Journal of Virological Methods, 2 (1980) 51-62 @ Elsevier/North-Holland BiomedicalPress

I~ROVED HEP~OS~~, AN ENZ~E-~M~OASSAY FOR HEPATITIS B

SURFACE ANTIGEN

G. WOLTERS, L.P.C. KUJJPERS and 3. CROESE

Two modifications of Hepanostika were studied in order to improve this enzyme-immunoassay with regard to duration of the test, sensitivity and reading of test results. Test conditions and composi- tion of some components of the test system were modified. With the first modi~cation the test could be performed within 3 h and was about 3X as sensitive as Hepanostika. However, this method was less specific than Hepanostika and it was not suitable for routine screening. A second modification was studied with two differently prepared conjugates. The duration of the test was less than 4 h and the sensitivity was at least twice that of Hepanostika. The test results could be measured directly with a suitable photometer. Preliminary results with more than 500 deep-frozen donor sera showed that the specificity was acceptable with a number of false positives being < 2%.

INTRODUCTION

Hepatitis B surface antigen (HBsAg) can be detected at low concentrations by an

enzyme-~unoassay which we developed some years ago (Walters et al., 1976). The

approximate lower detection limit of this test, which is commercially available (Hepa-

nostika Microelisa system, Organon Teknika) is about three units of HBsAg* per ml

or lower, for either subtype ad or ay. This method may be compared with the most

sensitive test thus far routinely available, namely radioimmunoassay. Since high sensi-

tivity is one of the most important requirements for an HBsAg screening test, this aspect

receives constant attention. The total test duration of e~yme-~munoas~y is 6-7 h,

so that the final test results can be obtained the same day if the samples are available

in the morning. Stute et al. (1979) described a procedure wherein the first and second

incubation steps were each reduced to 1 h in a waterbath at 5O’C without loss of sensi-

tivity. However, there is insufficient data that this procedure will have the same speci-

ficity with each Hep~ost~a batch as the original procedure. The results can be read with

the naked eye. An objective reading is also feasible, but this requires a transfer of the

liquid from each test-well to a photometer, which is rather laborious. For these reasons

*l unit corresponds with 1 ng HBsAg protein as found in untreated serum or plasma, according to the Paul Ehrhch Institute in West Germany.

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we studied the possibilities of improving Hepanostika in the following respects: a) sen-

sitivity; b) test duration; c) reading of the results. This paper describes our progress of

the development of an improved version of Hepanostika.

MATERIALS AND METHODS

Hepanostika test procedure was performed according to the instructions using a first

incubation overnight at room temperature, unless otherwise stated. This procedure is

approximately twice as sensitive as the incubation for 2 h at 37°C.

First modification

Conjugates of horseradish peroxidase (HRP) and sheep anti-HBs (Ig) were prepared

essentially as for Hepanostika by the ‘two-step’ glutaraldehyde method of Avrameas

and Ternynck (1971).

The optimum concentration of the conjugate was determined by testing HBsAg-

negative and HBsAg-positive sera with different dilutions of the conjugate. Normal

sheep plasma, diluted 1 : 4 in 0.2 mol/l Tris and 0.2 mol/l NaCl, was used as diluent for

the conjugate. Anti-HBs coated microtitre plates were essentially the same as for Hepa-

nostika. The enzyme activity was determined by adding 0.1 ml of a fresh solution of

1.6 mg/ml o-phenylenediamine and 0.6 mg/ml urea peroxide in a phosphate citrate buffer

of pH 5 .O. The reaction was stopped with 0.1 ml 2 mol/l sulphuric acid.

The test procedure consisted of the following steps: addition of 0.1 ml of sample to

the plate well, incubation for 30 min at 50°C in an incubator, addition of 0.05 ml of con-

jugate, incubation for 30 min at 50°C in an incubator, washing, addition of substrate

and stopping the reaction after 30 min at room temperature. Results were measured

according to the Hepanostika procedure.

Second modification

Conjugates were prepared as above or by using periodate according to Wilson and

Nakane (197X).

The diluent for the conjugate was normal bovine serum. Polystyrene microtitre plates

with 96 flat-bottom wells (Greiner) were coated with sheep anti-HBs as for Hepanostika.

