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Studies on HBsAg binding with polymerised human serum albumin by ELISA
Journal of Virological Methods, Elsevier
16 (1987) 75-85
75
JVM 00582
Studies on HBsAg binding with. polymerised human serum albumin by ELISA M. It-shad, B.M. Gandhi, Department of Gastroenterology
T.C. Chawla, S.K. Acharya, and B.N. Tandon
Y.K. Joshi
and Human Nutrition, All India institute of Medical Sciences, New Delhi, India (Accepted
8 January
1987)
Summary A simple and sensitive ELISA was developed to characterize the interaction between polymerised human serum albumin (pHSA) and HBsAg, using pHSA-coated polyvinylmicrotitre plates as solid phase and anti-HBs-coupled HRPO as the conjugate. The interaction was found to be specific and dependent on the size of albumin polymer. pHSA-binding activity (pHSA-BA) was studied in both HBsAgnegative and HBsAg-positive sera from various liver diseases including acute viral hepatitis, fulminant hepatitis, cirrhosis of liver, chronic active hepatitis, and healthy HBsAg carriers. pHSA-BA was detected only in HBsAg-positive sera. Analysis of HBsAg-positive sera indicated pHSA-BA in high proportions of patients sera as compared to sera from healthy HBsAg carriers. pHSA-BA was detected both in the presence and absence of HBe markers, though the mean BA was relatively high in presence of HBeAg. The effect of human serum immunoglobulins (IgG, IgA, and IgM) on the BA was investigated and a correlation between pHSA-BA and HBsAg-IgM complex positiGity in sera was established. Finally, the probable role of human serum IgM in facilitating the binding process was discussed. HBsAg; pHSA; Polymer; Binding
C’orrespondence to: M. Irshad, Department of Gastroenterology and Human stitute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. Olh6-0934/&7!$03.50
0
1987 Elsevier
Science
Publishers
B.V.
(Biomedical
Nutrition.
Division)
All India
In-
76
Introduction The binding between polymerised human serum albumin and HBsAg particles was first reported by Matuhashi and Hosokawa (1972). Since then, it has been substantiated by many investigators using various immunological techniques (Imai et al., 1979; Pontisso et al., 1983; Thung and Gerber, 1981; Neurath and Strick, 1979; Hansson and Purcell, 1979). An attempt was made to establish a relation between BA and HBe markers, however, the results varied widely (Hansson and Purcell, 1979; Milich et al., 1981, Pontisso et al., 1982; Hopp et al., 1984) and their relation remained controversial. Similarly, the biological significance of this interaction and the role of different host factors, such as complement and antibodies in the binding process, is not fully understood. The present study, therefore, was undertaken to develop a sensitive and specific ELISA for further ~hara~terisation of this binding between HBsAg and albumin polymer using sera samples from different HBV infections and also to find out the possible role of various host factors in the mechanism of binding.
Materials and Methods The present study was carried out by using HBsAg-negative (25) and HBsAgpositive sera from the patients of different HBV infections which included acute viral hepatitis (44), fulminant hepatitis (14), cirrhosis of liver (lo), chronic active hepatitis (4), and the asymptomati~ HBsAg carriers (31). The diagnosis in each group of patients was confirmed clinically, serologically, and, where necessary, histologically. HBsAg-positive sera were classified into three groups: HBeAg-positive, HBeAgianti-HBe-negative, and anti-HBe-positive. HBsAg, HBeAg, and anti-HBe were tested in sera samples by commercial ELISA kits from Abbott Laboratories, USA. The HBsAg-IgM complex was determined in the sera by ELISA (Gandhi et al., 1986) using rabbit anti-human IgM-coated microtitre plate as the solid phase and anti-HBs-HRPO from Abbott HBsAg EIA kit (AUSAZYME II) as conjugate. HBsAg
and antisera
Purification and quantitation of HBsAg from the pooled plasma of HBsAg-positive carriers was carried out by the method of Vyas et al. (1972). The IgG fractions of rabbit anti-human IgG, IgM, and IgA were obtained from Dakopat Laboratory, Denmark. Polymerization
of human serum albumin
The human albumin (Sigma Chemical, USA) was polymerised by cross-linking with glut~ald~hyde according to the method of Lenkei and Ghetie (1977). In brief, albumin (50 mg) was dissolved in 1.8 ml of 0.1 M phosphate buffer (pH 6.8) and
0.2 ml of 1% or 2.5% glutaraldehyde was added. The solution was mixed and incubated at room temperature (20°C) for 4 h and then dialysed extensively against 0.1 M phosphate-buffered saline (PBS) (pH 7.2) for 18 h at 4°C. After dialysis, the solution was centrifuged and chromatographed on a Sephadex G-200 column (2 x 85 cm). The column fractions were monitored for protein content by the method of Lowry et al. (1951) and leading protein peak was pooled, dialysed against 0.1 M carbonate-bicarbonate buffer (pH 9.6), and stored as pHSA at -20°C. The molecular weight of pHSA was determined by the method of Irshad et al. (1981). The average molecular weight of albumin polymers obtained by using I% and 2.5% glutaraldehyde were 200000 and 500000, respectively. ELLSA procedure
The titative phase. 0.1 M mg/ml
BA between pHSA and HBsAg in sera samples was determined by a quanELISA using flat bottom microtitre plate from Nunc, Denmark, as the solid To prepare plate, the pHSA (molecular weight 5~~) was diluted with carbon*ate-bicarbonate buffer (pH 9.6) to a final protein concentration of 1 and to each well of microplate 100 ~1 of diluted pHSA was added and plates
CUTOFF PT. --_I_
----w-c-
0.1
o-2 HBsAg
0.3
(uglml)
0.1
0.5
Fig. 1. The effect of HBsAg concentration on pHSA-BA. 100 (~1of HBsAg solution containing varying amounts of HBsAg was added to the wells of microplate and pHSA-BA was determined. The activity was expressed as OD value. Each point represents the mean of three values and dotted line shows cutoff point.
