Comparison of enhanced chemiluminescence and microparticle enzyme immunoassay for the measurement of hepatitis B surface antibody

R.A. McCartney, J. Harbour, A.P.C.H. Roome and E.O. Caul*

Two commercially available immunoassays for the quantitative measurement of antibodies to hepatitis B surface antigen (HBsA9) were compared. The Amerlite enhanced chemiluminescence assay (ECL), which utilizes capture antigen obtained from HBsAg-positive donors bound to microtitre wells, was compared with the Abbott Laboratories microparticle enzyme immunoassay ( IMx), which uses a recombinant antigen coated on to microparticle carriers. A total of 310 specimens, l l6 from naturally infected patients (group A), 93 from patients vaccinated with a human plasma-derived vaccine (9roup B) and 101 patients vaccinated with a recombinant vaccine (group C), were tested in both assays undiluted and diluted 1:11. Samples that showed discordant results were also tested in a conventional quantitative enzyme immunoassay ( Sorin ). The results show significant differences in the level of anti-HBsA9 in patient sera usin9 the two technologies. In 9eneral, IMx 9ave significantly higher values than ECL for all three patient categories tested. These differences may lead to conflictin9 reports bein9 issued by laboratories who change their assay system or when a sample from one patient is tested by different laboratories usin9 different assays.

Keywords: Anti-hepatitis B surface antigen; postvaccination testing; enzyme immunoassay

Hepatitis B vaccines have been available in the UK for the immunization of selected groups since 1981. Vaccine prepared from recombinant hepatitis B surface antigen (rHBsAg) has now replaced those derived from human plasma in the UK. Postvaccination testing is used to establish immunity and to identify non-responders or poor responders, particularly where subsequent clinical management may depend on knowledge of the immune status. It also allows assessment of the probable duration of protective levels of antibody, which is generally agreed to be ~>10mIUm1-1 (Refs 1 and 2). This is important in persons with impaired immunity such as renal dialysis patients 3,4.

Until 1989 laboratory assays available for measuring anti-hepatitis B surface antigen (HBsAg) had a saturation limit of 50 mIU ml- 1 when undiluted serum samples were tested. In this laboratory we used a combination of passive haemagglutination test (PHA, Serodia Mast Diagnostics, Bootie, Merseyside) and an

Regional Virus Laboratory, Public Health Laboratory, Myrtle Road, Kingsdown, Bristol BS2 8EL, UK. *To whom correspondence should be addressed. (Received 20 May 1992; revised 11 September 1992; accepted 15 September 1992)

enzyme-linked immunosorbent assay (EIA, Behring Diagnostics, Hounslow, Middlesex) at a dilution of 1 : 5. From previous comparative work with a radioimmuno- assay (RIA) we knew that samples which gave complete agglutination in the PHA assay contained ~> 100 mIU ml- 1

(unpublished results). Samples giving less than complete agglutination were tested by EIA after a 1:5 predilution step as the limit of this assay was 30 mIU ml-1

In recent years two commercial assays capable of measuring anti-HBsAg levels up to 1000 mIU m1-1 in undiluted serum became available. These had an advantage over previous tests in that they used new technology which allowed the streamlining of laboratory testing, particularly in the hepatitis field. This was especially attractive at a time when work-loads were increasing rapidly as a result of more comprehensive vaccination policies. These two new assays, enhanced chemiluminescence (ECL, Amerlite Diagnostics) and the microparticle enzyme immunoassay (IMx, Abbot Laboratories) were compared to investigate apparent discrepancies in the postvaccine response reported by many laboratories, and to establish whether these differences could be associated with the assay used rather than any other factor.

In this paper data are presented showing the scale of the discrepancies in three groups of patients - naturally

0264-410)(/93/09/0941-05 ~, 1993 Butterworth-Heinemann Ltd Vaccine, Vol. 11, Issue 9, 1993 941

Anti-HBs testing: R.A. McCartney et al.

infected (group A ), those vaccinated with plasma-derived vaccine (group B) and those vaccinated with recombinant antigen (group C). Those samples giving discrepant results were further tested using a conventional enzyme immunoassay ( EIA ) (Sorin).

