an enzyme immunoassay for hepatitis b e-antigen and antibody

Journal of Vimlogical Methods, 3 (1981) 89-97 Elsevicr/North-Holland Biomedical Press

89

AN ENZYME IMMUNOASSAY FOR HEPATITIS B e-ANTIGEN AND A~IBODY

ISA K. MUSHAHWAR and LACY R. OVERBY

Hepatitis Research Laboratory, Abbott Diagnostics Division, Abbott Laboratories, North Chicago, IL 60064, U.S.A.

(Accepted 20 March 1981)

A solid-phase enzyme-linked immunoas~y for the detection of hepatitis B e-antigen (HBeAg) and antibody (anti-HBe) was developed and compared with rheophoresis and radioimmunoas~y (RIA). The enzymeimmunoassay (EIA) was similar to RIA in ~usit~~ty and was ap~rox~mateIy 1000.foId more sensitive than rheophoresis for HBeAg, and approximately 6000-fold more sensitive than rheophoresis for anti-HBe.

INTRODUCTION

Hepatitis 3 e antigen (HBeAg) and its antibody [anti-HBe) are found in serum in association with type B viral hepatitis (Magnius et al., 1975). The presence or absence of these markers provides useful ~fo~ation on the status of individuals found to be hepatitis B surface antigen (HBsAg~-positive (Miyakawa and Mayumi, 1978). HBeAg is found during the early phase of virus replication. There is a seroconversion to anti-HBe with resolution of virus replication (Krugman et al., 1979; Ling et al., 1979). Serum HBeAg correlates with an increased number of infectious viruses (Dane particles), with core particles in the nucleus of the hepatocytes, and with viral specific DNA polymerase in serum(Nordenfelt andKjellen, 1975; Tong et al., 1977; Werner et al., 1977; Mushahwar et al., 1978). Other serological indicators of liver pathology are also present during this period (Krugman et al., 1979). During the HBeAg-positive stage, therefore, hepatitis B patients are at increased risk of t~nsmitting the virus to their contacts (Alter et al., 1976; Grady, 1976; Okada et al., 1976), and persistence of HBeAg in the hepatitis B virus carrier is often associated with chronic active hepatitis (~lefthe~ou et al,, 1975; El Sheikh et al., 1975; Trepo et al., 1976). The appearance of anti-HBe, on the other hand, indicates a reduced level of infectious virus due to a decrease of virus replication and this represents a better clinical prognosis (Trepo et al., 1976; Vogten et al., 1976,). This is not absolute and there are exceptions where patients have seroconverted to anti-HBe without resolution of chronic disease. For the above reasons, serological moni- toring of e-antigen status provides useful diagnostic and prognostic information during the course of hepatitis B virus infection.

0166-0934/81/0000-00~0/$02.50 ~Eise~er/North-Holland Biomedical Press

90

This paper describes an enzyme-immunoassay (EIA) (Waart et al., 1978; Bonino,

et al., 1980) for the detection of HBeAg and anti-HBe and compares its sensitivity and

detectability with rheophoresis and radioimmunoassay (RIA) (Mushahwar et al., 1978).

MATERIALS AND METHODS

Reagents

O-Phenylenediamine - 2 ‘HCl and horseradish peroxidase (HRP; RZ > 3 were purchased

from Sigma Chemical Co. (St. Louis, MO, U.S.A).

Source of anti-HBe

Human plasma containing anti-HBe with rheophoresis titers Z 1 : 32 were used as

starting material. The plasma selected gave double precipitin lines in immunodiffusion

with a reference serum designated as containing el and e2 antigens, according to the

nomenclature of Williams and Le Bouvier (1976). The plasma were recalcified, adjusted

to 50% saturation with (N&)*S04 and to pH 7.5 with NH40H. The immunoglobulin

(IgG) precipitate was collected and dissolved in a minimal volume of 0.01 M potassium

phosphate, pH 8.0, The solution was then dialyzed against two changes of the same

buffer overnight at 4”C, and passed through a DEAE-cellulose (DE-52, Whatman) column

equilibrated with the same buffer. The effluent fractions containing IgG were collected

and found to be free of IgM and albumin as determined by immunodiffusion techniques.