78
incubated overnight, usually 18-20 h at room temperature. The plate was washed 4 times with PBS (pH 7.2) containing 0.5% Tween-20 (PBS-T). Additional protein binding sites were saturated by addition of 200 ~1 of 0.5% gelatin in PBS (pH 7.2) to each well and incubating the plate for 4 h at 37°C. After washing, 100 l~,l of serum sample, after 1:lO dilution with PBS (pH 7.2), was added to each well and plate incubated at 37°C for 3 h. The plate was washed and 100 ~1 of antiHBs-HRPO conjugate obtained from Abbott Laboratories, USA, was added to each well and incubated at 37°C for 2 h. After additional washings, 100 p,l of a freshly prepared solution of o-phenylene diamine (0.4 mgiml) in 0.1 M phosin dark at phate-citrate buffer (pH 5.0) was added and the plate was incubated room temperature for 15 min. The enzyme reaction was stopped by adding 100 ~1 of 4 N sulphuric acid. The absorbance was measured at 492 nm with a mini ELISA reader. The pHSA-BA, as represented by the OD value, was calculated as follows: pHSA-BA = OD of test serum - OD of reagent blank. Reagent blank was
1.0
0.8
0.6
CUTOFF
Pt.
_--M------e--
0
20
LO
60
60
100
pH SA hg/ml)
Fig. 2; Inhibition of pHSA-BA by pHSA (molecular weight 500000). The two sera positive BA (S, and S,) were preincubated with varying concentrations of pHSA and then assayed pHSA-BA expressed as OD value.
for pHSAin ELISA.
79
the 0.1 ml of o-phenylene diamine solution ~ntaining 0.1 ml of 4 Based on OD values obtained for 25 sera from healthy persons, HBV markers, a cutoff point equivalent to mean ? SD of OD culated and found to be 0.23. This value was taken arbitrarily as to show pHSA-BA.
N sulphu~c acid. negative for all values, was cala base line value
Results The present ELISA was found to be simple, highly sensitive, and specific. The minimal concentration of HBsAg detectable by this technique was 0.1 pg/ml (Fig. 1). The specificity of interaction between HBsAg and pHSA in this assay was demonstrated by the quantitative inhibition of binding by the soluble pHSA (Fig. 2). Same time, there was no binding when non-polymerised HSA and other normal human serum proteins were used to coat the wells. In addition, the pHSABA was found to be dependent on the size of albumin polymer and increased with the increasing size of polymer (Fig. 3). HSA (66000)
60
0-
0
20
10 CONCENTRATION
60
80
too
(ug/ml)
Fig. 3. Effect of albumin polymer size on pHSA-BA. Albumin polymers with molecular weight 2OOOM and 500000 along with monomeric albumin (molecular weight 66000), in varying concentrations, were preincubated with HBsAg (1 pg/ml) and then assayed in ELISA. Percent binding was calculated using binding activity in the absence of inhibitor as 100%.
80 TABLE
1
pHSA-BA
with HBsAg
in healthy
persons
and patients
with various
No. of sera
Group
pHSA-BA % nositivitv
Healthy persons HBeAg Anti-HBe Both
(+I
liver diseases
Mean
p value 2 SD
4 10 17
25 20 29.4
0.26 2 0 0.25 k 0 0.37 k 0.13
NS NS
83.3 80.0 56.3
0.94 t 0.29 0.55 I? 0.22 0.36 2 0.13
NS NS
Acute viral hepatitis HBeAg Anti-HBe Both
(-)
10 12 22
Fulminant hepatitis HBeAg Anti-HBe Both
f’) f+) f-J
6 2 6
83.3 100 83.3
0.76 k 0.64 0.50 k 0.05 0.53 ? 0.26
NS NS
Liver cirrhosis HBeAg Anti-HBe Both
(+I f+) (-1
3 1 6
100 100 33.3
0.40 i 0.12 0.28 2 0 0.40 2 0.12
NS NS
Chronic active hepatitis HBeAg
(+I
2 0 2
100 _
0.43 k 0. 15
Anti-HBe Both
(+) (-)
Statistical significance in comparison dent’s t test. NS = not significant.
to HBeAg-positive
50 group
0.25 k
0
in the same disease,
NS determined
by Stu-
The pHSA-BA was negative in all the 2.5 HBsAg-negative sera. The results of percent positivity and mean BA in HBsAg-positive sera, both from the patients group as well as the asymptomatic carriers, in relation to HBe markers have been shown in Table 1. The majority of sera from patient group, both with and without HBe markers reacted with pHSA. The mean BA, as represented by mean 2 SD values of OD, was found to be higher with HBeAg as compared to anti-HBe or in absence of any of the HBe markers, though the difference was not statistically significant. Further. the BA decreased on diluting sera samples in a dose-dependent manner, in both HBeAg+ as well as anti-HBe+ sera samples. In asymptomatic carriers, both incidence as well as mean BA was low as compared to patient group. The role of serum immunoglobulins in the binding phenomenon between pHSA and HBsAg was investigated by using inhibition ELISA. The sera samples positive for pHSA-BA were assayed before and after preincubation with variable levels of rabbit anti-human IgG, IgA, and IgM. The results are presented in Fig. 4. These results indicate that whereas anti-IgG and anti-IgA had no effect, anti-&M significantly reduced the pHSA-BA values. However, normal rabbit IgG as such could not have an effect on BA. The sera samples analysed for pHSA-BA were also
81 ANTI
IgG
60
0
I
I
t
12
6
3
f-5
ANTIBODY
DILUTION
I
I
f
O-6
I
04
o*2xt~3
Fig. 4. Effect of rabbit anti-human IgG, IgA, and IgM on pHSA-BA. Each antibody, in varying dilutions, was preincubated with pHSA-BA positive serum and then BA was determined as described in methods. Percent binding activity was calculated using binding activity in absence of antibody as 100%.
tested for the presence of HBsAg-IgM complexes. The results of percent positivity of HBsAg-IgM complex are shown in Table 2. The relative incidence of HBsAg-IgM complex in sera with positive pHSA-BA, as compared to those with
TABLE
2
Relationship
between
pHSA-BA
and HBsAg-IgM
Q/c positivity
Acute viral hepatitis Fulminant hepatitis Liver cirrhosis Chronic active hepatitis Healthy carriers Statistical tion.
of HBsAg-IgM
Sera with positive
Group
significance
complex
positivity
complexes
pHSA-BA
in sera.
in sera
S&a with negative
pHSA-BA
No. of cases
o/o positivity
No. of cases
o/r positivity
P vsluc
26 10 3 2 8
72.2 60 100 IOU 75
10 2 4 1 22
14.3 0 25 0 27.2
0.05 NS NS NS NS
in comparison
to pHSA-BA
positive
cases was calculated
using test of propor-
82
negative binding, was quite high. In AVH, this difference was statistically significant (P ~0.05). However, in other groups it is not significant.
