Journal of Virological Methods, 3 (198 1) 1~ 11

Elsevier/North-Holland Biomedical Press

DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

FOR HEPATITIS B e ANTIGEN AND ANTIBODY

A.M. SMITH’.’ and R.S. TLDDER’

‘Department of Virology, Middlesex Hospital Medical School, London; and ‘Department of’ Medical

Microbiology, University College, Dublin, U.K.

(Accepted 5 December 1980)

Horseradish peroxidase has been conjugated with human immunoglobulin containing antibody to

hepatitis B e antigen (anti-HBe). This was used in a solid-phase ‘sandwich’ enzyme-linked immuno-

sorbent assay designed for reading by eye, to test sera for HBeAg and anti-HBe. Optimum conditions

for preparing the conjugate and for performing the test are described. Results of testing hepatitis B

surfAce antigen (HBsAg)-positive sera were compared with those obtained using a sensitive solid-

phase radioimmunoassay. The enzyme assay provided a simple and sensitive method of testing for

HBeAg and anti-HBe, and correlated well with the radioimmunoassay.

INTRODUCTION

The presence of hepatitis B e antigen (HBeAg) in serum is associated with numerous

Dane particles (Takahashi et al., 1976), high DNA polymerase levels (Cappel et al., 1977)

and, consequently, with high infectivity in vertical (Okada et al., 1976) and horizontal

transmission (Magnius et al., 1975). Antibody to HBeAg (anti-HBe), on the other hand,

indicates low infectivity.

Sensitive tests are needed to detect these clinically important markers in sera from

patients infected with hepatitis B. Immunodiffusion (ID) is the test most widely used, but

lacks sensitivity. In some surveys, less than half the HBsAg carriers could be classified

by this method. The greater sensitivity of radioimmunoassay (RIA) has resulted in a

marked increase in the number of sera in which either HBeAg or anti-HBe can be detected,

such that more than 90% of sera may now be classified. However, the high cost of gamma

counters, together with the short shelf-life of the reagents, may prohibit its wider use as a

routine diagnostic test. This has prompted the development of a solid-phase ‘sandwich’

enzyme-linked immunosorbent assay (ELISA), which is sensitive yet easy to perform

and which can be read by eye. Enzyme-linked immunoassays for HBeAg and anti-HBe

have recently been described (Van der Waart et al., 1978; Bonino et al., 1980) in which

results are determined spectrophotometrically. This involves a capital outlay which may

discourage the widespread use of ELlSA for detection of HBeAg and anti-HBe. The

methodi of testing described here eliminates the need for expensive equipment yet achieves

the required degree of sensitivity.

0166 ~0’934/81/0000-0000/$02.50 @Elsevier/North-Holland Biomedical Press

2

Because it has proven difficult to prepare specific anti-HBe IgG in sufficient quantity,

the whole IgG fraction from a human serum containing a high titre of anti-HBe is used.

The amount of anti-HBe in this fraction is not more than about 1% of the total IgG.

Therefore, to be able to detect specific binding of enzyme-linked anti-HBe to the solid

phase, it is essential that the level of non-specific binding is reduced to a minimum.

One common method of coupling IgG to enzymes uses glutaraldehyde (Avrameas,

1968; Engvall and Perlman, 1972). In addition to the desired formation of conjugates

between unlike proteins, this method causes (King and Kochoumian, 1979) the un-

wanted fo~ation ofconjugates of like proteins and the formation of conjugate polymers.

Both of these cause increased levels of non-specific binding. Among methods aimed at

avoiding these undesirable side reactions is that using sodium periodate (NaI04) (Nakane

and Kawaoi, 1974).

In this communication, we present those conditions of coupling, using NaIO,, most

suited to producing a horseradish peroxidase IgG(H~0 IgG) conjugate with a low level

of non-specific binding. The conditions necessary to optimize the performance of the

HRPO-conjugated anti-HBe are also described. Sera may contain either HBeAg or anti-

HBe. Since in clinical practice it is often desirable to obtain a comprehensive result as

soon as possible, the format of the assay comprises a test for both HBeAg and anti-HBe.

