51.

19. Osato, K. 1972. Antigen-antibody complexes in the im-mune response. I. Analysis of the effectiveness ofcomplexes on the primary antibody response. Immu-

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