Discussion The binding between pHSA and HBsAg has been studied in more detail during the last few years. Recent evidence suggests that pHSA binding with HBsAg plays a very vital role in the viral uptake by hepatocytes during the infectious process (Thung and Gerber, 1984). However, the exact mechanism of the binding and the role of various host factors in facilitating the attachment of virus on hepatocyte surface is not completely known. The present study, therefore, was planned to develop a new ELISA of high sensitivity to further characterise the BA using HBsAgnegative and HBsAg-positive sera from healthy persons and groups of patients with different HBV infections. This ELISA was found to be highly sensitive (0.1 kg/ml) with greater sensitivity even than radioimmunoassay as described by Milich et al. (1981). Binding between pHSA and HBsAg in this assay was observed to be specific and dependent on the size of albumin polymer. In agreement to the previous reports, BA increased with increasing size of polymer. Monomeric albumin as such did not show BA, thus pointing out that multisite attachment of pHSA with HBsAg is needed to stabilize binding between the two. The results of pHSA-BA in HBsAg carriers both from healthy groups as well as disease groups demonstrated that pHSA-BA positivity was present in large proportions in the latter group. Furthermore, the binding was recorded both in the presence and absence of HBe markers. This is in contrast to previous reports (Pontisso et al., 1983; Milich et al., 1981; Neill, 1979; Thung and Gerber, 1981) where pHSA-BA was detected mainly in association of HBeAg and was either absent or quite low with anti-HBe and in absence of HBe markers. One possibility of this difference was presumed to be the high sensitivity of the present test system and thus pHSA-BA could be demonstrated even in the absence of HBe-markers or presence of anti-HBe where HBsAg level is usually very low. This was further supported by the BA decreasing in a dose-dependent manner on diluting the sera samples (Irshad et al., 1986). Thus, BA seems to be a function of HBsAg level in serum. However, these results contradict the findings of Hopf et al. (1984) who demonstrated an inverse relation between the BA and serum level of HBsAg. According to them (Hopf et al.; 1984), BA depends on the size or type of HBsAg particles and not their serum concentrations. HBsAg particles showing BA, have definite receptors for pHSA (pHSA receptors) (Machida et al., 1983). Such a pHSA receptor was characterised and found to be the N-terminal extension of HBsAg (Machida et al., 1984). This polypeptide whose amino acid sequence is encoded by the pre-S2 region of HBV-DNA has 55 amino acids and a molecular weight of 8000 (William et al., 1984). The HBsAg particles with receptors were reported to be present in only HBeAg-positive sera and not anti-HBe-positive sera. But in few other reports (Milich et al., 1981) including our present series, pHSA binding was observed both with and without HBe markers. This implies that HBsAg molecules
83
having receptors are present with anti-HBe also but in comparatively low concentrations and could be detected only by the sensitive method. The presence of BA in absence of HBe markers points out that sampling time may correspond to the ‘e-window’ period where HBsAg is present in absence of HBe markers and shows BA. The high values of mean BA in presence of HBeAg as compared to anti-HBe emphasises that concentration of specific HBsAg is comparatively high during the period of viral multiplication. Similarly, high incidence of pHSA binding in disease groups, as compared to asymptomatic carriers, give rise to the speculation that only HBsAg particles with binding tendency are responsible for the pathogenesis of liver. The nature of pHSA binding with HBsAg was also found to be dependent on certain host factors in addition to the pHSA receptors. The majority of them were anti-pHSA (Lenkei et al., 1974; 1977) and complement system (Hopf et al., 1984). In the present study, we attempted to investigate the possible association of human serum immunoglobulins (IgG, IgA and IgM) in the binding process. The results of inhibition assay (Fig 4) explained that whereas human serum IgG and IgA are ineffective, IgM possibly participates in the interaction between pHSA and HBsAg. This was further supported by the high incidence of pHSA-BA in sera samples positive for HBsAg-IgM complex as compared to those negative for this complex (AVH: PcO.05) (Table 2). Hopf et al. (1984) demonstrated that HBsAg particles with positive pHSA-BA have associated proteins. Later, it was confirmed (Nowoslawski, 1979; Careoda et al., 1982) that HBsAg in sera positive for pHSABA circulate as HBsAg-IgM complexes. Since IgM antibody present in HBsAg-IgM complex has been characterised to be an antibody specific for serum proteins (Palla et al., 1983) and not the HBsAg, it was presumed that IgM represents antibody against pHSA that is formed mainly during liver diseases (Rothschild and Schreiher, 1969; Peters, 1970) and does not preexist in the blood. The IgM has high affinity to bind HBsAg or HBV nonspecifically and thus keeps HBsAg-HBV as HBsAg or HBV-IgM complex. In the binding process between HBsAg or HBV and pHSA, it further strengthens the linkage through its interaction with pHSA. Notably, IgM is not solely responsible for binding between HBsAg-HBV and pHSA, rather it helps partially in the binding phenomenon. It is proved by our data (Fig. 4) where BA could not be completely reversed even by the higher concentration of anti-IgM. Thus, the major binding between HBsAg and pHSA occurs through pHSA receptors and IgM further enhances this binding. This supports the hypothesis that human serum IgM plays a contributing role in the binding phenomenon. The findings of different workers (Hopf et al., 1984) that only large HBsAg particles bind to pHSA is also endorsed by this hypothesis and thus large particles were presumed to be HBsAg-HBV with associated proteins, i.e., HBsAgor HBV-IgM complex. However, this in vitro study does not confirm the occurrence of an identical in vivo binding between HBsAg and pHSA leading to the attachment of HBV to liver cells, as the pHSA level in. blood is usually very low. This shows that such binding may be possible in vivo also, in case adequate pHSA level is attained in blood. In conclusion, the ELISA described in this paper is sensitive enough to detect
84
the binding between HBsAg and pHSA in sera samples from different HBV infections. The binding activity is detectable both in the presence as well as absence of HBe markers. Binding occurs mainly through pHSA receptors but it is further strengthened by the serum IgM associated with HBsAg or HBV nonspecifically.