EIA for HBeAg and an ti-HBe

Polystyrene beads, 6 mm in diameter, were coated with anti-HBe serum as described

previously (Mushahwar et al., 1978). The beads were then used as a solid-phase antibody.

The anti-HBe IgG preparations were conjugated with horseradish peroxidase (HRP)

according to the method of Nakane and Kawaoi (1974). The HRP-conjugated anti-HBe

solution was diluted to approximately 1 .O lg/ml in a diluent of 0.1 M Tris-HCl saline,

pH 7.5, containing 15% normal human serum and 50% newborn calf serum.

HBeAg was determined by a direct solid-phase (sandwich) assay. The sample (0.2

ml) was incubated overnight with the antibody-coated bead at room temperature; after

washing with water the bead was further incubated in 0.2 ml of HRP-conjugated anti-HBe

solution at 40°C for 2 h. The bead was then washed with water. A substrate solution (0.3

ml of 0.3% O-phenylenediamine - 2 HCl in 0.1 M citrate phosphate buffer, pH 5.5,

containing 0.02% H,Oa) was added. The enzymic reaction was allowed to proceed for 30

min at room temperature in the dark. The reaction was stopped by adding 1 .O ml of 5%

HzS04. The intensity of the color that was developed as the result of the enzymic cata-

lysis of the substrate was measured at 492 nm by using a Quantum 1 Spectrophotometer

(Abbott Laboratories). Specimens with absorbancies equal to or greater than the cutoff

91

(negative control mean absorbance plus 0.06) were considered to be reactive by the

criteria of this test. For this calculation the mean of at least three sera was used as negative

control.

Anti-HBe was determined by a neutralization procedure. The test sample (0.05 ml)

was mixed with 0.20 ml of HBeAg-positive serum containing a predetermined quantity

of HBeAg. The solution was then incubated overnight with the antibody-coated bead

at room temperature. The beads were then washed and processed as described in the

HBeAg procedure. The quantity of HBeAg m the initial incubation was selected to give

an absorbance of 0.7-l .l in the presence of an anti-HBe-negative serum. Significant

(50% or more) reduction in absorbance indicated the presence of anti-HBe in the test

sample. When a test specimen contained HBeAg, an increase in absorbance was observed.

Therefore, this method actually was capable of detecting both HBeAg and anti-HBe,

depending upon whether the absorbancy at 492 nm was elevated or lowered. However,

the first described HBeAg method was more sensitive than the latter method.

Rheophoresis tests for HBeAg and anti-HBe were performed with agar gel plates

supplied by Abbott Laboratories. HBsAg, anti-HBs and anti-HBc were determined by

commercial RIA reagents (Abbott Laboratories). The rheumatoid factor was measured

by the rheumatoid slide test (Eosin Latex Test) of Difco Laboratories.

Specificit4, and con@mation

Two reference sera were identified for establishing specificity of positive EIA HBeAg

analyses: 1) a control serum containing HBsAg and antibody to hepatitis B core antigen

(anti-HBc) but negative for antibody to hepatitis B surface antigen (anti-HBs), anti-

HBe, and HBeAg by all available ~mLln~)assays; 2) a neutralizing serum containing

HBsAg and anti-HBc and also positive for anti-HBe in rheophoresis against a panel of

HBeAg sera.

For confirmation of HBeAg-positive analyses, the specimens were reanalyzed separately

with the control serum or the neutralizing serum added to the specimen at a level of 25%.

After incubating at room temperature for 1 h the mixtures were analyzed for HBeAg. The

absorbances were compared for the two analyses. A reduction in absorbancy of 50%

or greater indicated a confirmation of HBeAg.

