Journal of Medical Virology 3:43-49 (1978)

Enzymelmmunoassay in the Diagnosis of Hepatitis With Emphasis on the Detection of “e” Antigen (HBeAg) M.v.d. Waart, A. Snelting, J. Cichy, G. Wolters, and A. Schuurs Organon, Scientific Development Group, Oss, The Netherlands

A brief survey of the application of enzS.Ine-iinmunoas~y(EIA) for the detection of hepatitis B surface antigen (HBsAg), hepatitis A, and their corresponding antibodies is given. The prelirninary results of a similar EIA for detection of hepatjtis B-related “e” antigen (HBeAg) and its antibody (anti-HBe) are reported. This EIA is much more sensitive than immunodiffusion: a t least 128 times for HBeAg and at least 512 times for anti-HBe. HBsAg and its antibody do not interfere with the test. Only a few sera strongly positive for rheumatoid factor gave rise to false-positive results, as was demonstrated by a confirmatory test. Key words: hepatitis, enzyme-immunoassay, ELISA. “e” antigen, HBeAg

INTRODUCTION A new technique called enzyme-immunoassay (EIA) 01- enzyme-linked imrnunosorbent assay (ELISA), analogous to radioimmunoassay, was reported in 1971 [ l , 21 . Enzymes were used in place of radioisotopes as a label to determine compounds or substances present in biological specimens. As summariLed in several reviews [3-51 , EIA can be applied in many areas, and assays for hormones, serum proteins, antibodies against pathogens, and viral antigen-antibody systems have been developed. The first applications of ETA in the field of hepatitis were tests for hepatitis B surface antigen (HBsAg) and its corresponding antibody, anti-HBs, in serum [6-81 . The HBsAg test as developed in our laboratory makes use of the “sandwich” principle. Undiluted serum under test is incubated in a well of a polystyrene microtitration plate coated with sheep anti-HBs. Horseradish peroxidase (HRP)-labeled anti-HBs is then used to react with bound HBsAg. Finally, a colorless enzyme substrate is added which is converted into a colored product b y HRP. This test turned out to b e about as sensitive as a radioimmunoassay for HBsAg and was superior to reversed hemagglutination [6,9] .

Address reprint requests to M. v.d. Waart, Organon, Scientific Development Group, Oss, The

Netherlands. 0146-6615/78/0301-0043$01.70 0 1978 Alan R. Liss, Inc

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v.d. Waart

e t a1

Anti-HBs can be detected by preincubating the antibody-containing sera with a fixed amount of HBsAg-positive serum (blocking principle). The remaining HBsAg is assayed according to the procedure described above. With respect to sensitivity, this EIA compares favorably with the commercially available passive hemagglutination test for anti-HBs [unpublished results] . Meanwhile, other tests for HBsAg and anti-HBs based on the EIA principle have been reported [ 10-121 . Recent progress was made in our laboratory with the detection of hepatitis A antigen (HAAg) in fecal extracts and its antibody, anti-HAV, in serum [ I 31 . Essentially thc same method as outlined above was employed, except for the use of polyvinylchloride, instead of polystyrene, microtitration plates and human, instead of sheep, antibodies. This EIA for detection of IIAAg and anti-HAV has proved to be as sensitive as radioirnmunoassay and immune electron microscopy [ 141 . Similar results were obtained by Mathiesen et a1 [ 1S] , Because of its sensitivity in detection of HRsAg and HAAg, we wondered if EIA could also be applied to detect HBeAg, the “e” antigen associated with type B hepatitis. The presence of this antigen in HBsAg-posjtive blood seems to be correlated with a poor prognosis [16] and with high risk of both horizontal [ 171 and vertical [ 181 transmission. The lack of sufficiently sensitive detection methods has hampered the full understanding of the significance of this antigen and its corresponding antibody (anti-HBe). We developed a solid-phase sandwich EIA for H3eAg and anti-HBe and compared the results with those of immunodiffusion (ID).