References Careoda, F., De Franchis, R., Monforte, A.A., Vecchi, M., Rossi, E., Primignani, M., Palla, M. and Dioguardi, N. (1982) Persistence of circulating HBsAgiIgM complexes in acute viral hepatitis Type B; an early marker of chronic evolution. Lancet ii 358-360. Gandhi, B.M., Gupta, H., Irshad, M., Joshi, Y.K. and Tandon, B.N. (1986) Significance of circulating HBsAgiIgM complexes in viral hepatitis type B. Indian J. Med. Res. 84, 27-31. Hansson, B.G. and Purcelf, R.H. (1979) Sites that bind polymerised albumin on hepatitis B surEace antigen particles. Detection by radioimmunoassay. Infect. Immun. 26, 125-130. Hopf, U., Moller, B., Schermer, M. and Lobeck, If. (1984) Binding activity of HBsAg particles from chronic HBsAg carriers to polystyrene beads coated with polymerised human serum albumin: diagnostic relevance of the assay. Liver 4, 372-378. Imai, M., Yanese, Y., Nojiri, T., Miyakawa, Y. and Mayumi, M. (1979) A receptor for polymerised human and chimpanzee albumin on hepatitis B virus particles occuring with hepatitis B antigen. Gastroenterology 76, 242-247. Irshad, M., Khan, M.Y. and Salahuddin, A. (1981) Salting out behaviour of buffalo immunoglobulin C. Indian J. Biochem. Biophys. 18, 264-268. Irshad, M., Gandhi, B.M., Acharya, S.K. and Tandon, B.N. (1986) Relation between HBsAg binding with polym~rised human serum albumiq {poly-HSA) and H3V-replication. Intervirology, in press. Lowry, O.H., Rosebrough, NJ., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Bioi. Chem. 193, 265-275. Lenkei, R., Mota, G., Dan, M.E. and Kaky, M. (1974) The polymerised albumin and antialbumin autoantibodies in patients with hepatic diseases. Rev. Roum. Biochim. 11, 271-276. Lenkei, R., Babes, V.T., Dan, M.E., Mustea, A. and Dobre, I. (1977) Correlation between anti-albumin antibodies and HBsAg in hepatic patients. J. Med. Virol. 1, 29-34. Lcnkei, R. and Ghetie, V. (1977) Methods for detection of anti-albumin autoantibodies in hepatic diseases. J. Immunol. Methods 16, 23-30. Matuhasi, T. and Hosokawa, 2. (1972) Reactants to human serum albumin-coated red cells in Au (l)positive sera. Jpn. J. Exp. Med. 42, 183-185. Milich, D.R., Gottfied, T.D. and Vyas, G.N. (1981) Characterization of interaction between polymerised human albumin and hepatitis B surface antigen. Gastroenterology 81, 218-225. Machida, A., Kishimoto, S. and Ohnuma, H. (1983) A hepatitis B surface antigen polypeptide (P31) with the receptor for polymerised human as well as chimpanzee albumin. Gastroenterology 85, 26&274. Machida, A., Kishimoto, S. and Ohnuma, H. (1984) A polypeptide containing 5.5 amino acid residues coded by the pre-S-region of hepatitis B virus deoxyribonucleic acid bears the receptor for polymerised human as well as chimpanzee albumins. Gastroenterology 86, 910-918. Neurath, A.R. and Strick, N. (1979) Radioimmunoassay for albumin binding sites associated with HBsAg: Correlation of results with the presence of e-antigen in serum. Intervirology 11, 128. Neill, S.P. (19’79) Interaction of hepatitis B surface antigen with polymerised human serum albumin. J. Med. Viral. 4, 177-185. Nowoslawski, A. (1979) Hepatitis B virus induced immune complex disease In: Progress in Liver Disease (Popper, I-I., Schaffner, F., eds.), pp. 393-406, Grune and Stratton, New York and London. Peters, Jr., T. (1970) Serum albumin. Adv. Clin. Chem. 13, 37-111. Pontisso, P., Albert, A.. Schiavon, E. and Realdi, G. (1982) Receptors for polymerised human serum albumin (#ISA) on hepatitis B virus (HBV) particles. Abstracts of 17th Meeting of the European Association for the Study of the Liver, Goteborg, Sweden, 1982.
85 Pontisso, P., Albert, A., Bortolotti, F. and Realdi, G. (1983) Virus associated receptors for polymerised human serum albumin in acute and in chronic hepatitis B virus infection. Gastroenterology 84, 220-226. Palla, M., Rizzi, R., Almi, P., Rizzetto, M., Bonino, F. and Purcell, R. (1983) Complexes of hepatitis B surface antigen and immunoglobulin M in the sera of patients with hepatitis B viral infection. Infect. Immun. 41, 950-958. Rothschild, M.A. and Schreiber, S.S. (1969) Serum albumin. Am. J. Dig. Dis. 14, 711-744. Thung, S.N. and Gerber, M.A. (1981) HBsAg associated albumin receptors and anti-albumin antibodies in sera of patients with liver disease. Gastroenterology 80, 260-264. Thung, S.N. and Gerber,_M.A. (1981) Specificities of albumin receptors and albumin antibodies. Infect. Immun. 32, 1292-94. Thung, S.N. and Gerber, M.A. (1984) Polyalbumin receptors: their role in the attachment of hepatitis B virus to hepatocyte. Semin. Liver Dis. 4, 69-75. Vyas, G.N., Williams, E.W., Klaus, G.G.B. and Bond, H.E. (1972) Hepatitis associated Australia antigen - protein, peptides and amino acid composition of purified antigen with its use in determining sensitivity of the hemagglutination tests. J. Immunol. 108, 111+1116. William, J.R., Marilyn, Z., James, O., Yousef, S., Orgad, L., Pablo, G. and David, N.S. (1984) Transcription units of hepatitis B virus genes and structure and expression of integrated viral sequences. In: Viral Hepatitis and Liver Disease (Vyas, G.N., Dienstag, J.L. and Hoofnagle, J.H., eds.), pp. 67-86, Grune and Stratton, New York and London.
