Vox Sang. 36: 307-31 1 (1979)

Application of Microtiter Solid-Phase Radioimmunoassay to the Determination of Hepatitis B e Antigen Y . Miyakawa, F . Tsuda, Y . Akahane and M . Mayumi Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hepatitis Division, the Tokyo Metropolitan Institute of Medical Science, and Immunology Division, Jichi Medical School, Tokyo

Abstract. Microtiter solid-phase radioimmunoassay (micro-SPRIA) was applied to the detection of hepatitis B e antigen (HBeAg) in the serum. Antibody against HBeAg to coat the microtiter plate and for radiolabeling was obtained from the serum of asymptomatic carriers of hepatitis B surface antigen by an affinity column of partially purified HBeAg. The micro-SPRIA was sensitive and could be performed without prior concentration of the test serum. HBeAg was detected in 21 (35%) out of 60 serum samples positive for hepatitis B surface antigen, all of which were negative by the conventional immunodiffusion method even after they had been concentrated threefold.

Introduction Three antigens are presently identified in association with hepatitis B virus (HBV) infection, i.e., hepatitis B surface antigen (HB,Ag), hepatitis B core antigen (HB,.Ag) and hepatitis B e antigen (HB,Ag). Early studies disclosed a close correlation of HB,Ag in the serum with infectivity and also with the prognosis of liver disease [6, 81. The study of HB,Ag, however, has been greatly restricted by the low sensitivity of methods available for its determination. For the past 5 years since its discovery, immunodiffusion has practically remained the sole method for the determination of HB,Ag. We now describe a simple and sensitive

method for the determination of HBeAg utilizing microtiter solid-phase radioimmunoassay (micro-SPRIA).

Materials and Methods A total of 100 serum samples containing HBHAg were obtained from 100 subjects who were seropositive for HBxAg. They included 60 asymptomatic carriers of HBIIAg and 40 patients with type B hepatitis. HBeAg and antibody to HBrAg (anti-HBe) in them were tested by the immunodiffusion method [6] after they had been concentrated threefold by adding Lyphogel (Gelman Instruments Co., Ann Arbor, Mich.) at a ratio of 50 mg to 1 ml of the serum. There were 20 serum samples positive for HBeAg ( 8 asymptomatic carriers and 12 cases of hepatitis), and 20 positive

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for anti-HB,, (16 asymptomatic carriers and 4 cases hepatitis). The remaining 60 serum samples were negative for HBrAg or anti-HBl, by the immunodiffusion method. Control serum samples were obtained from 30 apparently healthy individuals without HBdAg o r antibody to HBsAg (anti-HBd), 20 patients with serologically diagnosed type A hepatitis, and 20 patients who contracted chronic hepatitis after transfusion of one or more units of blood, but did not develop any serum marker of hepatitis A o r B virus infection. Anti-HBI, to coat microtiter plates and for radioiodination was purified from a pool of sera from several asymptomatic carriers of HBSAg as described previously [14]. Briefly, y-globulin fractions of pooled human sera containing antiHB,, were coupled with Sepharose 4-B to prepare an affinity column of anti-HBl,. Sera of asymptomatic carriers of HBsAg containing a high titer of HBI.Ag (reversed passive hemagglutination titer 1:64 [14]) were applied onto the anti-HB,, column, and then the column was eluted with 3 M NaI. Partially purified HBI,Ag thus obtained was coupled with Sepharose 4-B and an affinity column of HBl,Ag was prepared. This column was used for the specific purification of anti-HBIs y-globulin. 100 ml of pooled sera with a high titer of antiHB,\ (passive hemagglutination titer 1:4,096 [14]) were passed through the HB1,Ag column. The column was washed with 50 ml of 1 M MgCI, and then eluted with 50 ml of 5 M MgCI,. The eluate was extensively dialysed against Tris-HCI buffer (0.01 M, pH 7.5) containing 0.1 M NaCl and 0.01 M EDTA (Tris-buffered saline) and concentrated by negative pressure ultrafiltration. 5 ml of the preparation containing anti-HBI, was then applied to a Sephadex (3-200 column (2.6 x 90 cm, bed volume 480 ml), eluted with Tris-buffered saline, and fractions corresponding to the position of IgG were collected. This anti-HB,, ).-globulin preparation (concentration 3 mg/ml) was diluted 100-fold with Tris-buffered saline and a 50-!t1 portion was delivered to each well of a polyvinyl V-bottom microtiter plate (Cook Engineering Co., Alexandria, Va.) by means of an Eppendorf pipette. The plate was allowed to stand at room temperature for 30 min, and anti-HBILsolution was recovered for reuse by aspiration. After washing the plate 5 times with 0.15 M NaCI, it was flooded with Tris-buffered sa-