M A T E R I A L S A N D M E T H O D S

Anti-HBsAg assays

Amerlite Diagnostics ECL. This is an enzyme immunoassay using the antigen ant ibody-antigen sand- wich principle. Human plasma-derived HBsAg subtypes Ad and Ay are coated on to the surface of enhanced- binding capacity white plastic, flat-bottomed microtitre wells. Specimens containing anti-HBs will form an immune complex with the 'capture' HBsAg where the subsequent addition of horseradish peroxidase (HRPO)- conjugated HBsAg completes the sandwich. The addition of signal reagent then produces a luminescent signal which is proportional to the concentration of the bound HRPO. The assay is calibrated against the World Health Organization (WHO) 1st International Reference Preparation (IRP) (1977) for anti-HBs (International Laboratory for Biological Standards, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) and has a working range of 0 1000 mIU ml- 1.

The manufacturer's control panel of six standards from a single batch at concentrations of 0, 8.4, 44.2, 211,415 and 1100 mIU ml- 1 was run in duplicate on each plate. The graph obtained from these standards was then used to calculate the concentration of anti-HBs in each of the samples. All assays were carried out over a 2-day period on a single ECL processor/reader.

Abbott Laboratories lMx. This is based on the sandwich principle described above. A rHBsAg is coated on to the surface of 2 20 #m microparticles which will capture anti-HBs in the sample, and an alkaline phosphatase-conjugated rHBsAg is added to form the sandwich. The reaction mixture is then transferred to a glass fibre matrix, the complex attaches to the glass fibres and the excess reagents and other unbound material are washed away from the detection area. A fluorescent substrate is then added; the rate of fluorescence produced is proportional to the concentration of anti-HBs in the sample. This assay is also calibrated against WHO 1st IRP (1977) and has a working range of 0 1000 mIU ml 1

The manufacturer's control panel of six standards at concentrations of 0, 10, 50, 100,500 and 1000 mIU ml l was run in duplicate in a calibration protocol before testing the patients' serum. A calibrator containing 100 mIU ml- 1, a positive control containing 80 mIU m l l of anti-H Bs and a negative control were included in each run. The readings obtained from the calibrator were compared with the stored standard curve and a computed value for each of the serum samples was calculated. All the assays were carried out over 3 days with a single batch of reagents and on a single IMx processor.

Sorin quantitative EIA (Sorin Biomedica, Vercelli, Italy). This is a conventional sandwich EIA. Heat- inactivated human plasma-derived HBsAg is coated on to the wells of standard microtitre plates, captured anti-HBs is then detected by HRPO-conjugated

plasma-derived HBsAg. A chromogenic substrate is then added and the concentration of anti-HBs in the sample is proportional to the intensity of the colour produced. This assay is also calibrated against WHO 1st IRP ( 1977 ) and has a working range of 0 - 1 5 0 m l U m1-1 on undiluted serum samples.

The manufacturer's control panel of six standards from a single batch at concentrations of 5, 10, 20, 40, 80 and 160 mIU ml- 1 was run in duplicate in each plate. A graph was generated and the results of the test serum were read directly from it. A Dynatech MR7000 programmable reader was used for all the assays, which were run over 2 days.

Patients

In all, 310 serum samples comprising 116 from naturally infected patients (group A: 80 men, 36 women, age range 18 82 years), 93 from patients vaccinated, for occupational reasons, with a human plasma-derived vaccine (H-B-Vax, Merck, Sharp and Dohme) (group B: 23 men, 66 women, four unknown gender, age range 19 56 years) and 101 from patients vaccinated, for occupational reasons, with a recombinant vaccine (Engerix-B, SmithKline-Beecham) (group C; 43 men, 57 women, one unknown gender, age range 15 61 years) were investigated. All samples were assayed both undiluted and after a 1 : 11 dilution in both the IMx and ECL assays according to the manufacturers' instructions. Where sample volume permitted, discordant samples were tested in the Sorin assay. Serum samples had been stored at -20~'C for up to 4 years.