16 (1987) 75-85
75
JVM 00582
Studies on HBsAg binding with. polymerised human serum albumin by ELISA M. It-shad, B.M. Gandhi, Department of Gastroenterology
T.C. Chawla, S.K. Acharya, and B.N. Tandon
Y.K. Joshi
and Human Nutrition, All India institute of Medical Sciences, New Delhi, India (Accepted
8 January
1987)
Summary A simple and sensitive ELISA was developed to characterize the interaction between polymerised human serum albumin (pHSA) and HBsAg, using pHSA-coated polyvinylmicrotitre plates as solid phase and anti-HBs-coupled HRPO as the conjugate. The interaction was found to be specific and dependent on the size of albumin polymer. pHSA-binding activity (pHSA-BA) was studied in both HBsAgnegative and HBsAg-positive sera from various liver diseases including acute viral hepatitis, fulminant hepatitis, cirrhosis of liver, chronic active hepatitis, and healthy HBsAg carriers. pHSA-BA was detected only in HBsAg-positive sera. Analysis of HBsAg-positive sera indicated pHSA-BA in high proportions of patients sera as compared to sera from healthy HBsAg carriers. pHSA-BA was detected both in the presence and absence of HBe markers, though the mean BA was relatively high in presence of HBeAg. The effect of human serum immunoglobulins (IgG, IgA, and IgM) on the BA was investigated and a correlation between pHSA-BA and HBsAg-IgM complex positiGity in sera was established. Finally, the probable role of human serum IgM in facilitating the binding process was discussed. HBsAg; pHSA; Polymer; Binding
C’orrespondence to: M. Irshad, Department of Gastroenterology and Human stitute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. Olh6-0934/&7!$03.50
0
1987 Elsevier
Science
Publishers
B.V.
(Biomedical
Nutrition.
Division)
All India
In-
76
Introduction The binding between polymerised human serum albumin and HBsAg particles was first reported by Matuhashi and Hosokawa (1972). Since then, it has been substantiated by many investigators using various immunological techniques (Imai et al., 1979; Pontisso et al., 1983; Thung and Gerber, 1981; Neurath and Strick, 1979; Hansson and Purcell, 1979). An attempt was made to establish a relation between BA and HBe markers, however, the results varied widely (Hansson and Purcell, 1979; Milich et al., 1981, Pontisso et al., 1982; Hopp et al., 1984) and their relation remained controversial. Similarly, the biological significance of this interaction and the role of different host factors, such as complement and antibodies in the binding process, is not fully understood. The present study, therefore, was undertaken to develop a sensitive and specific ELISA for further ~hara~terisation of this binding between HBsAg and albumin polymer using sera samples from different HBV infections and also to find out the possible role of various host factors in the mechanism of binding.
Materials and Methods The present study was carried out by using HBsAg-negative (25) and HBsAgpositive sera from the patients of different HBV infections which included acute viral hepatitis (44), fulminant hepatitis (14), cirrhosis of liver (lo), chronic active hepatitis (4), and the asymptomati~ HBsAg carriers (31). The diagnosis in each group of patients was confirmed clinically, serologically, and, where necessary, histologically. HBsAg-positive sera were classified into three groups: HBeAg-positive, HBeAgianti-HBe-negative, and anti-HBe-positive. HBsAg, HBeAg, and anti-HBe were tested in sera samples by commercial ELISA kits from Abbott Laboratories, USA. The HBsAg-IgM complex was determined in the sera by ELISA (Gandhi et al., 1986) using rabbit anti-human IgM-coated microtitre plate as the solid phase and anti-HBs-HRPO from Abbott HBsAg EIA kit (AUSAZYME II) as conjugate. HBsAg
and antisera
Purification and quantitation of HBsAg from the pooled plasma of HBsAg-positive carriers was carried out by the method of Vyas et al. (1972). The IgG fractions of rabbit anti-human IgG, IgM, and IgA were obtained from Dakopat Laboratory, Denmark. Polymerization
of human serum albumin
The human albumin (Sigma Chemical, USA) was polymerised by cross-linking with glut~ald~hyde according to the method of Lenkei and Ghetie (1977). In brief, albumin (50 mg) was dissolved in 1.8 ml of 0.1 M phosphate buffer (pH 6.8) and
0.2 ml of 1% or 2.5% glutaraldehyde was added. The solution was mixed and incubated at room temperature (20°C) for 4 h and then dialysed extensively against 0.1 M phosphate-buffered saline (PBS) (pH 7.2) for 18 h at 4°C. After dialysis, the solution was centrifuged and chromatographed on a Sephadex G-200 column (2 x 85 cm). The column fractions were monitored for protein content by the method of Lowry et al. (1951) and leading protein peak was pooled, dialysed against 0.1 M carbonate-bicarbonate buffer (pH 9.6), and stored as pHSA at -20°C. The molecular weight of pHSA was determined by the method of Irshad et al. (1981). The average molecular weight of albumin polymers obtained by using I% and 2.5% glutaraldehyde were 200000 and 500000, respectively. ELLSA procedure
The titative phase. 0.1 M mg/ml
BA between pHSA and HBsAg in sera samples was determined by a quanELISA using flat bottom microtitre plate from Nunc, Denmark, as the solid To prepare plate, the pHSA (molecular weight 5~~) was diluted with carbon*ate-bicarbonate buffer (pH 9.6) to a final protein concentration of 1 and to each well of microplate 100 ~1 of diluted pHSA was added and plates
CUTOFF PT. --_I_
----w-c-
0.1
o-2 HBsAg
0.3
(uglml)
0.1
0.5
Fig. 1. The effect of HBsAg concentration on pHSA-BA. 100 (~1of HBsAg solution containing varying amounts of HBsAg was added to the wells of microplate and pHSA-BA was determined. The activity was expressed as OD value. Each point represents the mean of three values and dotted line shows cutoff point.
