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A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies
Journal oflmmunologicalMethods, 87 (1986) 203-210
203
Elsevier JIM03808
A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies Sadakazu Usuda, Fumio Tsuda, Tohru Gotanda, Katsumi Tachibana, Motozumi N o m u r a *, Hiroaki Okamoto, Mitsunobu Imai, Tetsuo Nakamura, Yuzo Miyakawa and Makoto Mayumi ** Institute of lmmunology, Tokyo 113, Hepatitis Division, the Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japanese Red Cross Blood Center, Saitama-Ken 367, and Immunology Division, Jichi Medical School, Tochigi-Ken 329-04, Japan (Received 8 October 1985, accepted 30 October 1985)
Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype. Key words: Hepatitis B surface antigen; Subtype," Monoclonal antibody
Introduction
There are different strains of hepatitis B virus (HBV) that are distinguishable by the subtypic antigenic determinants expressed on hepatitis B surface antigen (HBsAg) particles. Using immunodiffusion, Le Bouvier (1971) found 2 distinct, mutually exclusive determinants on HBsAg particles, which he named d and y, in addition to the
* Dr. Nomura is presently affiliated to the Second Department of Internal Medicine, Fukui Medical College, Fukui-Ken 91011, Japan. ** Please address correspondence to: Makoto Mayumi, M.D., Immunology Division, Jichi Medical School, Minami-kawachiMachi, Tochigi-Ken 329-04, Japan.
commonly shared determinant, a. Bancroft et al. (1972) identified, also by immunodiffusion, w and r determinants, one or the other of which is carried by individual HBsAg particle in addition to d or y. Consequently, there are 4 major subtypes of HBsAg, i.e., adw, adr, ayw and ayr. Subtypes of HBsAg differ in their epidemiological distributions (Feinman et al., 1973; Mazzur, 1974; Yamashita et al., 1975), and are helpful in tracking the route of HBV infection in accidental and perinatal transmission of HBV (Mayumi and Nakajima, 1973; Okada et al., 1976). Traditionally, subtyping of HBsAg has been performed by immunodiffusion. This technique, though convenient in establishing the identity, or the lack of identity, of antigenic determinants, is
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
204 not very sensitive; it has not always been possible to characterize the subtypic specificities in materials of known HBsAg positivity. We raised monoclonal antibodies against the common and subtypic determinants of HBsAg, and developed a sandwich-type enzyme-linked immunoassay for their detection. Owing to high sensitivity in detection and assured availability of antibodies with desired specificities as required, the assay would become a useful tool in epidemiological and immunochemical studies of HBsAg.
Materials and methods
Monoclonal antibodies HBsAg particles of 3 different subtypes
(adw, adr and ayw) were isolated from plasma of asymptomatic carriers of HBV by the method described elsewhere (Takahashi et al., 1976). They were dissolved in 100 ~tl of saline at 2 mg/ml, emulsified in an equal volume of Freund's complete adjuvant and given intraperitoneally to BALB/c mice. After 2 months, mice were boosted by intraperitoneal injection with the same amount of HBsAg particles but without adjuvant. On the third day, their spleens were removed and immune spleen cells were harvested. Hybridization, subsequent culture and cloning were performed in accordance with a modification (Oi and Herzenberg, 1980) of the method originally described by Krhler and Milstein (1975). Briefly, immune spleen cells were mixed with mouse myeloma (NS-1) cells, at a ratio ranging from 5:1 to 10:1, and fused in the presence of 50% (w/v) polyethylene glycol-1500 (Wako Pure Chemicals, Osaka, Japan). Fused cells were distributed into wells of a plastic culture plate (Nunc, Roskilde, Denmark) and subjected to the selection in the medium containing hypoxanthine, aminopterine and thymidine. After 9 days of culture, the medium from each well was tested for the antibody (anti-HBs) against the common or subtypic specificity of HBsAg by the following method. Wells of an Immunoplate (Nunc) received 50 ~tl of phosphate-buffered (0.01 M, pH 7.6) saline (PBS) containing rabbit antimouse -/-globulin antibody (20 txg/ml). The plate was incubated at 4°C overnight, washed with saline
and then exposed to 40% (v/v) fetal calf serum (FCS) in order to mask unsaturated binding sites. Culture medium (50/~1) was delivered to each of 4 wells, and the plate was incubated at 37°C for 2 h. The plate was washed with saline and each well received 50/~1 of PBS supplemented with 25% FCS and containing one or other of HBsAg/adw, HBsAg/adr, HBsAg/ayw and HBsAg/ayr, purified from plasma of asymptomatic carriers, with a reversed passive hemagglutination (RPHA) titer for HBsAg of 21° (Vyas and Shulman, 1970). The plate was incubated at 37°C for 2 h and then washed with saline. Thereafter, each well received 50 /~1 of PBS supplemented with 25% FCS and containing polyclonal horse anti-HBs linked to horseradish peroxidase (Toyobo, Osaka, Japan) by the method of Nakane and Kawaoi (1974). The plate was incubated at 37°C for 2 h and washed with saline. Thereafter, each well received 50 ~1 of citrate buffer (pH 5.0) containing 0.33 /zl of 1% (v/v) HzO 2 solution and 30 ~tg of o-phenylene diamine. After 5 min, the reaction was terminated by adding 50 /xl of 4 N HzSO4, and the absorbance at 492 nm was determined. The specificity of monoclonal antibody was judged by its reaction with HBsAg particles of 4 different subtypes. Clones secreting anti-HBs of desired specificity were propagated in the peritoneal cavity of mice that had been made ascitic by the injection with 2,6,10,14-tetramethylpentadecane. Ascites fluid was harvested about 10 days after the implantation, and y-globulin fractions were precipitated with 2 M (NH4)2804, and then purified by gel filtration in Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden). Monoclonal antibodies were tested for their immunoglobulin classes by immunodiffusion against a panel of monospecific rabbit antisera (Miles Laboratories, Elkhart, IN).
