Journal of Medical Virology 30:253-257 (1990)

Comparison of Enzyme Immunoassay With Radioimmunoassay for the Detection of Antibody to Hepatitis B Core Antigen as the Only Marker of Hepatitis B Infection in a Population With a High Prevalence of Hepatitis B

Alan J. Parkinson, Brian J. McMahon, David Hall, Donald Ritter, and Mary Anne Fitzgerald Arctic Investigations Program, Center for Infectious Disease, Centers for Disease Control, Anchorage, Alaska (A.J.P., D.H., M.A.F.); Alaska Native Medical Center, Indian Health Seruice, Anchorage, Alaska (B.J.M.); State Public Health Laboratory, Department of Health and Social Seruices, State o f Alaska, Fairbanks, Alaska (D.R. ) .

Enzyme immunoassay (EIA) and radioimmu- noassay (RIA) for the detection of antibody to hepatitis B core antigen (anti-HBc) were com- pared using serum specimens f rom Alaska Na- tives screened during a hepatitis B control pro- gram that were initially positive by EIA for only anti-HBc. Of 36 specimens f rom persons previ- ously HBsAg positive but who were now only anti-HBc positive by EIA, 94.4% were anti-HBc positive by both assays, wi th anti-HBc levels ex- ceeding 93% inhibition. Low-level antibody to hepatitis B surface antigen (anti-HBs) (<I0 SRU) and antibody to hepatitis Be (anti-HBe) were also present in 50% and 48% of specimens positive for anti-HBc, respectively. Of 148 specimens f rom persons initially positive for only anti-HBc by EIA who had no previous documentation of any hepatitis B virus (HBV) infection, 64.5% were positive by repeat testing for anti-HBc by both assays, and anti-HBc levels in this sample ex- ceeded 70% in 91.6% and 80.2% o f specimens by EIA and RIA, respectively. Low-level anti-HBs and anti-HBe were present i n 45.8% and 15.6%, respectively. EIA detection of anti-HBc was found to be less specific than RIA. Of specimens positive for anti-HBc by EIA, 14.8% were nega- tive by RIA. The specificity of the EIA could be improved wi th respect to RIA by increasing the cut-off f rom 48% t o 68%. In samples wi th low- level anti-HBc (270% inhibit ion) as measured by either method, the anti-HBc result was less likely to persist upon retesting, whereas sample wi th anti-HBc levels of >70% inhibit ion the anti-HBc was a reproducible finding frequently accompa- nied by either low-level anti-HBs or anti-HBe.

KEY WORDS: HBV, anti-HBc, EIA, RIA

INTRODUCTION Assays for hepatitis B virus (HBV) infection are now

used widely for clinical diagnosis as well as to screen populations at risk and identify potential candidates for hepatitis B vaccine. Infection with HBV is usually signaled by the presence of both hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core an- tigen (anti-HBc) in persons with active infection, or anti-HBc and antibody to HBsAg (anti-HBs) in persons previously infected who are immune. Because anti- HBc is found in virtually all patients who have been or are currently infected with HBV, investigators believe that it is the best single marker of HBV infection and is therefore widely advocated for screening purposes (Kane et al., 1985; Lander et al., 1978; Dienstag et al., 1987). Because of the significant association between anti-HBc positivity in blood donors and development of non-A non-B hepatitis (NANB) in recipients (Delaris et al., 1986; Stevens et al., 1984), anti-HBc is now used as a surrogate test for NANB hepatitis in regional and national blood banks in the United States. Presence of anti-HBc alone, however, without HBsAg or anti-HBs, has been detected in 2-1354 of persons tested (Hu et al., 1984; Lok et al., 1988), and the interpretation of anti- HBc alone is uncertain. Anti-HBc alone could mean that a person has been previously infected but has ei- ther had a poor initial anti-HBs response or has lost anti-HBs years after acute infection and is therefore

Accepted for publication January 8, 1990. Address reprint requests to Dr. Alan J. Parkinson, Arctic In-

vestigations Program, Center for Infectious Diseases, Centers for Disease Control, Anchorage, AK 99501.

( 1990 WILEY-LISS, INC.