The reagents used for the enzyme reaction were the same as for the first modification

described above. The test procedure comprised the following steps: addition of 0.1 ml

of sample to the plate well, incubation for 1 h at 50°C in an incubator, washing, addition

of 0.1 ml conjugate, incubation for 1 h at 50°C in an incubator, washing, addition of

substrate and stopping after 30 min at room temperature. The results were measured

with a Multiskan (Flow Labs). This photometer reads all colours in one plate directly

through the bottom of the wells and the results are printed automatically on a paper

tape in 1 min.

59

~he~mato~ factor was determined by a latex agglutination test (~eumanosticon, Or-

ganon Teknika).

RESULTS AND DISCUSSION

An essential difference of this modification with Hepanostika was that the washing

step after the incubation of the sample with the solid-phase antibody was omitted. It

was found that this change reduced considerably the background of the non-specifically

bound labelled antibody. Therefore the conjugate concentration could be increased and

the incubation times shortened. An additiona reduction of the incubation times was

obtained by increasing the temperature of the incubator to 50°C.

Sera containing HBsAg were diluted in normal human sera free from HBsAg and

anti-HBs and subsequently tested with Hepanostika by the first modification. If 2.1

times the mean negative control was taken as the cut-off value for both test systems,

the modi~ed test was shown to be about three times as sensitive as Hepanost~a when

using a first incubation of 2 h at 37°C. Since an excess of HBsAg in the sample was not

removed before the addition of enzyme-IabeIled anti-HBs, the reactions of strongly positive samples with the modified test were relatively suppressed. This phenomenon

was observed at HBsAg concentrations of above 10,000 units/ml, but complete inhibition

was never seen. Up to IO6 units of HBsAg per ml reacted still positively. As this is an un-

fikefy high concentration, no problems were to be expected in practice.

A more serious drawback of the first modification was that factors in the test sample

which interfered with the enzyme label or affected the conjugate in some other way

were not removed. For instance, reducing agents such as sodium azide could give false

negative reactions, whereas peroxidase-like substances such as haemoglobin could cause

false positive reactions. Moreover, some clotting factors present in plasma or in serum

from insufficiently clotted blood gave rise to false positive reactions. In general, the test

performance was also more open to technical errors than Hepanostika. Thus, it was found

in a trial performed at three different centres that the percentage of false positive sera in

a blood donor population could be as high as 8~5, whereas the corresponding figure for

the original Hepanostika was in the order of 1%. The disappointing specificity of the first

modification was the most important reason for not developing this test further as a

routine screening test for HBsAg.

Second modification

The test procedure of the second modification was, in principle, the same as for Hepa-

nostika, except that the first and second incubations were reduced to 1 h each and the

incubation temperature was increased to 50°C. As in the first modification, the conjugate

concentration was increased to improve the sensitivity. From our experience with the

first modification it was found that the non-specific binding of conjugate at a high con-

60

centration could be reduced by a high protein concentration. This was done by dilution

of the conjugate in bovine serum. In order to find the best conjugate for the second

modification conjugates prepared according to two different methods were compared.

Dilution series of HBsAg-containing sera in normal human serum were tested with the

second modi~catjon using the two different conjugates and Hepanostika. Typical ex-

amples of the dose-response curves obtained are shown in Fig. 1. In the same experi-

ment 30 different normal human sera were also tested. The responses of the HBsAg

dilutions were expressed as the number of standard deviations above the mean of the

negative sera. In this way, the same criteria for sensitivity were taken for both test sys-

tems. In practice, 5 standard deviations above the mean negative correspond with 2.1

times the mean negative for Hepanostika. If 5 standard deviations were taken as the cut-

off level, the second modification was at least twice as sensitive as Hepanostika using the

overnight incubation for the sample. In this experiment, the conjugate prepared according

to Avrameas gave a slightly higher sensitivity than the conjugate prepared according to

Wilson and Nakane. However, in some other experiments the Avrameas conjugate was

found slightly less sensitive. In general, a lower detection limit of 0.5-l unit/ml was

found for subtype ad and ay .