RESULTS AND DKUSSION

Tiihatiorr of HBeAg by EIA and RIA

A human serum reactive fo: HBeAg by rheophoresis with a titer of 1 : 4 was serially

diluted in normal serum and assayed fo- Tr!?eAg by RIA and EIA. The results, expressed

92

as counts per minute (c.p.m.), absorbance at 492 nm, and ratios of these values to the

negative control values, are given in Table 1. The last dilution found positive by either

RIA or EIA tests was I : 6400, indicating equivalent sensitivity for HBeAg and represent-

ing a 1600-fold increase over rheophoresis.

Titration of an ti-HBe by EIA and RIA

A human serum reactive for anti-HBe by rheophoresis with a titer of 1 : 2 was serially

diluted in normal human serum and assayed for anti-HBe by RIA and EIA. The results,

expressed as c.p.m., absorbance at 492 nm, and percent inhibition for the competitive as-

says, are given in Table 2. At the 50% in~bition point, the titer of the test specimen was

1 : 12,800 by both RIA and EIA procedures. Thus, the RIA and EIA tests show equiva-

lent sensitivity for anti-HBe and are approximately 6400 times more sensitive than rheo-

phoresis.

Detectability of HBeAg and anti-HBe by RI/i, E-IA and rheophoresis

The detectability of HBeAg and anti-HBe in a large population of HBsAg-reactive

sera was determined by the RIA, EIA, and rheophoresis procedures. The results for this

group of 653 randomly selected HBsAg-positive sera are given in Table 3. Only 4% of the

specimens were HBeAg-positive by rheophoresis, whereas, 41.2% were positive by RIA,

TABLE I

Comparison of RIA and EIA for the detection of HBeAg

Reciprocal

dilution X 1 O-’

Abbott-HBe RIA Abbott-HBe EIA

C.p.m. S/Na Absorbance Absorbance-NC?

1 10172 21.6 1.256 1.205

2 9906 26.9 1.147 1.096

4 9626 26.2 1.178 1.127

8 7718 21.0 0.897 0.828

16 5655 15.4 0.677 0.626

32 3076 8.4 0.457 0.406

64 1718 4.7 0.265 0.214

128 733 2.0 0.098 0.047

Vontrols values:

Negative (NC)

Positive (PC)

368 1.0 0.05 1 0

9040 24.6 1.107 0.996

Cutoff: RIA -- a sample is considered reactive if S/N is 3 2.1

EIA - a sample is considered reactive it’ absorbance--NC is > 0.06.

93

TABLE 2

Comparison of RIA and EIA for the detection of anti-HBe

Reciprocal RIA

dilution X 10m2 C.p.m, % Inhibitiona

EIA

Absorbance (492 nm) % Inhibitiona

1

2

4

8

16

32

64

128

433 100.0 0.059 100.0

454 99.7 0.065 99.3

446 99.8 0.082 96.6

486 99.2 0.080 97.0

560 98.0 0.097 94.5

791 94.3 0.132 89.3

1535 82.6 0.184 81.6

3700 48.4 0.413 47.5

5295 23.3 0.552 26.9

6408 5.7 0.612 18.0

Control values

Negative 6768 0 0.733 0

Positive 433 100.0 0.060 100.0

NC2 - sample Y

a % Inhibition = x 100,

NCY - PC?

Where NCR is the mean absorbance of the negative control, PCE is the mean absorbance of the positive

control, and sample Y is the mean absorbance of the sample.

TABLE 3

Comparison of rheophoresis, RIA and EIA technqiues for the detection of HBeAg and anti-HBe

in HBsAg-positive sera

No. of Seraa

Total HBeAg- Anti-HBe-

positive positive

HBeAg and Anti-HBe-

negative

Rheophoresis 653 (100)

RIA 653 (100)

EIA 653 (100)

a Percentage given in parentheses.