234

immune, hence need not be offered HBV vaccine (Kane et al., 1985). Anti-HBc in the absence of other markers can also be found in persons recovering from acute HBV infection who have cleared HBsAg and have not yet developed anti-HBs. These persons are usually pos- itive for anti-HBc IgM., (Lindsey et al., 1987). Recent evidence suggests that some individuals positive for only anti-HBc may be chronic HBV carriers, with lev- els of HBsAg too low to detect by commercial assays and could be a t risk of transmitting HBV to others (Ben-Porath et al., 1984). Alternatively, the finding of anti-HBc as the only marker of HBV infection could be a false-positive result, due to either a cross-reactive IgG or IgA antibody (Sallberg et al., 1989) or interfer- ing substance in serum.

The detection of anti-HBc by enzyme immunoassay (EIA) has largely replaced radioimmunoassay (RIA), because of advantages that include longer shelf-life of EIA systems, absence of special radioisotope safety is- sues, licencing, and disposal regulations required for RIA but not EIA. Early studies in blood donors have demonstrated the equivalence of EIA and RIA sensi- tivity and specificity for the detection of anti-HBc (Hyo-Suk et al., 1987; Troisi et al., 1987). However, recent studies demonstrating that of persons identified by EIA as having anti-HBc as the only marker of HBV infection may have a primary response to HB vaccine have raised questions as to specificity of the EIA for the detection of anti-HBc, particularly in regions of high HBV prevalence (Lok et al., 1988).

Alaska Natives have a high prevalence of HBV in- fection (Schreeder et al., 1983). In 1982, a statewide program to control HBV among Alaska Natives was initiated (McMahon et al., 1987). This program in- cludes the screening of all Alaska Natives for HBV serologic markers by EIA and immunization of those who are seronegative. Anti-HBc was used as an initial screening test. Persons who were Serum positive for anti-HBc were then tested for HBsAg and anti-HBs. To date, 53,000 Alaska Natives have been screened, and the prevalence of HBsAg in different geographical re- gions has ranged from 0.5% to as high as 7.8%. Of those screened by EIA, 3.0% have been found to possess anti- HBc as the sole marker of HBV infection. Since many of these subjects reside in remote regions, an accurate interpretation of the test result is important to deter- mine whether these persons need immunization. In this study, we compared EIA and RIA for the detection of anti-HBc in sera of Alaska Natives screened during the hepatitis B control program who were initially identified by EIA as being anti-HBc only.

MATERIALS AND METHODS Two groups of serum samples were selected. The first

consisted of 36 samples from 33 persons who had doc- umentation of previous HBsAg and anti-HBc positiv- ity, but subsequently (0.25-13 years; mean 3.8 years) were positive for anti-HBc but negative for anti-HBs and HBsAg by EIA. In the second group, 148 serum

I’arkiiison et al.

samples from 130 Alaska Natives randomly selected from 1,500 persons, with no documentation of previous HBV infection, were positive for anti-HBc but negative for anti-HBs and HBsAg by EIA. Serum samples in this group were selected geographically by Indian Health Service Units. All regions of Alaska, including areas with both high and low HBV prevalence, were represented. Serum samples obtained on more than one occasion were available for 13 persons. Kits used for HBV markers were purchased from Abbott Laborato- ries (North Chicago, IL). All assays were performed strictly as described by the manufacturer’s instruc- tions. Serum samples selected were initially screened between January 1983 and June 1985 by EIA for the presence of anti-HBc (Corzyme’ ). Positive samples were tested for HBsAg (Auszyme 11) and anti-HBs (Ausab EIA). Serum samples were then stored a t -20°C. Kits for anti-HBc detection before June 1985 used HBcAg derived from plasma (PD-HBcAg). Serum samples were retested for the presence of anti-HBc by EIA (Corzyme) and RIA-(Corab). Kits for all anti-HBc retests used the highly purified HBcAg produced by recombinant DNA (rDNA) methods. Anti-HBc levels were quantitated by calculating the percentage inhibi- tion using the following formula (Hadler et al., 1984): Percentage inhibition = 100 ~ [test reading (cpm or O.U.)/negative