Subtyping of HBsAg Wells of an Immunoplate received 100 ~l of PBS containing monoclonal anti-HBs/a (monoclonal no. 3207, 50 t~g/ml). The plate was postcoated with 40% FCS. The test sample (100 /~1) was delivered to each of 5 wells, and individual wells received 100 /zl of PBS supplemented with 25% FCS and containing monoclonal antibody with a specificity of anti-HBs/a (824), anti-HBs/d
205
(3423), a n t i - H B s / y (3457), a n t i - H B s / w (4111) or a n t i - H B s / r (313) that had been linked to peroxidase. The plate was incubated at 37°C for 2 h and washed with saline: Then, 100 /~1 of citrate buffer containing substrates were delivered to each well, and the plate was incubated at 25°C for 30 min. The reaction was terminated by adding 50/~1 of 4 N H2SO 4, and the intensity of developed color was determined by the absorbance at 492 nm. For the quantification of the determinant a of HBsAg, the amount of a required to induce an absorbance of 1.0 in the enzyme immunoassay was defined as one arbitrary unit of a. The serum containing HBsAg particles was serially diluted and tested by enzyme immunoassay, and the volume of serum that contained 1 U of a was determined from plots of the obtained absorbance. Then, the number of units contained in 1 ml of the test serum was calculated. Similarly, defining 1 U of d by an absorbance of 0.3, 1 U of y by that of 1.0, and 1 U of r by that of 0.5 in the respective enzyme immunoassay, the number of units contained in 1 ml of serum was determined for each subtypic determinant.
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. The Japanese antigen panel comprised 35 sera containing HBsAg particles subtyped by the hemagglutination inhibition method (Imai et al., 1974). All sera revealed an R P H A titer for HBsAg of 2 x2 or greater.
Results
Monoclonal antibodies against common or subtypic determinants of HBsAg Six monoclonal antibodies with a high avidity for binding to HBsAg particles were selected. Each of them was directed to the common or a subtypic antigenic determinant of HBsAg. The binding of monoclonal antibodies to HBsAg particles of 4 major subtypes that had been fixed on a solidphase support, as well as their immunoglobulin classes, are given in Table I.
Subtyping of the N I H and Japanese HBsAg panels by the enzyme immunoassay with monoclonal antibodies The N I H panel, composed of 9 sera containing HBsAg, was tested by the enzyme immunoassay with monoclonal antibodies (Table II). It can be seen that the assay detected y and r determinants efficiently; HBsAg particles bearing these determinants invariably exhibited an absorbance greater than 2.00. The determinant d was detected with
HBsA g panels The N I H panel was composed of 9 HBsAgpositive sera and specified for the subspecificities of w determinant (Courouc6 and Soulier, 1976) for 4 out of 7 sera with HBsAg particles bearing w. It was distributed by Research Resources Branch,
TABLE I SPECIFICITY OF M O N O C L O N A L ANTIBODIES D I R E C T E D TO THE C O M M O N A N D SUBTYPIC D E T E R M I N A N T S OF HBsAg Monoclonal antibodies were captured by rabbit anti-mouse ],-globulin fixed on a solid support, and tested for the binding with HBsAg particles of 4 major subtypes. HBsAg particles bound to wells via monoclonal antibodies were then detected by horse anti-HBs linked to horseradish peroxidase. Values indicate the absorbance at 492 nm. The mean + SD value for culture media without anti-HBs was 0.10 _+ 0.05. Monoclonal
HBsAg particles of subtype
no.
adw
adr
ayw
ayr
classes
Immunoglobulin
3207 824 3423 3457 4111 313
> 2.00 > 2.00 > 2.00 0.03 > 2.00 0.15
> 2.00 > 2.00 > 2.00 0.06 0.08 > 2.00
> 2.00 > 2.00 0.11 > 2.00 > 2.00 0.08
> 2.00 > 2.00 0.10 > 2.00 0.08 > 2.00
IgM, lambda IgG3, kappa IgG1, kappa IgG1, kappa IgG1, kappa IgG1, kappa
206 TABLE II D E T E R M I N A T I O N OF A N T I G E N I C SPECIFICITIES OF THE N I H HBsAg PANEL BY THE ENZYME IMMUNOASSAY W I T H MONOCLONAL ANTIBODIES The N I H panel composed of 9 sera containing HBsAg was subtyped by the enzyme immunoassay that sandwiched HBsAg particles between monoclonal anti-HBs/a (no. 3207) fixed on a solid support and another monoclonal antibody, with the specificity as indicated, linked to horseradish peroxidase. Values indicate the absorbance at 492 nm. The mean + SD value for 30 normal sera without markers of HBV infection was 0.17 _+ 0.06 for the determinant a, 0.13 _+ 0.04 for d, 0.06 ± 0.02 for y, 0.11 ± 0.04 for w, and 0.09 + 0.05 for r. Subtypes of HBsAg
Determinants detected by monoclonal antibody (no.)