Statistical analysis

Demming's linear regression was performed to compare the values obtained by the two methods. Values reading offthe scale, > 1000 mIU ml- ~, in either method were not included in the regression calculations. The correlation between undiluted and dilution-corrected values was calculated for both methods for each patient category.

R E S U L T S

The statistical analysis of the correlation between the ECL assay and the IMx assay in undiluted and diluted samples from each patient group giving readings of 0 999 mlU ml i was performed using Demming's linear regression. The values are given in Table I.

These regression equations were then used to obtain comparative IMx values for neat samples for each patient category when ECL values of 50, 100, 250 and 500 mIU ml ~ were used, as shown in Table 2.

The correlation between neat and diluted samples for both methods are shown in Table 3 ( < 100 mlU ml 1 }

Table 1 Statistical analysis by patient group of the correlation between IMx and ECL assays

Group A Group B Group C

Variable Neat 1/11 Neat 1/11 Neat 1 ,'11

r value 0.806 0.863 0.671 0.769 0.737 0.845 a a 1.23 1.27 1.46 1.35 2.63 2.31 b a 52.4 55.9 90.3 62.4 42.3 39.6

aValues from regression equations IMx - a(ECL) 4- b

942 Vaccine, Vol. 11, Issue 9, 1993

Table 2 Comparative values of IMx and ECL assays by patient group

IMx (mlU m l - ' )

ECL (mlU ml -~) Group A Group B GroupC

50 114 163 174 100 175 236 305 250 360 455 700 500 667 820 1357

Table 3 Correlations calculated from examining undiluted and diluted samples in the range 0 - < 100 mlU m1-1

Group Assay Regression equation r n

A ECL A IMx

B ECL B IMx

C ECL C IMx

Diluted = 0.965 Neat +0.4 0.936 74 Diluted = 1.21 Neat + 1.3 0.962 58

Diluted = 1.93 Neat --3.9 0.819 21 Diluted = 2.25 Neat --1.9 0.679 18

Diluted = 1.43 Neat --4.6 0.888 45 Diluted = 1.08 Neat +12.6 0.795 33

Table 4 Correlations calculated from examining undiluted and diluted samples in the range 0 - < 1000 mlU m l - '

Group Assay Regression equation r n

A ECL A IMx

B ECL B IMx

C ECL C IMx

Diluted = 1.06 Neat +11.2 0.966 116 Diluted = 1.01 Neat +11.3 0.993 116

Diluted = 0.936 Neat +97 0.943 93 Diluted = 0.978 Neat + 12.4 0.985 93

Diluted = 1.04 Neat +66 0.930 101 Diluted = 0.974 Neat + 16 0.993 101

Table 5 Selected examples of individual sera showing discrepant results by ECL and IMx

ECL(mlU ml ') I M x ( m l U ml 1)

< 1.0 15.6 4.3 228.8 6.4 20.9

21.1 121.7 33 86.4 40 90.9 54 109 67 228 88 > 1000

150 > 1000 485 > 1000 234 > 1000

and Table 4 (0 1000 mlU ml- 1). Although both assays show good dilution characteristics over the whole range, the IMx assay shows higher recovery rates at levels < 100 mIU m1-1, while the ECL assay shows higher recovery rates at levels > 100 mIU ml-

Table 5 shows three examples of the major discrepancies between the ECL and IMx assays in each of the ranges <10, l0 50, 51 100 and > 1 0 0 m I U m 1 - 1 . The comparative data distributions for ECL and IMx showing the frequency of samples falling into these ranges are given in Table 6. Samples giving discordant results between ECL and IMx were tested in a conventional quantitative EIA (Sorin); the results of these tests by range and patient group are shown in Table 7. Table 8

Anti-HBs testing: R.A. McCartney et al.

shows the IMx and Sorin results of the 13 patients who were shown to have < 10 mIU ml - 1 ofanti-HBs by ECL.

The results of testing the ECL standards in the IMx system are given in Figure 1. These show almost exactly the same values across the whole range. We cannot explain the discrepancies between patient sera in terms of the standard curve.