control reading (cpm or O.D.)I x 100

where O.D. is the optical density of the test or control sample. Serum samples demonstrating inhibition of >49% by RIA and >58% by EIA were considered pos- itive as per the manufacturer’s recommendation. In this study, samples with inhibition levels of >70% were considered to have high levels of anti-HBc. Those with levels between the assay cut off and 70% were termed low-level anti-HBc (Hadler et al., 1984). Sam- ples positive for anti-HBc by both assays were tested for the presence of HBsAg by RIA (Ausria). Those pos- itive for HBsAg on RIA were confirmed by EIA neu- tralization. Samples were then tested for anti-HBs (Ausab) by RIA and anti-HBe by EIA. Sample ratio units (SRU) for the HBsAg and anti-HBs assays were calculated as described by the manufacturer. Sample ratio units (SRUs) 22.1 were considered positive. All serum samples were tested for IgM anti-HBc (Corzyme M).

RESULTS Anti-HBc Alone in Serum Samples of Previously

HBsAg-Positive Persons Thirty-six samples from 33 persons aged 6-76 years

(mean 28 years of age) who were positive for only anti- HBc by EIA 3 months to 13 years after documentation of HBsAg positivity were retested for anti-HBc by EIA and RIA. Of these samples, 34 (94.4%) were positive for

‘Use of tradenames is for identification only and does not imply endorsement by the U.S. Public Health Service or by the U.S. Department of Health and Human Services.

EIA and R I A Detwtion of Anti-HIk Alone 255

I 0 00 h

34 I

EIA cut off (58%)

'c Y- U

20 I O I

10 3' ~

Q Z ' 0 .I--T-.-- ,. r~-,-----I 4

0 10 20 30 40 50 60 70 80 90100 Percent Inhibition (RIA)

Fig. 1. Comparison of anti-HBc levels (percentage inhibition) deter- mined by radioimmunoassay (RIA) and repeat enzyme immunoassay (EIA) in serum samples tha t were initially positive (by EIAi for only anti-HBc. These persons had a previous sample tha t tested positive for HBsAg.

anti-HBc by both assays and all possessed levels of anti-HBc exceeding 93% inhibition by EIA and RIA (Fig. 1). Two samples were negative for anti-HBc by EIA but positive by RIA. When the remaining 34 sam- ples positive for anti-HBc by both EIA and RIA were tested for HBsAg, anti-HBs, and anti-HBc IgM, two (5.8%) were confirmed positive for HBsAg (SRU 2.2, 3.6) and one other (2.9% ) possessed anti-HBc IgM. Sev- enteen (50.0% ) contained low-level anti-HBs (SRU 2.3-6.4) by RIA and, of 29 of the anti-HBc positive by EIA and RIA, 14 (48.2%) were positive for anti-HBe ( 7 of these also had low-level anti-HBs). Of the 29 samples tested for all markers, anti-HBc, HBsAg, anti-HBs, anti-HBc IgM, and anti-HBe 9 (31.0%) had no other marker of HBV infection other than anti-HBc (Table I) .

Anti-HBc Alone in Serum Samples of Persons With no Documentation of Previous

HBV Infection Of 148 serum samples from persons with no docu-

mentation of any previous HBV infection but who had anti-HBc in the absence of HBsAg and anti-HBs by the initial EIA screen, 96 (64.8% ) were confirmed anti-HBc positive upon retesting by both EIA and RIA, 27 (18.2c% were negative by both assays, 22 (14.8%) were positive by EIA but negative by RIA, and 3 (2.0% were negative by EIA but positive by RIA (Fig. 2).

Levels of anti-HBc were evaluated in 96 serum sam- ples positive for anti-HBc by both assays. High levels were found in 88 (91.6%) by EIA and 77 (80.2%) by RIA. By contrast, only 6 (27.2%) of 22 samples anti-

TABLE I. Markers of Hepatitis B Infection Positive After Retesting of Serum Samples Initially Positive for Anti-HBc

but Negative for Anti-HBs, HBsAg, by EIA

Persons Persons with no previously documented

N (% 1 N (% 1 HBsAg positive HBsAg

Anti-HBc" 34136 94.4 961148 64.85

HBsAg" 2134 5.8 1196 1.0 Anti-HBc IgM" 1134 2.9 2196 2.0 Anti-HBs' 17134 50 44196 45.8

Anti-HBe" 14129 48.2 13183 15.6 Anti-HBc only 9129 31.0 43183 51.8

.'Retested by RIA and EIA "Retested by EIA. 'Retested by RIA.