adw adw 4 adr ayw ayw 1 ayw 2 ayw 3 ayr adyw
(824) a
(3423) d
> > > > > > > > >
> 2.00 1.84 1.93 0.16 0.18 0.21 0.14 0.20 0.79
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
an absorbance greater than, or close to, 2.00 except when it was expressed along with the determinant y in a single sample of subtype adyw. The absorbance for determinant w was rather variable. It was greater than 2.00 for 1 of 3 samples bearing
(3457) y
(4111) w
(313) r
0.03 0.03 0.04 2.00 2.00 2.00 2.00 2.00 2.00
> 2.00 0.54 0.04 1.52 > 2.00 > 2.00 1.36 0.06 1.72
0.17 0.03 > 2.00 0.01 0.06 0.08 0.00 > 2.00 0.02
> > > > > >
50 2O .3 i
c~
lO
5
A ) Determinant d
oo
• adw
o
o odr • oyw zx a y r D adyr
o
2 [3 I
1.00
E
1o
K
zx A
5
O.50
eq oh ~t k 0.20
~
I
B) Determinant y
2 E3 I
0.10
LU C~ 2 0
I
C3 ~ 0.05
oZX
~ 0.02
I
C) D e t e r m i n o n t r
I
I
I
I
I
I
5
4
3
2
1
0
DILUTION OF SERUM,
3N
Fig. 1. Simultaneous expression of allelic determinants, d and y, on HBsAg particles of a hybrid subtype adyr. Serum containing H B s A g / a d y r was serially diluted and sandwiched between monoclonal antibody against d, fixed on a solid support, and monoclonal antibody against y, linked to horseradish peroxidase. The bound antibody was then determined by the conversion of added substrates that developed color with an absorbance at 492 nm.
zx~ © []o o
5 zx
2 ! 2'
5'
DETERMINANT a, u m t s / m t
!
'o
20
x 10 - 4
Fig. 2. Expression of subtypic determinants on HBsAg particles of ordinary or hybrid subtype. Subtypic determinants, expressed in arbitrary units, were measured for individual batches of HBsAg particles, and plotted against the quantity of the common determinant a that also was expressed in arbitrary units.
207
T A B L E III D E T E R M I N A T I O N O F A N T I G E N I C S P E C I F I C I T I E S O F T H E J A P A N E S E H B s A g P A N E L BY T H E E N Z Y M E I M M U N O A S SAY WITH MONOCLONAL ANTIBODIES H B s A g in the J a p a n e s e p a n e l was s u b t y p e d b y the e n z y m e i m m u n o a s s a y with m o n o c l o n a l antibodies. Values i n d i c a t e the a b s o r b a n c e at 492 rim. Subtypes and no.
D e t e r m i n a n t s detected b y m o n o c l o n a l a n t i b o d y (no.) (824) a
(3423) d
(3457) y
(4111) w
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
> > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 1.89 1.36 1.43
0.04 0.06 0.06 0.07 0.11 0.06 0.07 0.10 0.07 0.03
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
0.05 0.05 0.05 0.07 0.05 0.05 0.07 0.06 0.08 0.09
0.09 OA2 0.10 0.10 0.10 0.10 0.11 0.23 0.11 0.15
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
0.16 0.20 0.12 0.09 0.20 0.13 0.09 0.10 0.13 0.17
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
1.52 1.23 1.08 0.96 0.93 0.90 0.88 0.84 0.82 0.67
> > "> > >
2.00 2.00 2.00 2.00 2.00
0.11 0.12 0.08 0.08 0.09
> > > > >
2.00 2.00 2.00 2.00 2.00
0.08 0.08 0.08 0.05 0.13
(313) r
adw
1 2 3 4 5 6 7 8 9 10
> > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 1.50
0.17 0.17 0.16 0.08 0.10 0.05 0.05 0.11 0.15 0.06
adr
1 2 3 4 5 6 7 8 9 10
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
ayw
1 2 3 4 5 6 7 8 9 10
0.14 0.04 0.09 0.06 0.08 0.03 0.04 0.04 0.09 0.08
ayr
1 2 3 4 5
w,
as
well
as
subspecificities, samples
for
the
but
much
samples lower
with w 4 subspecificity;
with at
w1
0.54
the other
or for
w2
the
2 samples
with
w
determinant,
subspecificity, between
showed
these 2 extreme
> > > > >
as
well
an
as
2.00 2.00 2.00 2.00 2.00
the
absorbance
situations.
1
with
w3
somewhere
208
Table III lists the results of subtyping the Japanese panel composed of 35 HBsAg-positive sera. The determinants y and r were unambiguously detectable. The determinant d was efficiently detected when it co-occurred with the determinant r. It was detected with an absorbance greater than 2.00 in 7 out of 10 samples in which it co-existed with w. The determinant w was efficiently detected in samples containing HBs/adw. The absorbance for determinant w in HBsAg/ayw samples was very variable, however. It was less than 2.00 in all 10 samples tested, distributing in the range from 1.52 to 0.67.
The expression of subtypic determinants on hybrid HBsAg particles of subtype adyr One batch of HBsAg exhibited d, y and r determinants by the enzyme immunoassay. As is depicted in Fig. 1, HBsAg/adyr was sandwiched between a n t i - H B s / d and anti-HBs/y, thereby indicating that d and y determinants co-occurred on the selfsame HBsAg particle. The expression of d and y determinants on hybrid HBsAg particles, however, was much less than that on HBsAg particles of ordinary subtypes bearing either d or y determinant (Fig. 2A-C). On the basis of determinant a, the determinant d was expressed at approximately 1 / 1 0 and the determinant y at about 1 / 4 on HBsAg/adyr particles, as compared with these subtypic determinants on HBsAg particles with ordinary subtypes. In contrast, the expression of determinant r on HBsAg/adyr was in line with that on HBsAg particles with ordinary subtypes.