Because of the low amounts of anti-HBe present in ~-globu~n (used in the solid phase)

and IgG prepared from whole human serum, it is important to establish the specificity

of positive reactions. A confirmatory test for HBeAg is therefore contained within the

assay. The results of testing human sera by the ELISA are compared with the results of a

sensitive solid-phase radioimmunoassay (Barbara et al., 1978).

MATERIALS AND METHODS

Human serum

Sera showing the highest titres for either HBeAg or anti-HBe by ID and RIA were

chosen from blood donations obtained from persistent hepatitis B surface antigen (HBsAg)

carriers,

Normal human serum (NHS) consisted of pooled sera negative for HBsAg, anti-HBe

and antibodies to HBsAg (anti-HBs) and to the hepatitis B core antigen (HBcAg).

Specimens tested by the ELISA were 102 HBsAg-positive clinical sera, 100 consecutive

HBsAg-negative blood donor sera from the North London Blood Transfusion Centre

and I6 Haag-negative sera known to be strongly positive for rheumatoid factor by sheep

cell agglutination test (geometric mean titre 1 in 1612, range 320~-5 120) and latex ag

glutination test (Wellco-test, Wellcome Reagents Ltd., Beckenham, Kent).

The ELISA solid phase

Flat-bottomed polystyrene microtitre plates (Dynatech) or strips of 8 flat-bottomed

3

wells (Removawells) were coated with 100 1.11 volumes of the appropriate dilution in 0.02

M Tris buffer, pH 7.6, containing 0.1% sodium azide (TAB), of the y-globulin fraction

of a human, high-titre anti-HBe-positive serum prepared by double precipitation with

40% ammonium sulphate. The wells were sealed and left at room temperature for 72 h.

After washing three times with TAB, they were completely filled with TAB containing

0.5% bovine serum albumen (BSA-Tris) and left at room temperature for 1 h in order

to block all remaining free sites on the plastic. Wells were stored damp at 4°C in a moist

chamber until required and were aspirated just before use.

Human IgG

Sera strongly positive for anti-HBe, when tested unconcentrated by ID, were titrated

by RIA and the highest dilution of each which neutralized a reference HBeAg serum was

determined. Anti-HBe IgG was prepared from the serum showing the highest neutralizing

titre by ion-exchange chromatography on DE 52 (Whatman Ltd.) which had been equili-

brated previously with 20 mM phosphate buffer, pH 8.0. Only about 1 in 10 of such

high titre sera contained a sufficiently high proportion of specific anti-HBe IgG to pro-

duce a suitable HRPO IgG conjugate.

IgG was prepared from NHS (NHS IgG) in the same way.

The IgG concentration was calculated from the absorbance at 280 nm, using an _!$&

value of 1.4.

Optimum method of preparation of the conjugate

Four mg horseradish peroxidase Type VI (HRPO, Sigma Chemical Co.), dissolved

in 1 ml freshly prepared 0.3 M sodium bicarbonate, pH 8.1, were gently mixed at room

temperature for 1 h with 100 ~1 1% 2,4-dinitrofluorobenzene (DNFB, Sigma Chemical

Co.) in absolute alcohol. The mixture was oxidized with 200 ~1 of freshly prepared 0.1

M sodium m-periodate (NaI04, Sigma Chemical Co.) for 30 min at room temperature.

After overnight dialysis at 4°C against 1 litre of 1 mM acetate buffer, pH 4.4, the pH was

raised to 9.5 by adding 20 d 0.2 M sodium carbonate buffer, pH 9.5. Immediately,

1 ml of anti-HBe IgG (2 mg/ml), previously dialysed against 0.01 M sodium carbonate

buffer, pH 9.5, was added to an equal volume of enzyme and mixed gently at room

temperature for 2 h. The conjugate formed was stabilized by adding 100 ~1 of freshly

made 0.1 M sodium borohydride (NaBH4, Sigma Chemical Co.). The solution was allowed

to stand at 4°C for 2 h. The conjugate was then concentrated (Minicon B 15 Macro-

solute Concentrates, Amicon Corp.) and fractionated by downward flow on a 2.6 X 40

cm column of Sephacryl S-300 (Pharmacia Ltd.) previously equilibrated with 0.4% saline

in borate buffer, pH 8.0, containing 0.1% BSA and 5% (t)-mannose (BBS-mannose).