27 (4.1) 129 (19.8) 497 (76.1)

269 (41.2) 368 (56.3) 16 (2.5)

263 (40.3) 366 (56.0) 24 (3.7)

94

and 40.3% were positive by EIA. Assays for the presence of anti-HBe yielded 19.8%

positive by rheophoresis, 56.3% by RIA and 56.0% by EIA. All HBeAg- and anti-HBe-

positive specimens by EIA were also found to be positive by the RIA procedure. Thus,

the RIA and EIA procedures showed equivalent sensitivity and detectability i> 94%) for

HBeAg and anti-HBe in these specimens. This was nearly four times greater than that of

rheophoresis.

Co~~~atio~ of HBeAg by neu~ul~za~io~ {specificity)

The specificity of the EIA was investigated (Mushahwar et al., 1978) by reanalyzing

positive sera after incubation with 25% of 1) normal human serum with no evidence

of hepatitis B association; 2) control serum containing HBsAg and anti-hepatitis B core

antigen (anti-HBc); and 3) neutralizing serum containing HBsAg, anti-HBc, and anti-

HBe confirmed by rheophoresis. Table 4 shows a Sudan of typical con~rmato~

TABLE 4

Confirmation of HBeAg-positive analysesa

Serum

ident~cation

Rheophoresis Absorbance at 492 nm of serum added to specimen

HBeAg Normal Control Neutralizing Conclusion

human serum (anti-HBc) (anti-HBe) HBeAg

HBeAg-positive

279

310

334

379

Positive 0.733 0.819 0.088 Positive




390 Negative 0.369 0.381 0.090 Positive




Normal sera

07-364~EW

81-335-BW

6303-90

07-477

Negative 0.032 0.036 0.033 Negative




Rheumatoid factor

321(3+)

370 (2+)

Negative

Negative

0.038

0.043

0.033

0.047

0.044 0.041 Negative


484 (4+) Negative

79.5 (I+) Negative



a Specimens were reanalyzed after incubating with 2.5% normal human serum, a control serum con-

raining HBsA& and anti-HBc, and a neutralizing serum containing HBsAg, anti-HBc and anti-HBe.

Results are shown as absorbance at 492 nm.

95

results. All sera positive for HBeAg by rheophoresis were confirmably neutralized. A

second group of HBsAg carriers negative by rheophoresis for HBeAg but positive by EIA

was also confirmable by neutralization. Normal sera gave no significant differences in

absorbance in the confirmatory analyses. Upon analysis of 875 normal sera, three (0.3%)

were found with absorbances greater than the cutoff value. These sera were not con-

firmable for HBeAg in the neutralization assay. Twelve sera authentically positive for

rheumatoid factor were shown to be negative in the HBeAg EIA. Four typical analyses

from weak (l+) to strong (4+) rheumatoid factor are shown in Table 4. These sera were

also negative in the competitive anti-HBe analysis. Similarly, four anti-HBs-positive sera,

two anti-HBc-positive sera and three preparations of purified HBsAg were shown to be

negative for both HBeAg and anti-HBe by EIA.

Frequency distribution of HBeAg and anti-HBe in normal blood donor population

The frequency distribution of HBeAg and anti-HBe in 500 HBsAg and anti-HBc-

negative sera is shown in Figs. 1 and 2 respectively. For HBeAg, the analysis shows that

499 samples had a net absorbancy (absorbance of sample at 492 nm minus negative

180 -

160 -

140.

120.

100 -

80.

60.

40.

20.

O- 1

<-.02 -.01-O .01-.02 .oi-.04 as-.04 .oi-.os . .09-.l

-x)2/-.01 o-.01 .02-.03 .04-.05 .06-.07 x)8-.09 > .l

NEl ARSORRANCY AT 492 nm (ABSORR; SAMPLE-ABSORB NC)

Fig. 1. Histogram of the net absorbance at 492 nm (absorbance of sample minus absorbance of normal

control) values obtained when 500 HBsAg and anti-HBc negative sera were tested for HBeAg by EIA.

The net absorbance for each specimen was calculated, and results were grouped by number of sera

in each percentile.

96

100 i

4 s P a!