Discussion
Monoclonal antibodies were raised against the common, as well as subtypic antigenic determinants of HBsAg, and applied to Subtyping HBsAg particles. Subtypes of HBsAg are accepted to represent phenotypic expression of distinct genotypes of HBV (Le Bouvier, 1972). Various strains of HBV infect some 200 million people in the world persistently, and their epidemiologic distributions are under regional influence. Based on the combination of 2 sets of mutually exclusive antigenic determinants, 4 major subtypes of HBsAg have been identified, i.e., adw, adr, ayw and ayr. There
are variants of respective determinants, particularly of w specificity, which too may be under geographical influence (Couroucr-Pauty et al.~ 1983). In actuality, the heterogeneity of w determinant was revealed by the monoclonal anti-HBs/w (no. 4111) used in the present study, also; the antibody did not always detect the determinant w efficiently. From the results of subtyping NIH panel, in which subspecificities of w were partly characterized, the monoclonal anti-HBs/w could not detect w~ and w4 as efficiently as ~) and w,. Such heterogeneity of w will be more clearly disclosed by obtaining monoclonal antibody against the respective subspecificity of w for the development of immunoassay. The reagents for subtyping HBsAg particles have been mostly polyclonal antibodies of either human, rabbit or guinea pig origin. Those antibodies had to be absorbed to make monospecific reagent, and the supply of good antisera was always very limited. The predefined, narrow specificity, taken together with an unlimited availability as required, will make monoclonal antibodies ideal reagents for the study of HBsAg subtypes worldwide. There is a possibility that subtypes of HBsAg may be applied to anthropological and cultural origin of nations. This takes advantage of the transmission of HBV by the mother-to-baby route, or through close contacts within restricted community, at least until medical practices such as transfusion and injection became routine maneuvers. We have found a gradual decrease of HBsAg/adr from south to north, along the axis of Japan, which appears to indicate the origin of Japanese ancestors in the Asian continent, most likely departing from the Korean peninsula (Yamashita et al., 1975). The research on subtypes of HBsAg is now being extended in an attempt to locate the linguistic origin of Japanese somewhere in the Asian continent. 1V~onoclonal antibodies, especially those directed to subspecificities of major subtypic determinants such as w subdeterminants, would become useful tools for an added accuracy in such studies. HBsAg particles of a hybrid subtype, adyr, carried allelic determinants, d and y, on the selfsame particle, since they allowed themselves to be sandwiched between 2 monoclonal antibodies each
209
with the specificity of a n t i - H B s / d or anti-HBs/y. Mazzur, Burgert and Le Bouvier (1975), reported the largest collection of such hybrid HBsAg particles. Nodenfelt and Le Bouvier (1974/75) have found that the expression of d and y on hybrid HBsAg particles, on the basis of the common determinant a, is less than that on ordinary HBsAg particles. C o n f i r m i n g their o b s e r v a t i o n s , HBsAg/adyr particles we studied showed much less reactivity with a n t i - H B s / d or anti-HBs/y than ordinary HBsAg particles of any of the 4 major subtypes. It is not clear, as yet, how hybrid HBsAg particles may be produced. If 2 HBV strains of subtypes adr and ayr co-infect the same hepatocytes, HBsAg polypeptides with different subtypes will be assembled in the same HBsAg particle. The expression of d and y determinants in such a particle may not remain uniform, since a fixed ratio in the population of 2 HBV strains of different subtypes may hardly be maintained. Persons who are infected with HBV of certain subtype, when infected with HBV of another subtype, usually raise antibodies to the subtypic determinant not shared by the original HBV, and clear the newly introduced HBV (Le Bouvier et al., 1976; Sasaki et al., 1976). In rare cases, however, simultaneous infection with 2 different strains of HBV has been described (Van Kooten Kok-Doorshodt et al., 1972). The study at the molecular level will shed light to the expression of subtypic determinants on hybrid HBsAg particles. For this purpose, HBsAg particles would have to be split into constituent polypeptides coded for by the S gene, P22/P27 (Peterson et al., 1977), as well as those encoded by the region pre-S (Tiollais et al., 1981) such as P31/P35 (Stibbe and Gerlich, 1982; Machida et al., 1983) and larger polypeptides (Heermann et al., 1984; Wong et al., 1985; Ohnuma et al., 1986), to see if they can be sandwiched between monoclonal antibodies directed to 2 allelic determinants. If such proves to be the case, hybrid HBsAg particles could be accepted as the product of new viral genome resulting from the recombination of 2 distinct HBV genomes, just as Nodenfelt and Le Bouvier (1974/75) proposed more than 10 years ago. Recently, complete nucleotide sequence of HBV-DNA has been determined for HBV strains
of major subtypes (Galibert et al., 1979; Valenzuela et al., 1979; Ono et al., 1983). It would be worthwhile to clone DNA from HBV with a hybrid subtype, and compare the nucleotide sequence of the S gene, as well as the region Pre-S, with those already deciphered for HBV strains of ordinary subtypes.
Acknowledgement We thank National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD, USA, for providing us with their HBsAg panel.