Fractions containing HRPO-labelled IgG were pooled and concentrated to 1 ml. The con-

centrated preparation was absorbed for at least 3 h at room temperature over 2 ml of

Sepharose CL 4B (Pharmacia Ltd.) which had been previously activated with cyanogen

4

bromide (CNBr, Koch Light Ltd.) and coupled with NHS and normal human plasma

(Sepharose-NHP) (Axen et al., 1967). The column was pre-equilibrated with BBS-

mannose. The non-absorbed material was concentrated to 2 ml by dialysis against sucrose

and stored at 4°C in 5% sucrose and 4% BSA.

Substrate

8 mg of 2,2’-azino-di-(3-ethyl-benzthiazohne sulphonic acid, ABTS, Sigma Chemical

Co.) were dissolved in 10 ml of 0.05-0.1 M citrate phosphate buffer, pH 4.0, containing

100 1.11 HzOz (20 ~01s.). The colour reaction was stopped by adding 50 1.11 of sodium

fluoride solution (NaF, Sigma Chemical Co., 200 mg per 100 ml of distilled water).

Determination of the enzymatic activity of conjugates

Doubling dilutions in citrate phosphate buffer of conjugates (2.5 1.11 standard volumes)

were prepared in flat-bottomed microtitre plates. 100 1.11 ABTS were added and incubated

at room temperature for 30 min. The reaction was stopped by addition of 50~1 of NaF

and the absorbance determined at 650 nm. The dilution giving an A = 0.1 was considered

to contain an arbitrary 1 unit of enzymatic activity per standard volume, and the enzyme

concentration of a conjugate was expressed as the reciprocal of this dilution. 500 units

of enzymatic activity per standard volume was established as a suitable working concen-

tration of conjugate. Aliquots of conjugate preparations were adjusted to an activity of

500 units in buffer before use in ELISA.

HBeAg-speci’c chromogen release as an indication of conjugate performance

100 /.d of NHS or 100 ~1 HBeAg-positive serum diluted 1 in 10, 100 and 1000 in NHS

were added to wells coated with anti-HBe and incubated overnight at room temperature.

After washing with PBS containing 0.05% Tween 20 (PBS-Tween), 100 ~1 of conjugate

containing 500 enzyme units were added and the plate was incubated at room temperature

for 2 h. The wells were washed again with PBS-Tween and 100 1.11 of substrate were

added and incubated in the dark for 30 min at room temperature. The reaction was

stopped with 50 ~1 of sodium fluoride. The absorbance at 650 nm was determined for

four NHS replicates and for duplicate wells incubated with each of the dilutions of

HBeAg-positive serum. The performance of the conjugate indicated by the ‘e-specific

chromogen release’ was expressed by subtracting the mean absorbance value for NHS

from that obtained for each of the reference antigen dilutions.

Format of the ELISA

A column of three wells was used for each serum. The first well comprised a neutrali-

zation assay for anti-HBe. The remaining pair of wells detected HBeAg and confirmed the

5

specificity by neutralization with standard anti-HBe. Strips of wells were aligned in rows

three wells deep. 50 1.11 of test serum were placed in the first well of each triplicate (Fig. 3,

well A). To this was added an equal volume of NHS containing a known quantity of

HBeAg (the highest dilution of HBeAg giving maximum chromogen release, Fig. 2). A

further 50 ~1 of test serum were added to the second and third wells (Fig. 3, wells 2 and

3) which contained 50 /.d NHS and anti-HBe respectively. After overnight incubation at

room temperature, the wells were washed five times with PBS-Tween and 100 d of con-

jugate (diluted to 500 units per standard volume in PBS-Tween containing 1% BSA and

0.5 mg/ml of normal human y-globulin which had been heat-inactivated for 15 min at

63°C) were added for 2-4 h at room temperature. The wells were washed fourtimes with

PBS-Tween and 100 ~1 substrate added for 30 min at room temperature in the dark. The

reaction was stopped with 50 ~1 NaF, and the results were read by eye. HBeAg was

detected by a change in the substrate from colourless to green.