MATERIALS A N D METHODS

Most of the methods used were the same as described by Wolters et a1 [6] ,with some modifications. lmmunodiffusion (ID) was performed with 2 X 2.5 pl samples in 0 , 9 %agarose (gel thickness, 0.6 mm; diameter of and distance between the wells, 3 mm). The results were read after five days at room temperature. Antibody-coated microtitration plates (Microelisa, specially produced by Greiner for Organon Teknika, Oss, The Netherlands) were prepared by incubating the wells with 0.1 ml of the immunoglobulin fraction of the 18% Na2S04 precipitate derived from a human anti-HBe, /HBe2 -positive serum (ID titer 2 8). This serum was positive for HBsAg in the EIA for this antigen (Hepanostika, Organon Teknika, Oss, The Netherlands), but negative for both anti-HBs (Ausab? Abbott Laboratories, Chicago, Illinois, USA) and rheumatoid factor (RF) (Rheumanosticon, Organon Teknika, Oss, The Netherlands). HW-labeled anti-HBe was prepared from the same Na2 SO4 precipitate according to the two-step method of Avrameas [191. Screening for HBeAg was performed as follows: 0.1 ml test sample (or normal human serum, NHS, as a control) was incubated in a coated well of a microtitration plate for 2 h at 37°C. The wells were washed four times with 0.2 M Tris buffer pH 7.4. Each well was then incubated with 0.1 ml of enzyme-labeled anti-HBe for 2 h at 37°C. The wells were washed four times, and 0.1 ml orthophenylenediamine-ureaperoxide was added. After further incubation for 50 min at room temperature in the dark, the enzyme reaction was stopped with 0.05 m l 4 N H2 SO4. The absorbance was read at 492 nni and corrected for the reagent blank (A492). Samples were considered positive if the positive-negative (P/N) ratio was 22.1. For this calculation the mean of at least five sera was used as negative control.

Enzyme-Immunoassay for “e” Antigen

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All samples reacting positively in the screening test were tested with a confirmatory blocking test similar to the method described for HBsAg [20] . An 0.1 ml test sample was incubated overnight in antibody-coated plates at room temperature. Thereafter, 0.15 ml anti-HBe (or NHS, as a control) was added, and the screening test for HBeAg was performed as described before. If A4= was at least 50% lower than that of the negative control, the sample was considered positive for HBeAg. Detection of anti-HBe was carried out by preincubating 0.1 ml test serum (or NHS, as a control) with 0.025 mi of a fixed amount of HBeAg for 18 11 at 37°C. Then 0.1 ml of this mixture was transferred to wells of an anti-HBe-coated microtitration plate and tested in the screening test. If A492 was at least 50% lower than that of the control, the sample was considered positive for anti-HBe. Test samples as well as the fixed amounts of HBeAg and anti-HBe for the anti-HBe test and the confirmatory test, respectively, were diluted in physiological saline/NHS (9/l, v/v). NHS was always a mixture of at least five normal human sera. RESULTS Sensitivity Determined in Dilution Series

Sera positive in ID for either HBeAg or anti-HRe were serially diluted. Dilutions were tested both in EIA and ID. Typical examples are shown in Figures 1 and 2. EIA titers were much higher than ID titers: 128 times for HBeAg, 512 times for anti-HBe. Testing of Sera From HBsAg-Positiveand HBsAg-Negative individuals

Sixty-eight HBsAg-positive sera were tested for HBeAg and anti-HBe. Two sera in this group were positive for HBeAg and 18 for anti-HBe in ID (Table I). In EIA 19 and

k ig. 1. Dose-response curve of HBeAg in EIA. Pos/neg ratio 1 8 corresponded with A,, For NHS values of A492 = 0.200 werc found. The reagent blank was A492 = 0.055.

=

2.700.

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v.d. Waart et a1 % inhibitian

102

titre

lo4

lo3

d i l u i i o n o f onti-HBe-contoining s e r u m

Fig. 2. Dose-response curve of anti-HBe in FIA. Absorbance ranged I'rom A492 = 1 .000 (no inhibition) to A492 = 0.200 (100% inhibition).

TABLE I. HBeAg and Anti-HBe in 68 HBsAg-Carriers

(%I

EIA (70)

2 ( 3) 18 (26) 48 (71)

19 (28) 36 (53) 5 ( 7) 8 (12)

ID HBeAg Anti-HBe Negative Conflicting

~

36 sera were positive for HBeAg and anti-HBe, respectively. Five out of the 68 sera were negative for both HBeAg and anti-HBe, whereas the remaining eight gave conflicting results. The latter were negative for HBeAg in tile screening test. They showed a positive reaction in the anti-HBe test, but a control reaction (sample t NHS, but without added HBeAg) gave an increased absorbance at 492 nm, which suggests also the presence of HBcAg or HBeAg-like activity. In a group of 73 sera negative for both HBsAg and anti-HBs, no HBeAg activity could be demonstrated. However, seven out of 12 sera negative for HBsAg and anti-HBs but (Fig. 3). In a group of 73 sera negative for both HBsAg and anti-HSs no HBeAg activity could be demonstrated. However, 7 out of 12 sera negative for HBsAg and anti-HBs but positive for R F gave a presumptive positive reaction ol' HBeAg that could not be confirmed. In another group of I00 HBsAg-negative blood donors two sera were found positive for anti-IIBe; both were also found to be positive for anti-HBs. Longitudinal Studies of Acute Hepatitis 6 Patients