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210 Okada, K., I. Kamiyama, M. Inomata, M. Imai, Y. Miyakawa and M. Mayumi, 1976, N. Engl. J. Med. 294, 746. Ono, Y., H. Onda, R. Sasada, K. Igarashi, Y. Sugino and K. Nishioka, 1983, Nucleic Acids Res. 6, 1747. Peterson, D.L., I.M. Roberts and G.N. Vyas, 1977, Proc. Natl. Acad. Sci. U.S.A. 74, 1530. Sasaki, T., Y. Ohkubo, Y. Yamashita, M. Imai, Y. Miyakawa and M. Mayumi, 1976, J. lmmunol. 117, 2258. Stibbe, W. and W.H. Gerlich, 1982, Virology 123, 436. Takahashi, T., S. Nakagawa, T. Hashimoto, K. Takahashi, M. Imai, Y. Miyakawa and M. Mayumi, 1976, J, Immunol. 117, 1392.
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203
Elsevier JIM03808
A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies Sadakazu Usuda, Fumio Tsuda, Tohru Gotanda, Katsumi Tachibana, Motozumi N o m u r a *, Hiroaki Okamoto, Mitsunobu Imai, Tetsuo Nakamura, Yuzo Miyakawa and Makoto Mayumi ** Institute of lmmunology, Tokyo 113, Hepatitis Division, the Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japanese Red Cross Blood Center, Saitama-Ken 367, and Immunology Division, Jichi Medical School, Tochigi-Ken 329-04, Japan (Received 8 October 1985, accepted 30 October 1985)
Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype. Key words: Hepatitis B surface antigen; Subtype," Monoclonal antibody
Introduction
There are different strains of hepatitis B virus (HBV) that are distinguishable by the subtypic antigenic determinants expressed on hepatitis B surface antigen (HBsAg) particles. Using immunodiffusion, Le Bouvier (1971) found 2 distinct, mutually exclusive determinants on HBsAg particles, which he named d and y, in addition to the
* Dr. Nomura is presently affiliated to the Second Department of Internal Medicine, Fukui Medical College, Fukui-Ken 91011, Japan. ** Please address correspondence to: Makoto Mayumi, M.D., Immunology Division, Jichi Medical School, Minami-kawachiMachi, Tochigi-Ken 329-04, Japan.
commonly shared determinant, a. Bancroft et al. (1972) identified, also by immunodiffusion, w and r determinants, one or the other of which is carried by individual HBsAg particle in addition to d or y. Consequently, there are 4 major subtypes of HBsAg, i.e., adw, adr, ayw and ayr. Subtypes of HBsAg differ in their epidemiological distributions (Feinman et al., 1973; Mazzur, 1974; Yamashita et al., 1975), and are helpful in tracking the route of HBV infection in accidental and perinatal transmission of HBV (Mayumi and Nakajima, 1973; Okada et al., 1976). Traditionally, subtyping of HBsAg has been performed by immunodiffusion. This technique, though convenient in establishing the identity, or the lack of identity, of antigenic determinants, is
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
204 not very sensitive; it has not always been possible to characterize the subtypic specificities in materials of known HBsAg positivity. We raised monoclonal antibodies against the common and subtypic determinants of HBsAg, and developed a sandwich-type enzyme-linked immunoassay for their detection. Owing to high sensitivity in detection and assured availability of antibodies with desired specificities as required, the assay would become a useful tool in epidemiological and immunochemical studies of HBsAg.
Materials and methods
Monoclonal antibodies HBsAg particles of 3 different subtypes
(adw, adr and ayw) were isolated from plasma of asymptomatic carriers of HBV by the method described elsewhere (Takahashi et al., 1976). They were dissolved in 100 ~tl of saline at 2 mg/ml, emulsified in an equal volume of Freund's complete adjuvant and given intraperitoneally to BALB/c mice. After 2 months, mice were boosted by intraperitoneal injection with the same amount of HBsAg particles but without adjuvant. On the third day, their spleens were removed and immune spleen cells were harvested. Hybridization, subsequent culture and cloning were performed in accordance with a modification (Oi and Herzenberg, 1980) of the method originally described by Krhler and Milstein (1975). Briefly, immune spleen cells were mixed with mouse myeloma (NS-1) cells, at a ratio ranging from 5:1 to 10:1, and fused in the presence of 50% (w/v) polyethylene glycol-1500 (Wako Pure Chemicals, Osaka, Japan). Fused cells were distributed into wells of a plastic culture plate (Nunc, Roskilde, Denmark) and subjected to the selection in the medium containing hypoxanthine, aminopterine and thymidine. After 9 days of culture, the medium from each well was tested for the antibody (anti-HBs) against the common or subtypic specificity of HBsAg by the following method. Wells of an Immunoplate (Nunc) received 50 ~tl of phosphate-buffered (0.01 M, pH 7.6) saline (PBS) containing rabbit antimouse -/-globulin antibody (20 txg/ml). The plate was incubated at 4°C overnight, washed with saline
and then exposed to 40% (v/v) fetal calf serum (FCS) in order to mask unsaturated binding sites. Culture medium (50/~1) was delivered to each of 4 wells, and the plate was incubated at 37°C for 2 h. The plate was washed with saline and each well received 50/~1 of PBS supplemented with 25% FCS and containing one or other of HBsAg/adw, HBsAg/adr, HBsAg/ayw and HBsAg/ayr, purified from plasma of asymptomatic carriers, with a reversed passive hemagglutination (RPHA) titer for HBsAg of 21° (Vyas and Shulman, 1970). The plate was incubated at 37°C for 2 h and then washed with saline. Thereafter, each well received 50 /~1 of PBS supplemented with 25% FCS and containing polyclonal horse anti-HBs linked to horseradish peroxidase (Toyobo, Osaka, Japan) by the method of Nakane and Kawaoi (1974). The plate was incubated at 37°C for 2 h and washed with saline. Thereafter, each well received 50 ~1 of citrate buffer (pH 5.0) containing 0.33 /zl of 1% (v/v) HzO 2 solution and 30 ~tg of o-phenylene diamine. After 5 min, the reaction was terminated by adding 50 /xl of 4 N HzSO4, and the absorbance at 492 nm was determined. The specificity of monoclonal antibody was judged by its reaction with HBsAg particles of 4 different subtypes. Clones secreting anti-HBs of desired specificity were propagated in the peritoneal cavity of mice that had been made ascitic by the injection with 2,6,10,14-tetramethylpentadecane. Ascites fluid was harvested about 10 days after the implantation, and y-globulin fractions were precipitated with 2 M (NH4)2804, and then purified by gel filtration in Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden). Monoclonal antibodies were tested for their immunoglobulin classes by immunodiffusion against a panel of monospecific rabbit antisera (Miles Laboratories, Elkhart, IN).
