Enzyme-linked immunoassay for hepatitis B surface antigen An evaluation of commercial test kits

N. NATH, R. Y. DODD, AND C. T. FANG

A commercial, licensed test for hepatitis B surface antigen (HBsAg) based upon an enzyme-linked immunoassay principle was evaluated. The sensitivity, when tested under field conditions, was 0.65 and 0.9 n g h l for ad and ay subtypes of HBsAg, respectively. Only 0.03 percent of 61,798 units of blood screened were reactive and specific for HBsAg while an additional 0.01% gave nonspecific (nonneutralizable) reactivity. TRANSFUSION 1983;23:45-48.

A1.i. BLOOD donated to the American Red Cross (ARC) since 1973 has been screened for hepatitis B surface antigen (HBsAg) using tests based on solid phase radioimmunoassay (RIA) or other third genera- tion tests. High levels of sensitivity, reproducibility, and the lack of subjectivity of RIA tests have been responsible for a very successful experience with this technology by the various blood collection agencies and its use has resulted in the reduction of post- transfusion hepatitis (PTH) caused by hepatitis B viruses (HBV) in blood.' Recently, commercial kits, based on an enzyme-linked immunoassay (ELISA)

have become available in the United States. In this technique, anti-HBs immunoglobulin (IgG) linked with a n enzyme instead of a radioisotope is used as the probe t o indicate the presence of HBsAg. Elimination of the radioisotope and significant im- provement in the shelf-life of reagents are two attractive advantages of the ELISA test over RIA. After a n exhaustive laboratory evaluation established that the performance characteristics (sensitivity and specificity) of commercial test kits (Auszyme, Abbott Laboratories, North Chicago, IL) were more ap- propriate to the needs of ARC compared to other commercially available licensed ELISA kits in the U.S., more extensive field trials were undertaken. In order t o determine whether the advantages of this test were accompanied by satisfactory overall performance, a field evaluation of a modified commercial kit (Auszyme 11, Abbott Laboratories) was carried out to determine its sensitivity, reproducibility, and speci- ficity. The results of this evaluation are presented.

From the American Red Cross Blood Services Laboratories, Transmissible Diseases and Immunology Laboratory, Bethesda. Maryland.

Contribution Number 529 from the American Red Cross. Received for publication November 25, 1981; revision received

February I . 1982, and accepted February 10, 1982.

Materials and Methods ARC Blood Services regions located at Johnstown, PA;

Columbus, OH; Birmingham, AL; and Portland, OR. participated in this field trial for different lengths of time during the overall period of September 1980 to March 198 I . A total of 61,798 units of blood donated to the regions (representing only a fraction of total donated) of Johnstown, Birmingham, and Portland were tested for HBsAg using Auszyme I I test kits. Most of these blood samples were not tested by other tests for H BsAg. The Columbus, OH, region only participated in the determination of sensitivity of Auszyme 11. Samples were initially tested using serum from the tube containing blood clots; however, when a sample had to be retested, either serum or plasma citrate-phosphate- dextrose (CPD) was tested in duplicate. Aliquots of all units that were found reactive on screening and, in at least one of two subsequent retests, were submitted to the Transmissible Diseases and lmmunology(TD1) Laboratory (American Red Cross Blood Services Laboratories, Bethesda, Md.) for confirmation of reactivity and specificity of HBsAg. Regions were, however, encouraged to submit any sample they thought needed further testing.

An HBsAg dilution panel consisting of four serial two- fold dilutions each of human serum pools containing HBsAglad and HBsAg/ay was used as the reference panel throughout the study. The dilutions were made in recalcified human plasma nonreactive for HBsAg (Ausria 11, Abbott Laboratories), anti-H Bs (Ausab, Abbott Laboratories) and anti-HBc (Corab, Abbott Laboratories). A number of sets were prepared and stored at -3OOC. Panel samples were recoded each month. Each participating center was sent a panel under code monthly for inclusion in weekly test runs. The concentration of HBsAg in each dilution in the panel was determined by reference to a commercial hepatitis sensitivity panel (#4. Abbott Laboratories) which consisted of 24 samples with known amounts of HBsAglad and ay. The four dilutions with HBsAg/ad in the panel were determined to contain HBsAg equivalent to 2.25, I . I2,0.55 and 0.25 ng per ml. Samples with HBsAg/ ay had 6.0,3.2, 1.6 and 0.8 ng per ml of the antigen.

Procedures A or B as described in the product insert of Auszyme I1 were followed throughout. In brief, Auszyme I I employs the principle of solid-phase double-antibody sandwich technique^,^'^ but uses goat anti-HBs linked with horseradish peroxidase (HRPO) as the probe.