We tested sera of 12 acute hepatitis B patients. In most of these longitudinal studies the presence of HBeAg could be detected with EIA only. Although small differences were observed between individual patients, in general a profile BS shown in

Enzyme-Immunoassay for “e” Antigen

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?& 100-

50

-

n

12

68

49

HBrAgt

Anti-HBr+

NHS

rheumatoid foctort

0

HBeAg-negatives

0

HBeAg-false positives HBeAg-true positives

Fig, 3 . Specificity of EIA for IIReAg.

Figure 4 was obtained. Apparent clearance of HBeAg occurred 6-8 weeks before the disappearance of HBsAg. During HBe-antigenemia anti-HBs was never detected. Anti-HBe activity (250% inhibition) could be detected approximately three weeks after the disappearance o r HBeAg. Dl SCUSS ION Sensitivity

The preliminary results of this study show that BIA is superior to ID with respect to sensitivity. EIA for HBeAg is at least 100 times more sensitive than lD, as can be concluded from serial dilutioIi series (Fig. 1). P/N ratios of about 20 and initial absorbances of about 3.0 for undiluted sera are maximally obtained. The EIA for anti-HBe appears to be at least 500 times more sensitive than ID. A further impression of the sensitivity of EJA was obtained by testing a panel of HBsAg-carriers (Table I). Only five of 68 HBsAg-positive sera were definitely negative for both HBeAg and anti-HBe. Longitudinal studies of acute hepatitis B patients revealed that anti-HBe was only definitely positive - that means gave more than 50% inhibition - about three weeks after disappearance of HBeAg. However, in the intermediate period we found between 15% and 50% inhibition, suggesting the presence of small amounts of anti-HBe. A further increase of the sensitivity of our tests might lead to a reduction of this intermediate phase in which HBeAg or anti-HBe failed to be detected. This might be achieved by increasing incubation times as done in a radioimmunoassay for HBeAg and anti-HBe, which was described recently [21]. With the latter test almost 100% of HBs-Ag-positive samples could be classified as positive for either HBeAg or anti-HBe. A hemagglutination test for HBeAg and anti-HBe [22] seems to be about as sensitive as the EIA, as both techniques detected either HBeAg or anti-HBe in about 80% of asymptomatic carriers of HBsAg.

48

v.d. Waart et a1 P I N ratio

% inhibition

I

HBeAg

0

i 50

-2

6

I

10

14

weeks

Fig. 4. Longitudinal study of an acute hepatitis B patient. “Weeks” on the absciss means “weeks after initial symptoms of hepatitis B.” In the screening test the saniple of the 11th week reacted weakly positive for HBeAg (pos/neg ratio 2.61, hut appeared to be false-positive in the confirmation test.

Specificity

On the basis of the present data the following conclusions seem justified. HBsAg did not interfere with the lest for HBeAg. The response in EIA of HBeAg/HBsAg-positive sera could be completely abolished b y adding sera positive for anti-HBe. Anti-HBs in test sera, even in high titers, did no1 inlerfere either, although some sera gave rise to falsepositive results in the screening test (Fig. 3). False-positive reactions due to R F activity were detected by the confirmation test. Twenty of 68 HBsAg-positive sera were found positive for HBeAg, 19 of which could be confirmed. The one false-positive serum demonstrated RF activity. In the group of 68 HBsAg-positive sera all sera positive for either HBeAg or antiHBe by ID reacted in EIA strongly positive for HBeAg and anti-HBe, respectively. The conflicting results obtained with eight sera can not yet be explained. Longitudinal Studies

HBeAg could be detected a t an early stage of infection, and it disappeared 6-8 weeks before the clearance of HBsAg. According to our criterion of 50% or mure inhibition, anti-HBe activity could be detected only about three weeks after the disappearance of HBeAg. From the shape of the anti-HBe curve, however, it could be deduced that anti-HBe activity could be detected as soon as HBeAg became negative. In future studies it therefore seems reasonable to use less severe criteria for anti-HBe positivity. The results of our longitudinal studies are in agreement with the KIA results of Sugg et a1 [21]. The results obtained so far with EIA for detection of HBeAg and anti-HBe are promising. They prove that human sera can be used for the preparation of antibodycoated plates and conjugates. Further work will be needed to improve sensitivity and t o investigate specificity in more detail.