Subtyping of HBsAg Wells of an Immunoplate received 100 ~l of PBS containing monoclonal anti-HBs/a (monoclonal no. 3207, 50 t~g/ml). The plate was postcoated with 40% FCS. The test sample (100 /~1) was delivered to each of 5 wells, and individual wells received 100 /zl of PBS supplemented with 25% FCS and containing monoclonal antibody with a specificity of anti-HBs/a (824), anti-HBs/d
205
(3423), a n t i - H B s / y (3457), a n t i - H B s / w (4111) or a n t i - H B s / r (313) that had been linked to peroxidase. The plate was incubated at 37°C for 2 h and washed with saline: Then, 100 /~1 of citrate buffer containing substrates were delivered to each well, and the plate was incubated at 25°C for 30 min. The reaction was terminated by adding 50/~1 of 4 N H2SO 4, and the intensity of developed color was determined by the absorbance at 492 nm. For the quantification of the determinant a of HBsAg, the amount of a required to induce an absorbance of 1.0 in the enzyme immunoassay was defined as one arbitrary unit of a. The serum containing HBsAg particles was serially diluted and tested by enzyme immunoassay, and the volume of serum that contained 1 U of a was determined from plots of the obtained absorbance. Then, the number of units contained in 1 ml of the test serum was calculated. Similarly, defining 1 U of d by an absorbance of 0.3, 1 U of y by that of 1.0, and 1 U of r by that of 0.5 in the respective enzyme immunoassay, the number of units contained in 1 ml of serum was determined for each subtypic determinant.
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. The Japanese antigen panel comprised 35 sera containing HBsAg particles subtyped by the hemagglutination inhibition method (Imai et al., 1974). All sera revealed an R P H A titer for HBsAg of 2 x2 or greater.
Results
Monoclonal antibodies against common or subtypic determinants of HBsAg Six monoclonal antibodies with a high avidity for binding to HBsAg particles were selected. Each of them was directed to the common or a subtypic antigenic determinant of HBsAg. The binding of monoclonal antibodies to HBsAg particles of 4 major subtypes that had been fixed on a solidphase support, as well as their immunoglobulin classes, are given in Table I.
Subtyping of the N I H and Japanese HBsAg panels by the enzyme immunoassay with monoclonal antibodies The N I H panel, composed of 9 sera containing HBsAg, was tested by the enzyme immunoassay with monoclonal antibodies (Table II). It can be seen that the assay detected y and r determinants efficiently; HBsAg particles bearing these determinants invariably exhibited an absorbance greater than 2.00. The determinant d was detected with
HBsA g panels The N I H panel was composed of 9 HBsAgpositive sera and specified for the subspecificities of w determinant (Courouc6 and Soulier, 1976) for 4 out of 7 sera with HBsAg particles bearing w. It was distributed by Research Resources Branch,
TABLE I SPECIFICITY OF M O N O C L O N A L ANTIBODIES D I R E C T E D TO THE C O M M O N A N D SUBTYPIC D E T E R M I N A N T S OF HBsAg Monoclonal antibodies were captured by rabbit anti-mouse ],-globulin fixed on a solid support, and tested for the binding with HBsAg particles of 4 major subtypes. HBsAg particles bound to wells via monoclonal antibodies were then detected by horse anti-HBs linked to horseradish peroxidase. Values indicate the absorbance at 492 nm. The mean + SD value for culture media without anti-HBs was 0.10 _+ 0.05. Monoclonal
HBsAg particles of subtype
no.
adw
adr
ayw
ayr
classes
Immunoglobulin
3207 824 3423 3457 4111 313
> 2.00 > 2.00 > 2.00 0.03 > 2.00 0.15
> 2.00 > 2.00 > 2.00 0.06 0.08 > 2.00
> 2.00 > 2.00 0.11 > 2.00 > 2.00 0.08
> 2.00 > 2.00 0.10 > 2.00 0.08 > 2.00
IgM, lambda IgG3, kappa IgG1, kappa IgG1, kappa IgG1, kappa IgG1, kappa
206 TABLE II D E T E R M I N A T I O N OF A N T I G E N I C SPECIFICITIES OF THE N I H HBsAg PANEL BY THE ENZYME IMMUNOASSAY W I T H MONOCLONAL ANTIBODIES The N I H panel composed of 9 sera containing HBsAg was subtyped by the enzyme immunoassay that sandwiched HBsAg particles between monoclonal anti-HBs/a (no. 3207) fixed on a solid support and another monoclonal antibody, with the specificity as indicated, linked to horseradish peroxidase. Values indicate the absorbance at 492 nm. The mean + SD value for 30 normal sera without markers of HBV infection was 0.17 _+ 0.06 for the determinant a, 0.13 _+ 0.04 for d, 0.06 ± 0.02 for y, 0.11 ± 0.04 for w, and 0.09 + 0.05 for r. Subtypes of HBsAg
Determinants detected by monoclonal antibody (no.)