RESULTS

The conditions for the formation of the enzyme IgG conjugate and for the perfor-

mance of the ELISA test were optimized. As the test was to be read by eye, optimum

conditions for each stage were considered to be those which gave a maximum difference

in colour production between positive and negative controls (e-specific chromogen

release) rather than those which gave maximum ratios between positive and negative

absorbance values. The initial end-product obtained after coupling of enzyme and IgG

under each of the conditions examined was fractionated by gel filtration on Sephacryl

S-300. PBS used initially as eluting buffer was replaced by BBS because of the bacterio-

static properties of borate buffer. Three protein peaks (A,,,,,) were detected (Fig. 1)

Fig. 1. E.lution pattern after fractionation on a Sephacryl S-300 column (2.6 X 40 cm) of an HRPO

IgG conjugate prepared using 4 mg/ml IgG. The conjugate (1 ml volume) was applied to the top of

the gel. Proteins were eluted with a downward flow of 0.1% BSA in BBS containing 5% mannose

in accordance with the manufacturer’s instructions and 60 fractions of 4.3 ml collected.

6

corresponding to aggregates of conjugate (peak I), IgG and IgG enzyme conjugate (peak

II) and free enzyme (peak III). The fractions corresponding to the major part of peak II

were pooled and absorbed over Sepharose--NHP. The enzymatic activity and the e-

specific chromogen release were determined for each condition examined.

Optimization of conjugate formation and of pe$ormance of the ELlSA

An Important factor in conjugate formation was the concentration of IgG used (Table

I)_ Increasing the concentration of IgG led to more efficient coupling of the enzyme, and

a consequent decrease in size of peak III (free enzyme). However, higher concentrations

of IgG gave substantial and visible aggregate formation. Before fractionation, these were

removed by centrifugation (3000 g for 1.5 min). As a result, the enzymatic activity in

peak II was reduced. The optimum IgG concentration was 1 mg/ml, and 50% of the en-

zymatic activity of the product was contained in peak II. The HRPO/IgG ratio, cal-

culated from the absorbance measurements for peak II at 280 and 403 nm was 1.35.

The quality of the conjugate was also affected by the length of time allowed for conjugate

formation. While maximum incorporation of enzyme into peak II was seen at 4 h, the e-

specific chromogen release was greatest in conjugates formed over a 2 h period.

The conditions of oxidation of HWO further ~fluenced conjugate formation. The

conditions yielding a max~um amount of enzyme for coupling with minimal enzyme in-

activation were 0.016 M Naf04 for 30 min at room temperature, followed by a gradual

stopping of the reaction by removal through dialysis of the excess periodate.

For coating wells, a 1 in 100 dilution of y-globulin, containing anti-HBe in 0.02 M

TAB, proved optimum. At this dilution, approximately lo-20 c(g y-globulin were added

to each well.

Although the optimum conditions for incubation of the conjugate on the solid phase

were 4 h at room temperature, a shorter and more convenient 2 h incubation proved

adequate.

TABLE I

Effect of IgG concentration on coupling efficiency

w concentration

(m&l)

Enzymatic

activity

(Peak II)

Conjugate performance

Absorbance e-specific chromogen releasea

NHS eAg X 10-l eAg X 10.’ eAg X lo-”

1 4,480 0.06 0.42 0.29 0.12

2.5 19,840 0.10 0.32 0.24 0.09

5 5,540 0.08 0.28 0.26 0.10

10 1,472 0.07 0.17 0.12 0.04

a This is the derived absorbance value: (A standard HBe) - (A NHS).

Interpretation of the ELISA

As the assay was designed to be read by eye, a cut-off or reference colour standard

was included in each test. The amount of colour release caused by 1 in 1000 dilution of

a reference HBeAg-positive serum in NHS was within the limits of visual discrimination

(Fig. 2) and provided this standard. Wells producing chromogen release equal to or

greater t.han this (Fig. 3, column 10) were deemed to have contained HBeAg.