78
incubated overnight, usually 18-20 h at room temperature. The plate was washed 4 times with PBS (pH 7.2) containing 0.5% Tween-20 (PBS-T). Additional protein binding sites were saturated by addition of 200 ~1 of 0.5% gelatin in PBS (pH 7.2) to each well and incubating the plate for 4 h at 37°C. After washing, 100 l~,l of serum sample, after 1:lO dilution with PBS (pH 7.2), was added to each well and plate incubated at 37°C for 3 h. The plate was washed and 100 ~1 of antiHBs-HRPO conjugate obtained from Abbott Laboratories, USA, was added to each well and incubated at 37°C for 2 h. After additional washings, 100 p,l of a freshly prepared solution of o-phenylene diamine (0.4 mgiml) in 0.1 M phosin dark at phate-citrate buffer (pH 5.0) was added and the plate was incubated room temperature for 15 min. The enzyme reaction was stopped by adding 100 ~1 of 4 N sulphuric acid. The absorbance was measured at 492 nm with a mini ELISA reader. The pHSA-BA, as represented by the OD value, was calculated as follows: pHSA-BA = OD of test serum - OD of reagent blank. Reagent blank was
1.0
0.8
0.6
CUTOFF
Pt.
_--M------e--
0
20
LO
60
60
100
pH SA hg/ml)
Fig. 2; Inhibition of pHSA-BA by pHSA (molecular weight 500000). The two sera positive BA (S, and S,) were preincubated with varying concentrations of pHSA and then assayed pHSA-BA expressed as OD value.
for pHSAin ELISA.
79
the 0.1 ml of o-phenylene diamine solution ~ntaining 0.1 ml of 4 Based on OD values obtained for 25 sera from healthy persons, HBV markers, a cutoff point equivalent to mean ? SD of OD culated and found to be 0.23. This value was taken arbitrarily as to show pHSA-BA.
N sulphu~c acid. negative for all values, was cala base line value
Results The present ELISA was found to be simple, highly sensitive, and specific. The minimal concentration of HBsAg detectable by this technique was 0.1 pg/ml (Fig. 1). The specificity of interaction between HBsAg and pHSA in this assay was demonstrated by the quantitative inhibition of binding by the soluble pHSA (Fig. 2). Same time, there was no binding when non-polymerised HSA and other normal human serum proteins were used to coat the wells. In addition, the pHSABA was found to be dependent on the size of albumin polymer and increased with the increasing size of polymer (Fig. 3). HSA (66000)
60
0-
0
20
10 CONCENTRATION
60
80
too
(ug/ml)
Fig. 3. Effect of albumin polymer size on pHSA-BA. Albumin polymers with molecular weight 2OOOM and 500000 along with monomeric albumin (molecular weight 66000), in varying concentrations, were preincubated with HBsAg (1 pg/ml) and then assayed in ELISA. Percent binding was calculated using binding activity in the absence of inhibitor as 100%.
80 TABLE
1
pHSA-BA
with HBsAg
in healthy
persons
and patients
with various
No. of sera
Group
pHSA-BA % nositivitv
Healthy persons HBeAg Anti-HBe Both
(+I
liver diseases
Mean
p value 2 SD
4 10 17
25 20 29.4
0.26 2 0 0.25 k 0 0.37 k 0.13
NS NS
83.3 80.0 56.3
0.94 t 0.29 0.55 I? 0.22 0.36 2 0.13
NS NS
Acute viral hepatitis HBeAg Anti-HBe Both
(-)
10 12 22
Fulminant hepatitis HBeAg Anti-HBe Both
f’) f+) f-J
6 2 6
83.3 100 83.3
0.76 k 0.64 0.50 k 0.05 0.53 ? 0.26
NS NS
Liver cirrhosis HBeAg Anti-HBe Both
(+I f+) (-1
3 1 6
100 100 33.3
0.40 i 0.12 0.28 2 0 0.40 2 0.12
NS NS
Chronic active hepatitis HBeAg
(+I
2 0 2
100 _
0.43 k 0. 15
Anti-HBe Both
(+) (-)
Statistical significance in comparison dent’s t test. NS = not significant.
to HBeAg-positive
50 group
0.25 k
0
in the same disease,
NS determined
by Stu-
The pHSA-BA was negative in all the 2.5 HBsAg-negative sera. The results of percent positivity and mean BA in HBsAg-positive sera, both from the patients group as well as the asymptomatic carriers, in relation to HBe markers have been shown in Table 1. The majority of sera from patient group, both with and without HBe markers reacted with pHSA. The mean BA, as represented by mean 2 SD values of OD, was found to be higher with HBeAg as compared to anti-HBe or in absence of any of the HBe markers, though the difference was not statistically significant. Further. the BA decreased on diluting sera samples in a dose-dependent manner, in both HBeAg+ as well as anti-HBe+ sera samples. In asymptomatic carriers, both incidence as well as mean BA was low as compared to patient group. The role of serum immunoglobulins in the binding phenomenon between pHSA and HBsAg was investigated by using inhibition ELISA. The sera samples positive for pHSA-BA were assayed before and after preincubation with variable levels of rabbit anti-human IgG, IgA, and IgM. The results are presented in Fig. 4. These results indicate that whereas anti-IgG and anti-IgA had no effect, anti-&M significantly reduced the pHSA-BA values. However, normal rabbit IgG as such could not have an effect on BA. The sera samples analysed for pHSA-BA were also
81 ANTI
IgG
60
0
I
I
t
12
6
3
f-5
ANTIBODY
DILUTION
I
I
f
O-6
I
04
o*2xt~3
Fig. 4. Effect of rabbit anti-human IgG, IgA, and IgM on pHSA-BA. Each antibody, in varying dilutions, was preincubated with pHSA-BA positive serum and then BA was determined as described in methods. Percent binding activity was calculated using binding activity in absence of antibody as 100%.
tested for the presence of HBsAg-IgM complexes. The results of percent positivity of HBsAg-IgM complex are shown in Table 2. The relative incidence of HBsAg-IgM complex in sera with positive pHSA-BA, as compared to those with
TABLE
2
Relationship
between
pHSA-BA
and HBsAg-IgM
Q/c positivity
Acute viral hepatitis Fulminant hepatitis Liver cirrhosis Chronic active hepatitis Healthy carriers Statistical tion.
of HBsAg-IgM
Sera with positive
Group
significance
complex
positivity
complexes
pHSA-BA
in sera.
in sera
S&a with negative
pHSA-BA
No. of cases
o/o positivity
No. of cases
o/r positivity
P vsluc
26 10 3 2 8
72.2 60 100 IOU 75
10 2 4 1 22
14.3 0 25 0 27.2
0.05 NS NS NS NS
in comparison
to pHSA-BA
positive
cases was calculated
using test of propor-
82
negative binding, was quite high. In AVH, this difference was statistically significant (P ~0.05). However, in other groups it is not significant.