D I S C U S S I O N

One objective of post hepatitis B vaccination testing is to identify poor or non-responders so that appropriate action may be recommended. More recently, the level of anti-HBsAg has been used by some workers as a predictor of the duration of protective levels, which is generally agreed to be 10 mlU m1-1 (Ref. 1 ). The Public Health Laboratory Service (PHLS) currently accepts a minimum protective response of 100 mlU ml- 1 following primary

Table 6 Data distribution demonstrating the frequency of samples in different ranges (mlU ml 1)

IMx (mlU ml -~) ECL (mlU ml ') <10 10 50 51-100 >100 Total no.

< 10 51 26 0 1 78 10-50 4 11 14 13 42 51-100 0 0 1 19 20

> 100 0 1 1 168 170 Total no. 55 38 16 201 310

Table 7 Discordant samples by range and group tested in the Sorin assay

ECL IMx Sorin

mlU ml -~ A B C A B C A B C

<10 4 4 5 0 0 0 6 4 6 10-50 7 2 14 3 5 5 6 3 9 51 100 6 1 2 5 1 7 4 0 6

> 100 0 1 0 9 2 9 1 1 0 Total 17 8 21 17 8 21 17 8 21

Values are number of specimens in each patient group, for example, in Group A a total of 17 samples gave discordant results between IMx and ECL, six of these were negative by Sorin, four by ECL and none by IMx. In Group C, of 21 samples, none gave levels of > 100 mlU ml - ' by ECL or Sorin but nine did so in the IMx assay

Table 8 Comparison of all patients who gave values of < 10 mlU ml ' by ECL and > 10 mlU m1-1 by IMx which were tested using Sorin

Patient Patient ECL IMx Sorin group no. (mlU m l - ' ) (mlU m1-1) (mlU m l - ' )

A 1 8 14 <1 2 1 16 2 3 3 13 8 4 5 21 5

B 5 4 224 3 6 3 13 <1 7 0 11 <1 8 4 28 1

C 9 6 36 6 10 6 33 8 11 9 40 11 12 7 30 10 13 6 21 3

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Anti-HBs testing: R.A. McCartney et al.

x

1000 -

9O0

8O0

700

60O

500

400

3O0

200

100

0

-100

-100

I I I I I I I l I ] I

0 100 200 300 400 500 600 700 800 900 1000

Amerl i te values

Figure 1 Amerlite ECL standards tested in the Abbott IMx system. Function relation y - m x + c where m = 0.996117392; c = 3.6432491418; correlation coefficient=0.9997569017; standard error of s l o p e - 0.0109814705; standard error of intercept=4.7434433056; t value (slope/standard error of slope) = 90.708925448; significance of slope p - 8.8552202E-8; degrees of freedom = 4

vaccination of normal healthy subjects so that a booster need not be administered for 4 - 5 years.

These results highlight significant differences in the level of anti-HbsAg in patients' sera measured using the two technologies. The correlation coefficient obtained between the two methods for undiluted and diluted samples respectively were; r = 0.806 and 0.863 (group A), r = 0.671 and 0.769 (group B) and r = 0.737 and 0.845 (group C) (Table l). The anti-HBs levels ( m I U m 1 - 1 ) for neat samples for all three groups obtained with the IMx assay were significantly higher than the corresponding results obtained with the ECL assay as shown by the regression equations in Table 1 and the calculated equivalent value shown in Table 2. These differences may lead to conflicting reports being issued by laboratories who change their assay system or when the same serum sample is tested by different laboratories using different technologies.

The reasons for the differences in measured anti-HBs levels in the two assays are unclear. The fact that the largest difference occurs in the group vaccinated with the recombinant vaccine (Engerix-B, Tables 1 and 2) would suggest that the presentation of the detecting antigen in the assay may play a role. As the IMx assay uses a recombinant antigen and the ECL assay uses naturally occurring antigen, epitope affinity may make the IMx assay more sensitive to antibodies raised against recombinant antigen (Engerix-B) than antibodies induced by natural infection. In contrast, when rHBsAg was compared with plasma-derived antigen as detector antigen in an enhanced chemiluminescence assay good correlation between the two assays was obtained 5.