With sera negative for HBeAg and anti-HBe there was colour formation in well A but

not in wells B and C (Fig. 3, sera 1, 2, 5 and 6). If the serum contained anti-HBe at a

significant level, the colour in well A was reduced to below the cut-off level. Frequently,

anti-HBe-positive sera would render this well colourless (Fig. 3, sera 4 and 7). If sera con-

tained HBeAg, well A would remain coloured and well B would develop colour greater

than the cut-off level. The specificity of the colour release in well B was confirmed by its

inhibition in well C by the anti-HBe (Fig. 3, sera 3 and 9). Occasionally, a serum which

contained very high levels of HBeAg would not be completely neutralized by the anti-

HBe in well C and colour would develop (Fig. 3, serum 8). When such sera were retested

at a 1 in 10 dilution, this breakthrough was avoided.

102 HBsAg-positive sera, 100 HBsAg-negative sera and 16 rheumatoid factor-positive

sera were screened for HBeAg and anti-HBe by the ELISA. The 100 HBsAg-negative

sera and the 16 rheumatoid factor-positive sera all gave negative results for HBeAg by

ELISA. The results of testing 102 sera by ELISA were compared with the results of the

radioimmunoassay (Table 2). The y-globulin used in the solid-phase of both assays was pre-

pared from the same anti-HBe-positive serum, but the IgG used in the enzyme and radio-

labels were obtained from two different sera.

1 0,006 .‘NE - - - - - _ - I,

NEAT IO’ 10’ IO3 104 Serum dllutlon

Fig. 2. The amount of colour release caused by a series of dilutions of the reference HBeAg-positive

serum in NHS. The mean absorbance values at 650 nm for the dilutions and NHS are shown.

I 1 2 3 415 6 7 8 9110

Fig. 3. Photograph of sera tested (columns l-9 inclusive) by the ELISA. 50 ~1 of serum and 50

_ul of HBeAg were added to well A. To we& B andC, containing 50 ~1 NHS and 50 ~1 anti-HBe respec-

tively, 50 ~1 of test serum were added. The first 4 sera were NHS (1 + 2), HBeAg-positive (3) and

anti-HBe (4) controls. Sera 5-9 were tested. The wells of column 10 provided the cut-off reference

colour. Interpretation of the results of sera 5-9 is described in the text.

DISCUSSION

The ELISA proved to be a suitable test for HBeAg and anti-HBe. Optimizing conditions

provided a test which could be read by eye while meeting the required degree of sensi-

tivity and eliminating the need for expensive equipment to interpret the results, Conjugate

preparations, stored under the conditions indicated, were stable for at least 6 months (the

TABLE 2

102 selecteda HBsAg-positive sera from carriers and acute infections tested by ELBA and RIA for

HBe and anti-HBe

RIA

Total eAg eAb Neg.

Total 102 33 46 23

I?ilisA

eAg 32 32 0 0

eAb 53 0 46 7

Neg 17 1 0 16

a Selected to include a greater proportion of sera negative for HBeAg and anti-HBe by RIA

9

longest storage period to date). Similarly prepared conjugates stored at 4’C in 50% glyc-

erol have been stable for up to one year (Dr. G.W. Rook, personal communication).

One of the major requirements in developing the ELISA was that it could be read by

eye. Therefore, the choice of substrate was important. Origin~y, ~-phenylenediam~e,

which changes from colourless to yellow, was used. However, the detection by eye of a

weak positive reaction was not always easy. ABTS was investigated as a possible alternative

and found to be easy to read by eye. In particular, weak positive reactions were more

readily detected. The pH of the buffer used with ABTS was critical. Optimum pH for the

substrate was 4.0. However, at values below 4.0 there was a rapid loss in enzymatic

activity, and even at pH 3.9 the decrease was notable. Thus it was important to ensure

that the enzyme-substrate reaction took place at, or just above 4.0. This was easily

achieved by adjusting the pH of the stock supply of buffer to between 4.0 and 4.1. Both

ABTS and o-phenylenediamine were light-sensitive. Therefore, freshly made substrate

solutions were prepared in ‘universal’ bottles from which light was excluded. Wells to

which substrate had been added were incubated in the dark for 30 min.