All samples submitted to the TDI Laboratory for HBsAg confirmation/ specificity assay were routinely tested by Ausria I I and Auszyme I I tests. Since it was not always

0041-1 132/83/0100/0045 SO1.00 @ J. B. Lippincott Co.

45

46 NATH ET AL. TRANSFUSION Vol. 23. No. I - 1983

Table 1. Summary of Data from Centers

Reactive

On Screening On Retesting' No.

Region Tested No. (O/o)t No. (Yo)?

Birrningham,AL 9,424 105 (1.11) 114 (0.12) Johnstown, PA 29,193 202 (0.69) 17 (0.06) Portland, OR 23,117 105 (0.45) 11 (0.05) TOTAL 61,804 412 (0.67) 39 (0.06)

t Percent of total tested. 4 Includes 6 donors who were found reactive on previous

Reactive at least once on retest.

donation.

immediately known if the sample submitted was plasma, recalcified plasma or serum, one to two drops of a solution (1000 NIH units per ml) of topical bovine thrombin (Parke, Davis & Co.. Detroit, MI ) were added to each sample. Fibrin clots were removed with a wooden applicator stick.

All samples that were nonreactive in both Ausria 11 and Auszyme 11 were tentatively called nonreactive for HBsAg. If the test values obtained in the region were above the cutoff in all three tests (screen and retest in duplicate), such samples were further tested for the presence of anti-HBc (Corab. Abbott Laboratories) and/or tests in the TDI Lab were repeated. A sample was finally labeled nonreactive only when all tests showed the clear absence of HBsAg and/ or anti-HBc. I f a sample was reactive either in Ausria 11 or Auszyme I1 but not in both, it was tested by the neutralization in the test that gave a positive result. A sample was called reactive for HBsAg when both Ausria I1 and Auszyme I 1 were positive or if reactivity was specifically neutralized by anti-HBs in one or both tests.

Results As shown in Table I , a total of 61,804 units of blood were

tested using Auszyme I 1 test kits in the three regions during this study. Of the numbers tested, there were I . I I , 0.69 and 0.45 percent reactive on screening a t Birmingham, Johns- town, and Portland, respectively. However, on retesting by regions.'only about 10 percent of samples remained reactive.

A total of 33 samples were submitted to the TDI Laboratory by the three regions for HBsAg specificity/con- firmation testing (Table 2) during the period of this evaluation. An additional six samples from Birmingham were from the donors who where already confirmed reactive for HBsAg on a previous donation; these samples were not resubmitted for confirmation testing. These reactive donors represent a small percentage of donors who insist on donating blood in spite of being informed of nonsuitability of their blood for transfusion purposes.

The prevalence of HBsAg reactives in the total donor population of these three regions was 0.06 percent. However, after excluding the number of donors that had been found reactive on previous donations, the prevalence in Birming- ham dropped to 0.05 percent which was not significantly different from Portland (0.05%) and Johnstown (0.06%).

Twenty (60.6%) were confirmed reactive for HBsAg and 6 ( 18.2%) reacted as nonspecific; these could not be neutralized by treatment with specific human anti-HBs sera. Seven

Table 2. Results of HBsAg ConfirmationlSpecificity Testing by TO/ Laboratory on Samples Found Repeatably

Reactive by the Regions

HBsAg Reactivity

No. Non- Non- Region Tested reactive Specific specific

Birmingham, AL 5' 2 2 1 Johnstown, PA 17 5 7 5 Portland, OR 11 0 11 0 TOTAL 33 7 20 6

-

Does not includesamples that were from donors who had been found reactive for HBsAg on previous donations.

(21.2%) of the 33 samples were clearly nonreactive for HBsAg when tested by Auszyme I1 and Ausria I1 test kits a t the TDI Laboratory. The corrected prevalence of HBsAg was 20 in 61,798 (0.03%) units of blood collected at these ARC regions.

The overall sensitivity of Auszyme I1 was estimated by plotting the mean of net absorption (AA) on each of the eight samples in the HBsAg dilution panel (Fig. I ) . It is estimated that the sensitivity of Auszyme 11 as defined by the intercept with the cutoff value of 0.05 was 0.65 ng per ml for HBsAg/ad and 0.9 ng per ml for HBsAg/ay. One of the regions found the sensitivity of the test consistently a t I .O and 1.5 ng per ml for ad and ay respectively. All other regions found the sensitivity below the I ng per ml level for both H BsAg/ ad and ay.

A hypothetical sample containing I ng per ml of ad or ay had 82 and 53 percent chances, respectively, of being found reactive when tested by Auszyme 11 (Fig. 2). For this estimate, a sample was considered reactive only when its net AA value was greater than 0.05. as recommended by the manufacturer. It is estimated that a sample must have a t least I .5 ng per ml of ad or 2.4 ng per ml of ay to be found reactive in 95 percent of tests.