line containing 2% ( w h ) BSA at room temperature for 20 min. The plate was washed with 0.15 M NaCI, and stored at 4 ‘ C until used. The anti-HBl, y-globulin preparation was labeled with l25I in the presence of chloramine T at a specific activity of approximately 10 !tCi/ug after the method of Greenwood et ul. [3]. T h e labeled anti-HBl, (1251anti-HBI,) was diluted in normal human sera (Blood Group AB) so as to contain 0.02!cg of antibody in a 50-!tl portion. 25 !tl of the test serum were transferred into the well of a micro-SPRIA plate and allowed to stand for 2 h at room temperature and overnight at 4 C. The plate was washed 5 times with 0.15 M NaCI, and 50!tl of Ir51-anti-HBl, solution were delivered to each well. Then, the plate was successively incubated for 2 h at room temperature and overnight at 4 OC. After washing the plate 5 times with Tris-buffered saline containing 2% (w/v) BSA, wells were cut apart by scissors and the radioactivity was counted for 1 min in a gammacounter.

Results

The background binding for 30 serum samples of normal individuals without HB,Ag or anti-HB, was 1.42 f 0.24% (range 1.23-2.20%) of the added radioactivity. The serum samples obtained from patients with type A hepatitis and those with posttransfusion, non-A non-B hepatitis showed a similarly low binding. Figure 1 illustrates binding curves of 2 representative serum samples containing HB,.Ag. The serum samples were diluted in normal serum without HB,Ag or anti-HB,. A maximum binding of about 15% of the added radioactivity was obtained for both of them. The bound radioactivity decreased beyond 1 :10 dilution, but a binding exceeding 3 times that of background was still obtained at 1:30 dilution. All the other HB,,Ag-positive serum samples tested showed similarly steep binding curves.

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Radioimmunoassay for Hepatitis B e Antigen

Y

: m

v

H

. 10-0 10 Serum dilution



10

10.)

Fig. 1. Titration of hepatitis B e antigen in the serum by the microtiter solid-phase radioimmunoassay: bound radioactivity in percent as a function of serum dilution. Binding curves of two serum samples containing HBrAg are shown.

The results of micro-SPRIA tests performed on 100 serum samples containing HB,Ag are given in figure 2 in reference to the results of immunodiffusion tests carried out on the identical samples, but after three fold concentration. All of the 20 serum samples positive for HB,Ag by immunodiffusion (group A) showed binding values of 1*5I-anti-HB, clustering around 14%. In contrast, none of the 20 samples positive for anti-HB,. (group C) revealed any binding appreciably higher than controls; a perfect correlation of the results by the two methods was observed for these two groups. 60 serum samples negative for HB,Ag or anti-HB, (group B) were divided into two subgroups on the basis of their binding activity. 21 of them showed binding values ranging from 8.2 to 15.3%, but the binding of the remaining 39 was less than 2%. The specificity of the binding of 20 serum samples in group A, as well as that of 21 in group B which showed binding activities ex-

HB, Ag(+)

HB As(-) A&HB(-)

Anti-HB, (+)

lmmunodjffusion test

Fig. 2. Comparison of microtiter solid-phase radioimmunoassay and immunodiffusion in tests for hepatitis B e antigen in 100 serum samples containing hepatitis B surface antigen. Serum samples were divided into three groups according to the results of immunodiffusion: column A, HBcAg-positive; column B, negative for HBrAg or anti-HBp; column C, anti-HBc-positive. Closed circles represent the serum samples which specifically bound 1251-anti-HB,,.

ceeding those of controls, was ascertained by an inhibition test. When the binding by test serum samples in the presence of 10 pl of a preparation containing partially purified anti-HB,. was compared with that in the presence of 10 pl of Tris-buffered saline, a 50% or greater inhibition was achieved for all of them.

Discussion In 1972, Ling and Overhy [ 5 ] introduced the solid-phase radioimmunoassay for the detection of HB,Ag. Because of its high

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sensitivity, this tcchnique was also applied to the subtyping of HB,Ag [7]. Purcrll rt a/. have modified this technique to a small scale, and developed the methods to detect HB,Ag [ 111 and HB,.Ag [12] in a microtiter plate. The present communication describes the application of micro-SPRIA to the detection of HB,,Ag in the serum. The results of micro-SPRIA performed on 100 serum samples containing HB,Ag were in perfect agreement with those of immunodiffusion. Unlike immunodiffusion, the microSPRIA can be carried out on the test serum without prior concentration. It enabled the detection of HB,.Ag in 21 (35%) out of 60 serum samples in which neither HB,.Ag nor anti-HB,. was demonstrable by immunodiffusion even after they had been concentrated threefold. In our hands, the present micro-SPRIA has proved to be as sensitive as the passive hemagglutination method developed by Takahashi et a/. [14] for the detection of HB,Ag. However, higher sensitivity may be achieved by raising the specific activity of 1251-labeled anti-HB,. The clinical significance of identifying HB,,Ag among H B,Ag-positive serum samples has become increasingly evident. H B,,Ag has been correlated with the prognosis of the patients with type B hepatitis, its presence in the serum being a poor prognostic sign [S]. Even more importantly, its presence is thought to signal infectivity both in vertical [lo] and horizontal [ l ] transmission of HBV, especially when a smalldose inoculum is involved. A close correlation of HB,,Ag with the markers for Dane particles (presently accepted as HBV) [2], such as HB,.Ag and HB,Ag-associated DNA polymerase [4, 9, 131 lends strong support to the above view. Possible applications of the present method are to detect HB,.Ag

Miyakawa/Tsuda/Akahane/Mayumi

in low concentrations, in order to identify highly infectious serum samples and blood products likely to transmit HBV, and to study the nature of HB,3Ag which still remains undefined despite its obvious practical importance.