90.

RO-

70.

60-

50.

40.

30.

20.

10.

O- _ <-20 -w;-10 -5i-o s-10 15120 25-30 45-50 35-40

-2O/-15 -lO/-5 o-5 10-15 20-25 40-45 >50

PERCENT INHIBITION

Fig. 2. Histogram of the distribution of inhibition values obtained when 500 HBsAg and anti-HBc

negative sera were tested for anti-HBe by EIA. The percent inhibition value for each specimen was cal-

culated, and results were grouped by number of sera in each percentile.

control absorbance) of less than 0.06. Only one sample (0.2%) was positive for HBeAg.

This sample could not be neutralized with anti-HBe and is presumably false positive.

Similarly, analysis of 500 HBsAg-negative sera from healthy individuals showed that all

had inhibition values of less than 50% (Fig. 2) and therefore were considered non-

reactive for anti-HBe.

These results showed that the EIA was as simple, sensitive and as specific as the RIA

for the detection of both HBeAg and anti-HBe.

ACKNOWLEDGEMENT

We thank Carlos M. Cabal for competent technical assistance.

REFERENCES

Alter, H.J., L.B. Seef, P.M. Kaplan, V.J. McAuliffe, L.C. Wright, J.L. Germ, R.H. Purcell, P.V. Holland

and H. J. Zimmerman, 1976, N. Engl. J. Med. 295,909.

Bonino, F., S. Recchia, A. Ponzetto, B. Fillippone, M. Palla, A.R. Zanetti and P. Perroni, 1980, J.

Immunol. Methods 3 3. 195.

97

Eleftheriou, N., J. Heathcoat, H.C. Thomas and S. Sherlock, 1975, Lancet 2, 1171.

El Sheikh, N., LL. Woolf, R.M. Galbraith, A.L. Eddleston, I.W. Dymock and R. Williams, 1975, Br.

Med. J. 4, 252.

Grady, CF., 1976, Lancet 2,492.

Krugman, S., L.R.Overby,I.K. Mushahwar, C.-M. Ling, G.G. Frosner and F. Deinhardt, 1979, N. Engl.

J. Med. 300, 101.

Ling, C.-M., I.K. Mushahwar, L.R. Overby, K.R. Berquist and J.E. Maynard, 1979, Infect. Immun.

24, 352.

Magnius, L.O., A. Lindholm, P. Lundin and S. Iwarson, 1975, J. Am. Med. Assoc. 231, 356.

Miyakawa, Y. and M. Mayumi, 1978, in: Viral Hepatitis, eds. G.N. Vyas, S.N. Cohen and R. Schmid

(The Franklin Institute Press, Philadelphia) p. 193.

Mushahwar, I.K., L.R. Overby, G. Frosner, F. Deinhardt and C.-M. Ling, 1978, J. Med. Virol. 2, 77.

Nakane, P.K. and A. Kawaoi, 1974, J. Histochem. Cytochem. 22, 1084.

Nordenfelt, E. and L. Kjellen, 1975, Intervirology 5, 225.

Okada, K., I. Kamiyama and M. Inomata, 1976, N. Engl. J. Med. 294,746.

Tong, M.J., D. Stevenson and I. Gordon, 1977, J. Infect. Dis. 135,980.

Trepo, C.G., L.O. Magnius, R.A. Schaefer and A.M. Prince, 1976, Gastroenterology 71, 804.

Vogten, A., J.M. Schalm and S.W. Jummerskill, 1976, Lancet 2,126.

Waart, M., v.d., A. Snelting, J. Ciehy, G. Wolters and A. Schuurs, 1978, J. Med. Virol. 3,43.

Williams, A and G. Le Bouvier, 1976, Bibl. Haematol. 42, 71.

Werner, B.G., A.P. O’Connell and J. Summers, 1977, Proc. Natl. Acad. Sci. U.S.A. 74, 2149.

an enzyme immunoassay for hepatitis b e-antigen and antibody

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