100 ~

I ' 00 I

n Q w 70 6 60' 2 2 50 ' - C I . +-' 40

30 '

v I

c a,

a l 20

10 A .

0 I-

..... ..... ......... ........ ....... . . . . . . . . .

22 . , :.*' 96 ' . . . , . , . . .

. . . . . . . .......................... ' : * 'EIA cut off (58%) . . . .

. .

'c'

0 , % -

0 10 20 30 40 50 60 70 80 90100 Percent Inhibition (RIA)

Fig. 2. Comparison of anti-HBc levels (percentage inhibition) deter- mined by radioimmunoassay (RIA) and repeat enzyme immunoassay iEIAi in serum samples that were initially positive (by EIA) for only anit-HBc. These persons had no documentation of previous hepatitis I3 virus (HRVl infection.

HBc positive by EIA had inhibition levels >70%, whereas all three positive by RIA had levels of anti- HBc <70% (Fig. 2). The three anti-HBc RIA-positive/ EIA-negative specimens were RIA negative when retested.

All 96 serum samples positive for anti-HBc by both EIA and RIA were retested for the presence of HBsAg, anti-HBs and anti-HBc IgM (Table I). One sample was positive for HBsAg by RIA (3.4 SRU) and was con- firmed positive by EIA neutralization. Two additional samples (2.0% ) were positive for anti-HBc IgM, and 44 (45.8%) were positive for anti-HBs by RIA, all with SRUs of 2.1-10.5. Of 83 samples positive for anti-HBc

266

by both EIA and RIA tested for anti-HBe, 13 (15.6%) were positive. Of 83 samples tested for all markers, 43 (51.8%)) were positive for only anti-HBc. The levels of anti-HBc in these samples exceeded 70% inhibition in 92% of samples by EIA and 80% by RIA.

Thirteen persons initially positive by EIA for only anti-HBc later had serum drawn on two or more occa- sions. Two persons had initial serum specimens with low-level (<70%,) anti-HBc by EIA and were negative by RIA. Subsequent sera from these persons were ei- ther low-level anti-HBc positive or negative by either EIA or RIA upon retest. By, contrast, all other persons who had anti-HBc levels of >70% inhibition by both EIA and RIA in the initial serum tested were also pos- itive for anti-HBc by both EIA and RIA in subsequent serum. Similarly, of five persons with low-level anti- HBs, <10 SRUs, in initial serum specimens, all were positive for low-level anti-HBs in subsequent speci- mens tested.

DISCUSSION In this study, EIA and RIA were compared for the

detection of anti-HBc by retesting the original serum samples positive for anti-HBc but negative for HBsAg and anti-HBs when initially tested by EIA. Serum samples were selected from individuals both with and without documented previous HBsAg positivity. When sera from persons previously HBsAg positive were tested the EIA and RIA for the detection of anti-HBc were equivalent. The level of anti-HBc in these persons all exceeded 93% by both assays confirming the con- tinuing presence of high levels of specific antibody 3 months to 13 years following the last documented HBsAg-positive test. Furthermore, 50% of these per- sons possessed low-level anti-HBs detected by RIA, but not by EIA. The RIA for anti-HBs has been shown to be a more sensitive test for anti-HBs than the EIA, al- though the EIA correlates well with protection because the cutoff has been set to an RIA equivalent of 10 SRU (Racela et al., 1986). In a subset of sera from persons with a prior history of HBsAg who were tested for anti- HBe, 48.2% were positive, demonstrating the value of anti-HBe as an additional marker of natural HBV in- fection in persons identified with isolated anti-HBc. However, of those persons with a prior history of HBsAg positivity who were tested for all markers, 31% were positive for only anti-HBc. These results suggest, first, that some of these individuals may have had a poor or delayed initial response to HBV infection. A previous study in this population demonstrated that some patients did, indeed, have a delayed development of anti-HBs after acute symptomatic HBV infection and others had only transient presence of anti-HBs (McMahon et al., 1981). Most Alaskan Eskimos, how- ever, respond well to HBsAg (HB vaccine) and produce high levels of anti-HBs (McMahon et al., 1987). Second, anti-HBs and anti-HBe may decay with time after HBV infection. Anti-HBs decay was suggested by Draelos et al. (19871, who showed that persons with