However, when rHBsAg was used as the conjugate and as the capture antigen this resulted in false positives 5. Antigen epitope saturation is also a possible explanation for the observed discrepancies, however, a similar bias was observed when samples were assayed following dilution (Table 1). Over the whole range of results (0-1000 mIU ml - 1 ) linear dilution characteristics were observed in both assays with r always >0.9 (Table 4). However, non-linear dilution characteristics were observed in the IMx assay at anti-HBs levels below 100 mIU ml 1, with increased recovery on dilution of the serum samples for all three groups (Table 3). The reasons for this are unclear and the observation does not follow theoretical assumptions. Certain samples did show significant over-recovery on dilution in both methods as a result of epitope saturation, but there was no discernible trend to this phenomenon and it is less frequent in both these assays than in conventional EIA tests (personal observation). Further evidence for this is shown in Table 7 where discordant samples were tested by a conventional EIA (Sorin). Thirteen of these samples gave readings of < 10 m I U ml 1 by ECL and Sorin but all gave readings of > 10 mlU ml ~ ~ by IMx ( Table 8). These results will have serious implications if, as has been suggested, a cut-off of 10mIU ml -~ is introduced in this country.

From the present results it is apparent that the choice of assay used to measure antibody levels will have important implications in the final recommendations made for revaccination of poor responders which would occur less frequently when the IMx assay is used. This clearly has cost implications to health authorities who would use less vaccine doses if the IMx assay was routinely used. While this is economically attractive there remains a potential risk of a person becoming reinfected when they have been reported as immune if the recommended level of postvaccine antibody is reduced to 1 0 m I U m 1 - 1 However, there is no convincing evidence to support the theory that clinical hepatitis can occur in those who genuinely respond to the vaccine. Also there are more pressing problems in testing population groups such as renal dialysis patients, haemophiliacs and diabetics who are known poor responders and whose antibody levels decline rapidly postvaccination 4, if an immune cut-off of 10 m l U ml is universally applied. From these results it is clear that the IMx assay records both low and high levels of antibody when other tests are negative (TableS). Therefore we are concerned by the implications and the problems of interpretation when applied to high-risk groups and health-care workers where important clinical decisions may be affected by these results. An alternative approach has been suggested by Prince 6'v who states that patients who have previously been shown to seroconvert postvaccination do not require revaccination as immuno- logical memory is sufficient protection against clinical disease. We would agree with this hypothesis as applied to immunologically normal individuals but would remain concerned when considering immunocompromised high- risk groups where control of infection is as important as clinically apparent disease.

R E F E R E N C E S

1 Hadler, S.C., Francis, D.P., Maynard, J.E., Thompson, S.E., Judson, F.N., Eichenberg, D.F. e t a l . Long term immunogenicity and

944 Vaccine, Vol. 11, Issue 9, 1993

efficacy of hepatitis B vaccine in homosexual men. N. Engl. J. Med. 1986, 315, 209-214 Jilg, W., Schmidt, M. and Deinhardt, F. Vaccination against hepatitis B: comparison of three different vaccination schedules. J. Infect. Dis. 1989, 160, 766-769 CDC. Hepatitis B virus: A comprehensive strategy for eliminating transmission in the United States through universal childhood vaccination. Recommendations of the Immunization Practices Advisory Committee (ACIP). Morbid. Mortal. Week. Rep. 1991, 40, 1-25

Ant i -HBs test ing: R.A. McCar tney et al.

4 Stevens, C.E., Alter, H.J., Taylor, P.E, Zang, E.A., Harley, E.J., Szmuness, W. and the Dialysis Vaccine Trial Study Group. Hepatitis B vaccine in patients receiving haemodialysis. N. Engl. J. Med. 1984, 311,496-501

5 Ireland, D. and Samuel, D. Enhanced chemiluminescence ELISA for anti-HBs measurement. J. Biolumin. Chemilumin. 1989, 4, 159-163

6 Prince, A.M, Brotman, B., Purcell, R.H. and Gerin, J.L A final report on safety and immunogenicity of a bivalent aqueous subunit HBV vaccine. J. Med. Virol. 1984, 15, 399 419

7 Prince. A.M. Revaccination against hepatitis B. Lancet 1991, ii, 61

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