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ACKNOWLEDGMENTS

We are grateful to Dr. Biswas (Gottingen, Germany), Dr. CouroucC (Paris, France), and Dr. LeBouvier and Mr. Williams (New Haven, Connecticut, USA), who checked some of our presumptive HBeAg or anti-HBe positive sera and who made reference sera available. Prof. Mudric (Now Sad, Yugoslavia) was kind enough to provide us with the sera from longitudinally followed patients. Dr. Babes (Bucarest, Rumania) was very helpful by supplying us with HBsAg-positive sera. We are also indebted to Drs. Reesink and Schut (Amsterdam) who provided antiHBs-positive and normal human sera. ADDENDUM

With radioimmunoassay five of the eight conflicting sera (see Results) were found positive for anti-HBe, one was negative for HBeAg and anti-HBe, and two could not be tested. (Kindly performed by Dr. Biswas, Gottingen.) Thcreupon, we have retested the remaining six sera in EIA. Four out of the five sera positive for anti-HBe in RTA were found positive for anti-HBe in ETA as well. The other two were negative. Tt cannot be excluded that the different outcome wab at least partially due to repealedly freedng and thawing of the sera. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22.

Van Weemen BK, Schuurs AHWM (1971): PEBS Lett 15:232-235. Engvall E, Perlmann P (1971): Immunochemistry 8:871-874. Wisdom G B (1976): Clin Chem 22: 1243-1255. Scharph SL, Cooreman WM, Blomme WJ, Laekeman GM (1976): Clin Chem 22:733-738. Schuurs AHWM, Van Weemen BK (1977): Clin Chini Acta 81 :1-40. WoltersG, Kuijpers L, Kacaki J, Schuurs A (1976): J Clin Path01 29:873-879. Wolters G, Kuijpers LPC, Kacaki J , Schuurs AHWM (1977): J Infect Dis 136:(suppl) 31 1-317. Wolters G, Kuijpers L, Schuurs A (1977): XVIth Symposium of the European Association against Virus Diseases, Amsterdam. Kacaki J , Wolters G, Kuijpers L, Stulemeyer S (1978): v o x Sanguinis 35:65-74. Caldwell CW, Barrett JT (1977): C h i Chim Acla 81:305-309. Wei R, Knight GI, Zimmerman DH, Bond HE (1977): Clin Cliem 23:813-815. Halbert SP, Anken M (1977): J Infect Dis 136(Supplj318-323. Duermeyer U‘,Van der Veen-du Prie J , Koster B (1977): XVIth Symposium of the European Association against Virus Diseases, Amsterdam. Duermeyer W, Van der Van der Veen-du Prie J, Koster B (1978): Lancet 1:823-824. Mathiesen LR, Feinstone Shll, Skinhoej P, Purcell RH (1977): Abstracts o f the 12111 Meeting of the European Association for the Study of the Liver, Kavouri, 8- 10 September, page 6. Smith JL, Murphy BL, Auslander MO, Maynard JE, Schalm SS, Summerskill WHJ, Gitnick G L (1976): Gastroenterology 71: 208-209. Alter HJ, Seeff LB, Kaplan PM, McAuliffe VJ, Wright EC, Gerin JL, Purcell RH, Holland PV, Zimmerman HJ (1976): N Engl J Med 295:909 ~ 9 1 3 . Okada K, Kaniiyama I. Inomata M, Iniai M, hfiyakawa Y, Mayunii M (1976): N Engl J Med 294:746-749. Avrameas s, TernynckT (1971): Imrnunochemistry 8:1175-1179. Kacaki J, Wolters G, Kuijpers L, Schuurs A (1977): J Clin Pathol 30:894-898. Sugg U, Frosner GG, Schneider W, Haas H (1977): XVIth Symposium of the European Association against Virus Diseases, Amsterdam. Takahasi K, Fukuda M, Baba K , Imai M, Miyakawa Y,Mayumi M (1977): J Immun 119:1556-1559.

Enzyme-immunoassay in diagnosis of hepatitis with emphasis on the detection of "e" antigen (HBeAg).

Journal of Medical Virology 3:43-49 (1978) Enzymelmmunoassay in the Diagnosis of Hepatitis With Emphasis on the Detection of “e” Antigen (HBeAg) M.v...
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