Parkinson et al.

anti-HBc alone measured by RIA had an intermediate response to HB vaccine when compared to uninfected control person or persons with other markers of HBV infection. Although studies in Alaska and elsewhere show that anti-HBs decay does occur after HB vaccine with time, studies are not available to determine whether this is common after natural HBV infection. Third, some of these persons may be chronic HBsAg carriers with HBsAg levels too low to be detected by the commercial RIA or EIA used in this study. In one study using a sensitive monoclonal antibody RIA, HBsAg was detected in up to 26% of persons, with anti- HBc as the only marker of HBV infection (Ben-Porath et al., 1984). However, testing for HBsAg by a mono- clonal antibody assay with a sensitivity of 20 pgiml of a random sample of sera from persons in this study who were anti-HBc only by both EIA and RIA failed to detect any low-level HBsAg carriers in this group (Wands, JR, unpublished results). More sensitive methods such as the polymerase chain reaction may still identify low level HBV carriage as a cause of anti- HBc as the only marker of HBV infection (Liang et al., 1989).

In most instances, however, the prior serologic his- tory of persons identified with anti-HBc as the only marker of HBV infection is unknown. Within this group retested for anti-HBc by EIA and RIA in the present study, 18.2% of these persons were negative by both tests. It is possible that the high rate of initial false positives may be related to the use of PD HBcAg in test kits manufactured before June 1985. Today’s systems use highly purified rDNA HBcAg and may have a greater specificity than systems that used the PD HBcAg (Hyo-Suk et al., 1987). However, even with the current EIA and RIA anti-HBc tests that use rDNA HBcAg, 14.8% of serum samples retested were positive by EIA but negative by RIA, and of these, most (73%) possessed levels of anti-HBc of <70?Z inhibition by EIA. Yet, only 2% of these serum samples were anti- HBc positive by RIA, but negative by EIA. This sug- gests RIA has a greater diagnostic specificity for anti- HBc than EIA. Vaccination of Alaska Natives positive for anti-HBc by EIA alone resulted in only 3 of 41 (7.3%) developing a booster response to one dose of HB vaccine, suggesting false-positive results for anti-HBc in these patients (McMahon et al., 1987). These obser- vations are supported by other recent reports. Schmidt et al. (1988) found that of blood donors identified by EIA as anti-HBc positive, only 60% were positive by RIA. Similarly, Lok et al. (1988) also observed that of 32 subjects with anti-HBc only tested by EIA, 56% had a primary response to HB vaccine suggesting that some of these showed false-positive results.

In our study, with the cutoff set at 58% inhibition, the specificity of the EIA was only 55%. By increasing the EIA cutoff to 65%) inhibition, the specificity of the EIA could be improved to 83% with little loss in dignos- tic sensitivity in relationship to the RIA. The reason for the difference between these two assays, which use the

E I A and RIA Detection of Anti-HRc Alone

Same antigen Source and competitive assay format, is unclear but may be related to use ofan enzyme label in the EIA, which, unlike the isotopic label used in the RIA, may be subject to inactivation by inhibitors in

257

hepatitis A and B in the Shanghai area. Prevalence of serum markers. American Journal of Epidemiology 120:404-413.

Hyo-Suk L, Ra.jagopalar MS, Chien I), Cordell R, Perkins HA, Vyas GR (19x71: Specificity ofenzyme immunoassay for hepatitis B core antibody used in screening blood donors. Transfusion 27:103-108.

human serum and in this competitive format would result in a reproducible but false-positive test.

The results of this study suggest that RIA for the detection of anti-HBc is a more specific test for the detection of anti-HBc than the EIA. It was found that of samples with low-level anti-HBc (570% inhibition) measured by either method, anti-HBc was less likely to persist on retesting either the same or subsequent spec- imens from the same person. Conversely, sera with high levels of anti-HBc (>70% inhibition) by EIA or RIA persisted on retesting, and these specimens were more likely to contain low-level anti-HBs if measured by RIA or anti-HBe detected by EIA, indicating that these persons were probably previously infected with HBV.

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