Discussion The binding between pHSA and HBsAg has been studied in more detail during the last few years. Recent evidence suggests that pHSA binding with HBsAg plays a very vital role in the viral uptake by hepatocytes during the infectious process (Thung and Gerber, 1984). However, the exact mechanism of the binding and the role of various host factors in facilitating the attachment of virus on hepatocyte surface is not completely known. The present study, therefore, was planned to develop a new ELISA of high sensitivity to further characterise the BA using HBsAgnegative and HBsAg-positive sera from healthy persons and groups of patients with different HBV infections. This ELISA was found to be highly sensitive (0.1 kg/ml) with greater sensitivity even than radioimmunoassay as described by Milich et al. (1981). Binding between pHSA and HBsAg in this assay was observed to be specific and dependent on the size of albumin polymer. In agreement to the previous reports, BA increased with increasing size of polymer. Monomeric albumin as such did not show BA, thus pointing out that multisite attachment of pHSA with HBsAg is needed to stabilize binding between the two. The results of pHSA-BA in HBsAg carriers both from healthy groups as well as disease groups demonstrated that pHSA-BA positivity was present in large proportions in the latter group. Furthermore, the binding was recorded both in the presence and absence of HBe markers. This is in contrast to previous reports (Pontisso et al., 1983; Milich et al., 1981; Neill, 1979; Thung and Gerber, 1981) where pHSA-BA was detected mainly in association of HBeAg and was either absent or quite low with anti-HBe and in absence of HBe markers. One possibility of this difference was presumed to be the high sensitivity of the present test system and thus pHSA-BA could be demonstrated even in the absence of HBe-markers or presence of anti-HBe where HBsAg level is usually very low. This was further supported by the BA decreasing in a dose-dependent manner on diluting the sera samples (Irshad et al., 1986). Thus, BA seems to be a function of HBsAg level in serum. However, these results contradict the findings of Hopf et al. (1984) who demonstrated an inverse relation between the BA and serum level of HBsAg. According to them (Hopf et al.; 1984), BA depends on the size or type of HBsAg particles and not their serum concentrations. HBsAg particles showing BA, have definite receptors for pHSA (pHSA receptors) (Machida et al., 1983). Such a pHSA receptor was characterised and found to be the N-terminal extension of HBsAg (Machida et al., 1984). This polypeptide whose amino acid sequence is encoded by the pre-S2 region of HBV-DNA has 55 amino acids and a molecular weight of 8000 (William et al., 1984). The HBsAg particles with receptors were reported to be present in only HBeAg-positive sera and not anti-HBe-positive sera. But in few other reports (Milich et al., 1981) including our present series, pHSA binding was observed both with and without HBe markers. This implies that HBsAg molecules
83
having receptors are present with anti-HBe also but in comparatively low concentrations and could be detected only by the sensitive method. The presence of BA in absence of HBe markers points out that sampling time may correspond to the ‘e-window’ period where HBsAg is present in absence of HBe markers and shows BA. The high values of mean BA in presence of HBeAg as compared to anti-HBe emphasises that concentration of specific HBsAg is comparatively high during the period of viral multiplication. Similarly, high incidence of pHSA binding in disease groups, as compared to asymptomatic carriers, give rise to the speculation that only HBsAg particles with binding tendency are responsible for the pathogenesis of liver. The nature of pHSA binding with HBsAg was also found to be dependent on certain host factors in addition to the pHSA receptors. The majority of them were anti-pHSA (Lenkei et al., 1974; 1977) and complement system (Hopf et al., 1984). In the present study, we attempted to investigate the possible association of human serum immunoglobulins (IgG, IgA and IgM) in the binding process. The results of inhibition assay (Fig 4) explained that whereas human serum IgG and IgA are ineffective, IgM possibly participates in the interaction between pHSA and HBsAg. This was further supported by the high incidence of pHSA-BA in sera samples positive for HBsAg-IgM complex as compared to those negative for this complex (AVH: PcO.05) (Table 2). Hopf et al. (1984) demonstrated that HBsAg particles with positive pHSA-BA have associated proteins. Later, it was confirmed (Nowoslawski, 1979; Careoda et al., 1982) that HBsAg in sera positive for pHSABA circulate as HBsAg-IgM complexes. Since IgM antibody present in HBsAg-IgM complex has been characterised to be an antibody specific for serum proteins (Palla et al., 1983) and not the HBsAg, it was presumed that IgM represents antibody against pHSA that is formed mainly during liver diseases (Rothschild and Schreiher, 1969; Peters, 1970) and does not preexist in the blood. The IgM has high affinity to bind HBsAg or HBV nonspecifically and thus keeps HBsAg-HBV as HBsAg or HBV-IgM complex. In the binding process between HBsAg or HBV and pHSA, it further strengthens the linkage through its interaction with pHSA. Notably, IgM is not solely responsible for binding between HBsAg-HBV and pHSA, rather it helps partially in the binding phenomenon. It is proved by our data (Fig. 4) where BA could not be completely reversed even by the higher concentration of anti-IgM. Thus, the major binding between HBsAg and pHSA occurs through pHSA receptors and IgM further enhances this binding. This supports the hypothesis that human serum IgM plays a contributing role in the binding phenomenon. The findings of different workers (Hopf et al., 1984) that only large HBsAg particles bind to pHSA is also endorsed by this hypothesis and thus large particles were presumed to be HBsAg-HBV with associated proteins, i.e., HBsAgor HBV-IgM complex. However, this in vitro study does not confirm the occurrence of an identical in vivo binding between HBsAg and pHSA leading to the attachment of HBV to liver cells, as the pHSA level in. blood is usually very low. This shows that such binding may be possible in vivo also, in case adequate pHSA level is attained in blood. In conclusion, the ELISA described in this paper is sensitive enough to detect
84
the binding between HBsAg and pHSA in sera samples from different HBV infections. The binding activity is detectable both in the presence as well as absence of HBe markers. Binding occurs mainly through pHSA receptors but it is further strengthened by the serum IgM associated with HBsAg or HBV nonspecifically.