adw adw 4 adr ayw ayw 1 ayw 2 ayw 3 ayr adyw
(824) a
(3423) d
> > > > > > > > >
> 2.00 1.84 1.93 0.16 0.18 0.21 0.14 0.20 0.79
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
an absorbance greater than, or close to, 2.00 except when it was expressed along with the determinant y in a single sample of subtype adyw. The absorbance for determinant w was rather variable. It was greater than 2.00 for 1 of 3 samples bearing
(3457) y
(4111) w
(313) r
0.03 0.03 0.04 2.00 2.00 2.00 2.00 2.00 2.00
> 2.00 0.54 0.04 1.52 > 2.00 > 2.00 1.36 0.06 1.72
0.17 0.03 > 2.00 0.01 0.06 0.08 0.00 > 2.00 0.02
> > > > > >
50 2O .3 i
c~
lO
5
A ) Determinant d
oo
• adw
o
o odr • oyw zx a y r D adyr
o
2 [3 I
1.00
E
1o
K
zx A
5
O.50
eq oh ~t k 0.20
~
I
B) Determinant y
2 E3 I
0.10
LU C~ 2 0
I
C3 ~ 0.05
oZX
~ 0.02
I
C) D e t e r m i n o n t r
I
I
I
I
I
I
5
4
3
2
1
0
DILUTION OF SERUM,
3N
Fig. 1. Simultaneous expression of allelic determinants, d and y, on HBsAg particles of a hybrid subtype adyr. Serum containing H B s A g / a d y r was serially diluted and sandwiched between monoclonal antibody against d, fixed on a solid support, and monoclonal antibody against y, linked to horseradish peroxidase. The bound antibody was then determined by the conversion of added substrates that developed color with an absorbance at 492 nm.
zx~ © []o o
5 zx
2 ! 2'
5'
DETERMINANT a, u m t s / m t
!
'o
20
x 10 - 4
Fig. 2. Expression of subtypic determinants on HBsAg particles of ordinary or hybrid subtype. Subtypic determinants, expressed in arbitrary units, were measured for individual batches of HBsAg particles, and plotted against the quantity of the common determinant a that also was expressed in arbitrary units.
207
T A B L E III D E T E R M I N A T I O N O F A N T I G E N I C S P E C I F I C I T I E S O F T H E J A P A N E S E H B s A g P A N E L BY T H E E N Z Y M E I M M U N O A S SAY WITH MONOCLONAL ANTIBODIES H B s A g in the J a p a n e s e p a n e l was s u b t y p e d b y the e n z y m e i m m u n o a s s a y with m o n o c l o n a l antibodies. Values i n d i c a t e the a b s o r b a n c e at 492 rim. Subtypes and no.
D e t e r m i n a n t s detected b y m o n o c l o n a l a n t i b o d y (no.) (824) a
(3423) d
(3457) y
(4111) w
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
> > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 1.89 1.36 1.43
0.04 0.06 0.06 0.07 0.11 0.06 0.07 0.10 0.07 0.03
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
0.05 0.05 0.05 0.07 0.05 0.05 0.07 0.06 0.08 0.09
0.09 OA2 0.10 0.10 0.10 0.10 0.11 0.23 0.11 0.15
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
0.16 0.20 0.12 0.09 0.20 0.13 0.09 0.10 0.13 0.17
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
1.52 1.23 1.08 0.96 0.93 0.90 0.88 0.84 0.82 0.67
> > "> > >
2.00 2.00 2.00 2.00 2.00
0.11 0.12 0.08 0.08 0.09
> > > > >
2.00 2.00 2.00 2.00 2.00
0.08 0.08 0.08 0.05 0.13
(313) r
adw
1 2 3 4 5 6 7 8 9 10
> > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 1.50
0.17 0.17 0.16 0.08 0.10 0.05 0.05 0.11 0.15 0.06
adr
1 2 3 4 5 6 7 8 9 10
> > > > > > > > > >
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
ayw
1 2 3 4 5 6 7 8 9 10
0.14 0.04 0.09 0.06 0.08 0.03 0.04 0.04 0.09 0.08
ayr
1 2 3 4 5
w,
as
well
as
subspecificities, samples
for
the
but
much
samples lower
with w 4 subspecificity;
with at
w1
0.54
the other
or for
w2
the
2 samples
with
w
determinant,
subspecificity, between
showed
these 2 extreme
> > > > >
as
well
an
as
2.00 2.00 2.00 2.00 2.00
the
absorbance
situations.
1
with
w3
somewhere
208
Table III lists the results of subtyping the Japanese panel composed of 35 HBsAg-positive sera. The determinants y and r were unambiguously detectable. The determinant d was efficiently detected when it co-occurred with the determinant r. It was detected with an absorbance greater than 2.00 in 7 out of 10 samples in which it co-existed with w. The determinant w was efficiently detected in samples containing HBs/adw. The absorbance for determinant w in HBsAg/ayw samples was very variable, however. It was less than 2.00 in all 10 samples tested, distributing in the range from 1.52 to 0.67.