Acknowledgements This work was supported in part by grants from Japanese Ministry of Health and Welfare and Tokyo Metropolitan Government. We thank the Japan Red Cross Association for serum samples containing HBhAg.

References 1 Alter, H. J.; Seeff, L. B.; Kaplan, V. J.; McAuliffe, V. J.; Wright, W. C.; Gerin, J. L.; Purcell, R. H.; Holland, P. V., and Zirnmerman, H. J.: Type B hepatitis. The infectivity of blood positive for e antigen and D N A polymerase after accidental needlestick exposure. New Engl. J. Med. 295: 909-913 (1976). 2 Dane, D. S.; Cameron, C. H., and Briggs, M.: Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet i: 695-698 (1970). 3 Greenwood, F. C.; Hunter, W. M., and Glover, J. S.: The preparation of 131l-labelled human growth hormone of high specific radioactivity. Biochem. J. 89: 113-123 (1963). 4 Imai, M.; Tachibana, F. C.; Moritsugu, Y.; Miyakawa, Y., and Mayumi, M.: Hepatitis B antigen-associated deoxyribonucleic acid polymerase activity and e antigedanti-e system. Infect. Immunity 14: 631-635 (1976). 5 Ling, C. M. and Overby, L. R.: Presence of hepatitis B virus antigen as revealed by direct radioimmune assay with 125l-antibody. J. Immun. 109: 834-841 (1972). 6 Magnius L. 0. and Espmark, J. A,: New specificity in Australia antigen positive sera distinct from Le Bouvier determinants. J. Imrnun. 109: 1017-1021 (1972).

Radioimmunoassay for Hepatitis B e Antigen

7 Miyakawa, Y.; Imai, M., and Mayumi, M.: Application of the paired label radioantibody technique to detection and subtyping of hepatitis B surface antigen. J. Immun. 114: 11351137 (1975). 8 Nielsen, J. L.; Dietrichson, O., and Juhl, W.: Incidence and meaning of the ‘e’ determinant among hepatitis-B-antigen positive patients with acute and chronic liver disease. Lancet ii: 913915 (1974). 9 Nordenfeld, E. and Kjellen, L.: Dane particles, DNA polymerase, and e-antigen in two different categories of hepatitis B antigen carriers. Intervirology 5: 225-232 (1975). 10 Okada, K.; Kamiyama, I.; Inomata, M.; Imai, M.; Miyakawa, Y., and Mayumi, M.: e antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. New Engl. J. Med. 294: 746-749 (1976). 11 Purcell, R. H.; Wong, D. C.; Alter, H. J., and Holland, P. V.: Microtiter solid-phase radioimmunoassay for hepatitis B antigen. Appl. Microbiol. 26: 478-484 (1973). 12 Purcell, R. H.; Gerin, J. L.; Almeida, J. D., and Holland, P. V.: Radioimmunoassay for the detection of the core of the Dane particle and antibody to it. Intervirology 2: 231-243 (1973/ 74). 13 Takahashi, K.; Imai, M.; Tsuda, F.; Takahashi, T.; Miyakawa, Y.,and Mayumi, M.: Associa-

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tion of Dane particles with e antigen in the serum of asymptomatic carriers of hepatitis B surface antigen. J. Immun. 117: 102-105 (1976). 14 Takahashi, K.; Fukuda, M.; Baba, K.; Imai, M.; Miyakawa, M., and Mayumi, M.: Determination of e antigen and antibody to e by means of hemagglutination method. J. Immun. 119: 1556-1561 (1976).

Addendum Since preparation of this paper another SPRIA method for detection of HBrAg has been described by Fields et al. (J. Immunol. 121: 930-935, 1978). This uses HBeAg and anti-HBe obtained from chimpanzees, and its sensitivity relative to ID for HB(,Ag in sera containing HBHAg is similar to that of the method described here.

Received: September 21, 1978 Accepted: December 18, 1978 Yuzo Miyakawa, MD, The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo,Tokyo 113 (Japan)

Application of microtiter solid-phase radioimmunoassay to the determination of hepatitis B e antigen.

Vox Sang. 36: 307-31 1 (1979) Application of Microtiter Solid-Phase Radioimmunoassay to the Determination of Hepatitis B e Antigen Y . Miyakawa, F ...
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