A second important requirement for the ELISA was to produce an enzyme-IgG

conjugate with minimum non-specific binding. The conditions of coupling of HRPO to

the anti-HBe 1gG contributed in a large part to achieving this. It was essential to minimize

aggregate formation yet still provide efficient coupling. Prolonging the duration of con-

jugation to an extent which might have allowed either undesirable side reactions or even

the reversal of the initial coupling reaction had also to be avoided. Conjugation for 2 h

with IgG at a concentration of 1 mg/ml met these requirements.

For reading by eye, it was necessary to maximize the difference in colour released

by positive and negative sera, while keeping the non-specific binding of the conjugate

below levels detectable by eye so that negatives appeared colourless. Fractionation of the

conjugate on Sephacryl S-300 and subsequent absorbtion over Sepharose-NHP were

found to reduce effectively the level of non-specific binding. Further reduction was also

achieved by including normal serum proteins in the conjugate diluent. False reactions

caused by rheumatoid factor-positive sera were inhibited when 0.5 mg/mI heat-aggregated

~-~obu~in was added to this diluent. Theoretically, interference by rheumatoid factor is

best overcome by conjugating the HRPO with IgG-F(ab’),! fragments. l-fowever, addition

of heat-aggregated IgG or heat-aggregated y-globulin to the conjugate diluent was com-

pletely successful in preventing this interference. Since it was hoped that the method of

preparing the conjugate and of performing the ELISA could be repeated in Iaboratories

with only limited facilities, the procedure chosen at each stage of experimentation was

that which was effective yet economical and easy to perform. Addition of heat-aggregated

y-globulin met all of these requirements and was therefore included in the diluent.

The use of three wells per serum, with reference to the standard cut-off colour,

offered a clearly defined means of interpreting results, particularly in the case of sera

weakly positive for antigen or antibody. Of the 102 HBsAg-positive selected sera (Table

2) tested in this way, one reported negative by ELISA was HBsAg-positive by RIA at a

level just above the cut-off vaiue for that test. The ELISA showed only trace amounts

10

of colour in well 2, below the level determined as the cut-off colour. Seven sera reported

negative by RIA (Table 2) were positive for anti-HBe by ELISA. This increase in sensitivity

of ELISA for antibody detection by neutralization was a function of the lower sensitivity

of the ELISA for antigen. If the same absolute quantity of HBeAg was added to both

neutralization assays, the titre of antigen would be less in ELISA than in RIA, i.e. there

would be less detectable HBeAg in the ELISA test. The sensitivity of a neutralization is

inversely proportional to the quantity of detectable antigen used, and as a result the

ELISA anti-HBe test is more sensitive than the RIA. The addition of 25% extra HBeAg

to the ELISA anti-HBe test effectively reduced the sensitivity of the antibody test to that

of the RIA.

The infectivity of a serum may be defined indirectly by testing for anti-HBe. Although

patients whose sera contain anti-HBe are unlikely to be highly infectious, the value of this

test depends upon the precision with which anti-HBe is assayed. Unequivocal antigen or

antibody is detected by ID, and with such tests of low sensitivity there is little chance

of detecting mixtures of HBeAg and anti-HBe. However, where mixtures occur, highly

sensitive ELISA and RIA neutralization tests may detect antibody, and to avoid the

possibility of misclassifying an infectious patient, we conducted both neutralization

assays with a quantity of e antigen, which left some &IO% of carriers remaining unclassi-

fied. In practical terms, it would seem prudent to consider that such unclassified sera repre-

sent an infection risk. This caution is further supported by the occasional finding of sera

containing large numbers of hepatitis B virus particles in the absence of HBeAg or anti-

HBe detectable by the most sensitive assay (Dr. Dane, personal communication).

In conclusion, the ELISA proved a simple yet sensitive assay for HBeAg and anti-HBe.

The reagents were stable for months after production and could be used in a test where

the results were read by eye. The assay was specific and acceptable as a routine diagnostic

test.

ACKNOWLEDGEMENTS

We thank the members of the Department of Virology and Dr. John Barbara of the

North London Blood Transfusion Centre for their help and encouragement.

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