The expression of subtypic determinants on hybrid HBsAg particles of subtype adyr One batch of HBsAg exhibited d, y and r determinants by the enzyme immunoassay. As is depicted in Fig. 1, HBsAg/adyr was sandwiched between a n t i - H B s / d and anti-HBs/y, thereby indicating that d and y determinants co-occurred on the selfsame HBsAg particle. The expression of d and y determinants on hybrid HBsAg particles, however, was much less than that on HBsAg particles of ordinary subtypes bearing either d or y determinant (Fig. 2A-C). On the basis of determinant a, the determinant d was expressed at approximately 1 / 1 0 and the determinant y at about 1 / 4 on HBsAg/adyr particles, as compared with these subtypic determinants on HBsAg particles with ordinary subtypes. In contrast, the expression of determinant r on HBsAg/adyr was in line with that on HBsAg particles with ordinary subtypes.
Discussion
Monoclonal antibodies were raised against the common, as well as subtypic antigenic determinants of HBsAg, and applied to Subtyping HBsAg particles. Subtypes of HBsAg are accepted to represent phenotypic expression of distinct genotypes of HBV (Le Bouvier, 1972). Various strains of HBV infect some 200 million people in the world persistently, and their epidemiologic distributions are under regional influence. Based on the combination of 2 sets of mutually exclusive antigenic determinants, 4 major subtypes of HBsAg have been identified, i.e., adw, adr, ayw and ayr. There
are variants of respective determinants, particularly of w specificity, which too may be under geographical influence (Couroucr-Pauty et al.~ 1983). In actuality, the heterogeneity of w determinant was revealed by the monoclonal anti-HBs/w (no. 4111) used in the present study, also; the antibody did not always detect the determinant w efficiently. From the results of subtyping NIH panel, in which subspecificities of w were partly characterized, the monoclonal anti-HBs/w could not detect w~ and w4 as efficiently as ~) and w,. Such heterogeneity of w will be more clearly disclosed by obtaining monoclonal antibody against the respective subspecificity of w for the development of immunoassay. The reagents for subtyping HBsAg particles have been mostly polyclonal antibodies of either human, rabbit or guinea pig origin. Those antibodies had to be absorbed to make monospecific reagent, and the supply of good antisera was always very limited. The predefined, narrow specificity, taken together with an unlimited availability as required, will make monoclonal antibodies ideal reagents for the study of HBsAg subtypes worldwide. There is a possibility that subtypes of HBsAg may be applied to anthropological and cultural origin of nations. This takes advantage of the transmission of HBV by the mother-to-baby route, or through close contacts within restricted community, at least until medical practices such as transfusion and injection became routine maneuvers. We have found a gradual decrease of HBsAg/adr from south to north, along the axis of Japan, which appears to indicate the origin of Japanese ancestors in the Asian continent, most likely departing from the Korean peninsula (Yamashita et al., 1975). The research on subtypes of HBsAg is now being extended in an attempt to locate the linguistic origin of Japanese somewhere in the Asian continent. 1V~onoclonal antibodies, especially those directed to subspecificities of major subtypic determinants such as w subdeterminants, would become useful tools for an added accuracy in such studies. HBsAg particles of a hybrid subtype, adyr, carried allelic determinants, d and y, on the selfsame particle, since they allowed themselves to be sandwiched between 2 monoclonal antibodies each
209
with the specificity of a n t i - H B s / d or anti-HBs/y. Mazzur, Burgert and Le Bouvier (1975), reported the largest collection of such hybrid HBsAg particles. Nodenfelt and Le Bouvier (1974/75) have found that the expression of d and y on hybrid HBsAg particles, on the basis of the common determinant a, is less than that on ordinary HBsAg particles. C o n f i r m i n g their o b s e r v a t i o n s , HBsAg/adyr particles we studied showed much less reactivity with a n t i - H B s / d or anti-HBs/y than ordinary HBsAg particles of any of the 4 major subtypes. It is not clear, as yet, how hybrid HBsAg particles may be produced. If 2 HBV strains of subtypes adr and ayr co-infect the same hepatocytes, HBsAg polypeptides with different subtypes will be assembled in the same HBsAg particle. The expression of d and y determinants in such a particle may not remain uniform, since a fixed ratio in the population of 2 HBV strains of different subtypes may hardly be maintained. Persons who are infected with HBV of certain subtype, when infected with HBV of another subtype, usually raise antibodies to the subtypic determinant not shared by the original HBV, and clear the newly introduced HBV (Le Bouvier et al., 1976; Sasaki et al., 1976). In rare cases, however, simultaneous infection with 2 different strains of HBV has been described (Van Kooten Kok-Doorshodt et al., 1972). The study at the molecular level will shed light to the expression of subtypic determinants on hybrid HBsAg particles. For this purpose, HBsAg particles would have to be split into constituent polypeptides coded for by the S gene, P22/P27 (Peterson et al., 1977), as well as those encoded by the region pre-S (Tiollais et al., 1981) such as P31/P35 (Stibbe and Gerlich, 1982; Machida et al., 1983) and larger polypeptides (Heermann et al., 1984; Wong et al., 1985; Ohnuma et al., 1986), to see if they can be sandwiched between monoclonal antibodies directed to 2 allelic determinants. If such proves to be the case, hybrid HBsAg particles could be accepted as the product of new viral genome resulting from the recombination of 2 distinct HBV genomes, just as Nodenfelt and Le Bouvier (1974/75) proposed more than 10 years ago. Recently, complete nucleotide sequence of HBV-DNA has been determined for HBV strains
of major subtypes (Galibert et al., 1979; Valenzuela et al., 1979; Ono et al., 1983). It would be worthwhile to clone DNA from HBV with a hybrid subtype, and compare the nucleotide sequence of the S gene, as well as the region Pre-S, with those already deciphered for HBV strains of ordinary subtypes.
Acknowledgement We thank National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD, USA, for providing us with their HBsAg panel.
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