References Careoda, F., De Franchis, R., Monforte, A.A., Vecchi, M., Rossi, E., Primignani, M., Palla, M. and Dioguardi, N. (1982) Persistence of circulating HBsAgiIgM complexes in acute viral hepatitis Type B; an early marker of chronic evolution. Lancet ii 358-360. Gandhi, B.M., Gupta, H., Irshad, M., Joshi, Y.K. and Tandon, B.N. (1986) Significance of circulating HBsAgiIgM complexes in viral hepatitis type B. Indian J. Med. Res. 84, 27-31. Hansson, B.G. and Purcelf, R.H. (1979) Sites that bind polymerised albumin on hepatitis B surEace antigen particles. Detection by radioimmunoassay. Infect. Immun. 26, 125-130. Hopf, U., Moller, B., Schermer, M. and Lobeck, If. (1984) Binding activity of HBsAg particles from chronic HBsAg carriers to polystyrene beads coated with polymerised human serum albumin: diagnostic relevance of the assay. Liver 4, 372-378. Imai, M., Yanese, Y., Nojiri, T., Miyakawa, Y. and Mayumi, M. (1979) A receptor for polymerised human and chimpanzee albumin on hepatitis B virus particles occuring with hepatitis B antigen. Gastroenterology 76, 242-247. Irshad, M., Khan, M.Y. and Salahuddin, A. (1981) Salting out behaviour of buffalo immunoglobulin C. Indian J. Biochem. Biophys. 18, 264-268. Irshad, M., Gandhi, B.M., Acharya, S.K. and Tandon, B.N. (1986) Relation between HBsAg binding with polym~rised human serum albumiq {poly-HSA) and H3V-replication. Intervirology, in press. Lowry, O.H., Rosebrough, NJ., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Bioi. Chem. 193, 265-275. Lenkei, R., Mota, G., Dan, M.E. and Kaky, M. (1974) The polymerised albumin and antialbumin autoantibodies in patients with hepatic diseases. Rev. Roum. Biochim. 11, 271-276. Lenkei, R., Babes, V.T., Dan, M.E., Mustea, A. and Dobre, I. (1977) Correlation between anti-albumin antibodies and HBsAg in hepatic patients. J. Med. Virol. 1, 29-34. Lcnkei, R. and Ghetie, V. (1977) Methods for detection of anti-albumin autoantibodies in hepatic diseases. J. Immunol. Methods 16, 23-30. Matuhasi, T. and Hosokawa, 2. (1972) Reactants to human serum albumin-coated red cells in Au (l)positive sera. Jpn. J. Exp. Med. 42, 183-185. Milich, D.R., Gottfied, T.D. and Vyas, G.N. (1981) Characterization of interaction between polymerised human albumin and hepatitis B surface antigen. Gastroenterology 81, 218-225. Machida, A., Kishimoto, S. and Ohnuma, H. (1983) A hepatitis B surface antigen polypeptide (P31) with the receptor for polymerised human as well as chimpanzee albumin. Gastroenterology 85, 26&274. Machida, A., Kishimoto, S. and Ohnuma, H. (1984) A polypeptide containing 5.5 amino acid residues coded by the pre-S-region of hepatitis B virus deoxyribonucleic acid bears the receptor for polymerised human as well as chimpanzee albumins. Gastroenterology 86, 910-918. Neurath, A.R. and Strick, N. (1979) Radioimmunoassay for albumin binding sites associated with HBsAg: Correlation of results with the presence of e-antigen in serum. Intervirology 11, 128. Neill, S.P. (19’79) Interaction of hepatitis B surface antigen with polymerised human serum albumin. J. Med. Viral. 4, 177-185. Nowoslawski, A. (1979) Hepatitis B virus induced immune complex disease In: Progress in Liver Disease (Popper, I-I., Schaffner, F., eds.), pp. 393-406, Grune and Stratton, New York and London. Peters, Jr., T. (1970) Serum albumin. Adv. Clin. Chem. 13, 37-111. Pontisso, P., Albert, A.. Schiavon, E. and Realdi, G. (1982) Receptors for polymerised human serum albumin (#ISA) on hepatitis B virus (HBV) particles. Abstracts of 17th Meeting of the European Association for the Study of the Liver, Goteborg, Sweden, 1982.
85 Pontisso, P., Albert, A., Bortolotti, F. and Realdi, G. (1983) Virus associated receptors for polymerised human serum albumin in acute and in chronic hepatitis B virus infection. Gastroenterology 84, 220-226. Palla, M., Rizzi, R., Almi, P., Rizzetto, M., Bonino, F. and Purcell, R. (1983) Complexes of hepatitis B surface antigen and immunoglobulin M in the sera of patients with hepatitis B viral infection. Infect. Immun. 41, 950-958. Rothschild, M.A. and Schreiber, S.S. (1969) Serum albumin. Am. J. Dig. Dis. 14, 711-744. Thung, S.N. and Gerber, M.A. (1981) HBsAg associated albumin receptors and anti-albumin antibodies in sera of patients with liver disease. Gastroenterology 80, 260-264. Thung, S.N. and Gerber,_M.A. (1981) Specificities of albumin receptors and albumin antibodies. Infect. Immun. 32, 1292-94. Thung, S.N. and Gerber, M.A. (1984) Polyalbumin receptors: their role in the attachment of hepatitis B virus to hepatocyte. Semin. Liver Dis. 4, 69-75. Vyas, G.N., Williams, E.W., Klaus, G.G.B. and Bond, H.E. (1972) Hepatitis associated Australia antigen - protein, peptides and amino acid composition of purified antigen with its use in determining sensitivity of the hemagglutination tests. J. Immunol. 108, 111+1116. William, J.R., Marilyn, Z., James, O., Yousef, S., Orgad, L., Pablo, G. and David, N.S. (1984) Transcription units of hepatitis B virus genes and structure and expression of integrated viral sequences. In: Viral Hepatitis and Liver Disease (Vyas, G.N., Dienstag, J.L. and Hoofnagle, J.H., eds.), pp. 67-86, Grune and Stratton, New York and London.