Response

30

1

1

A Improved

W/N version

zo-

Hepanostiko

(overnight]

10-

fj____ --- --____ ----_-_

0,25 0,s 1,O 2,O

Concentration HBsAg U/ml (log scale)

Fig. 1. Dose-response of improved enzyme-immunoassay for HBsAg (second modification) compared with Hepanostika. The response is expressed as the No. of S.D. above the mean of 30 normal human sera. The positive cut-off level is defined as a response of 5 S.D. (broken line). The concentration of HBsAg is given in units/ml as defined by the Paul Ehrlich Institute in West Germany. A = conjugate prepared according to Avrameas; W/N = conjugate prepared according to Wilson and Nakane.

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In a preliminary study of the specificity 5 16 donor sera, which had been stored at

-2O”C, were tested. The results are shown in Table 1, part 1. The presumptive positives

were tested for rheumatoid factor. Hepanostika was reactive with three sera, which were

negative in the repeat test and also negative for rheumatoid factor. The modified test

with the Wilson and Nakane conjugate had 6 false positives on screening. The two false

positives that were negative for RF were not reactive when the test was repeated. The

other four sera were positive for rheumatoid factor and were repeatably reactive with the

modified Hepanostika, although three were only borderline positive. This reactivity could

not be neutralized with human anti-HBs. The same rheumatoid factor positive sera were

also reactive with the Avrameas conjugate in the modified test in addition to one rheu-

matoid factor positive that was only reactive with this conjugate. The rheumatoid factor

containing sera were found more reactive with the Avrameas conjugate than with the

Wilson and Nakane conjugate (data not shown). In addition to the five presumptive

positives which contained rheumatoid factor, the Avrameas conjugate showed another

four presumptive positives. All but one, which could not be neutralized with human anti-

HBs, were negative upon repeat.

The overall specificity of the second modification seemed acceptable in this experi-

ment with both conjugates (1.2% and 1.8% false positive results respectively), although

some more false positives may be expected than with Hepanostika because of possible

interference with rheumatoid factor. In the same experiment, an HBsAg positive control,

containing 4 units/ml of subtype ad in normal human serum, was included in each test

plate. The mean response of this control was determined for Hepanostika and for the

second modification with both conjugates (Table 1, part 2). Thus, it was found that the

specific response of the second modification using the Wilson and Nakane conjugate was

approximately twice as high as with Hepanostika with an overnight period of incubation.

The Avrameas conjugate showed a similar response as Hepanostika, indicating that a

TABLE 1

Screening of 516 stored and frozen donor sera with Hepanostika and a second modification using

different conjugates

Hepanostika

(overnight)

Modification

WIN A

1. No. of false positivesa

RF - 3 (0.6) 2 (0.4) 4 (0.8)

RF+ 0 (0.0) 4 (0.8) 5 (1.0)

2. Indication of sensitivity

(No. of S.D. above mean negative)

HBsAg (4 units/ml) 11 21 10

a Cut-off = mean (excluding the RF-positives) + 5 S.D.; W/N = Wilson and Nakane;A = Avrameas;

RF = rheumatoid factor.

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higher sensitivity of the second version was not obtained with this type of conjugate

in this experiment. However, this represents a better result than Hepanostika with a first

incubation period of 2 h at 37°C.

On the basis of the results with the second modification of Hepanostika, it is con-

cluded that the development of an improved Hepanostika is feasible: the sensitivity was

increased by a factor of at least 2, the total duration of the test was reduced by at least

2.5 h and the test results could be easily measured with a photometer. Although the first

results with stored and frozen donor sera are promising, further studies with fresh donor

sera are needed to obtain more information regarding the specificity of the second modi-

fication with both conjugates.

ACKNOWLEDGEMENTS

The authors thank Mr. T. Jacobs and Mrs. A. Siebelink-Liauw for their technical help.

REFERENCES

Avremeas, S. and T. Ternynck, 1971, Immunochemistry 8, 1175. Strute, R., Luxem and H.T. Bruster, 1979, Lab. Med. 3,47. Wilson, M.B. and PX. Nakane, 1978, in: Immunofluorescence and Related Staining Techniques, eds.

W. Knapp, K. Holubar and G. Wick (Elsevier/North-Ho~and BiomedicalPress, Amsterdam) p. 215. Wolters, G., L. Kuijpers, J. Ka&rki and A. Schuurs, 1976, J. Clin. Pathol. 29,873.