Discussion An extensive laboratory and field evaluation of

Auszyme 11 was conducted in four selected American Red Cross Blood Services regions. The estimated sensitivity of Auszyme I1 under field conditions was below 1 ng per ml of HBsAg. The sensitivity of Auszyme 11 was slightly greater for HBsAglad than for HBsAglay.

A total of 0.04 percent of all units o f blood donated to the participating regions were repeatedly reactive in Auszyme 11. This included 0.01 percent of all units tested which were found to be nonneutralizable (non- specifics). Among the units found repeatedly reactive with Auszyme II ,6 of 33 ( 1 8%) were nonspecific. The true prevalence of HBsAg was, therefore, 0.03 percent.

The long shelf-life and nonisotopic nature of the reagents used in Auszyme I1 testing are its main advantages over radioimmunoassays. RIA is cur- rently considered the most sensitive test for the detection of HBsAg in b l ~ o d . ~ - ~ However, the test

47 TRANSFUSION ELISA FOR HBsAg I983 - Vol. 23. No. 1

.30

.26

.20

.I6

.10

.05

1 1 I I I 1 I I I 1

a 5 4 3 2 1 0

mrAg nolml

FIG. I. Overall sensitivity of Ausiyme I I as estimated by plotting mean change in adsorbance ( A A ) (based on a total of 59 reports on HBsAg dilution panel from four Red Cross regions) and the estimated amount of HBsAg (ng per m l ) in each sample in the panel. Samples giving LA 20.05 are considered reactive. A . - - - A HBsAg ay:. ... 0 HBrAgiad.

based on ELISA may achieve a comparable level of sensitivity.Y-” Because Austyme I1 is read spectro- photometrically, it is free of the elements of subjectivity which are inherent in tests such as reversed passive hemagglutination and counter immunoelectrophoresis. Our data show that under field conditions, only about 10 percent of samples remain reactive on retesting. This is in agreement with the rate reported by Reesink, et al.’ using an earlier version of the Auszyme 11 test. Most false reactives are clearly due to technical errors and this is supported by the observation that the rate of false positives decreased with the amount of experience gained. However, it has been suggested that fresh sera give more false positive reactions than do sera that have been aged for 1 to 2 days a t 40C.13

Since the completion of this study, we have found that Auszyme I 1 was susceptible to false positive reactivity if blood was collected in the evacuated tube of a particular manufacturer. Apparently an unidenti- fied agent leaches out of the tube or stopper into the serum resulting in positive Auszyme I1 reaction. Tubes by two different manufacturers were in use a t the participating regions during this study and may account for some of the false reactives encountered. In

I I I I I I ! I

O y a u U I 100 -

-- 90-

80 -

c. 7 0 -

co b 0 -

- d

d 5 0 -

z 40 -

nsrAa ngm

Flci. 2. Percent of samples containing different levels of HBsAg found reactive by Ausiyme 11. The figuresshow the percentage of tests that were reactive in relation to the levels of HBsAg. The dotted line indicates the amount of HBsAg necessary in a sample to give positive reaction in 95 out of 100 tests. A--A HBsAglay: 0 --0 HBsAgiad.

addition, since the samples submitted to TDI Labora- tory for confirmation/ specificity testing were either serum or plasma, it is possible for an aliquot of serum to be found repeatedly reactive in the region but at the same time aliquots of plasma submitted to TDI Laboratory to be found clearly nonreactive. The difference may be due to the artifact introduced in the serum from the blood collection tubes. This may explain why only seven out of 17 samples from Johnstown and all I I from Portland were specific.

We have also found that the most common cause of the nonspecific reactivity in Auszyme I1 is most probably antibodies to goat serum in the blood of some donors. These antibodies were more prevalent in certain parts of the United States, particularly in the South. We understand that Abbott Laboratories modified the composition of the reagents in Auszyme I 1 since the completion of this study, and it has resulted in the reduction of the occurrence of nonspecific reactivity.

The prevalence of HBsAg detected using Auszyme 11 in Birmingham, Johnstown, and Portland was 0.06 percent before elimination of false positives and nonspecifics. The prevalence of HBsAg tested by

48 NATH E l

Ausria 11 in these regions during the same period in 1980 was 0.075 percent ( 1 19 reactives in 158,668 units of blood tested) which is not significantly different (p = 0.35). Nationwide H BsAg prevalence has been reported to be 0.07 percent.14 The true prevalence of HBsAg carriers among the units of blood was 0.03 percent after excluding false reactives, nonspecifics, and the donors who had been identified to be HBsAg carriers on the basis of previous blood donations. The levels of sensitivities of Auszyme I 1 and Ausria I 1 are close and we estimate that the vast majority of HBsAg carriers have comparatively high levels of HBsAg in their blood”; less than one in 50,000 donors may be expected to carry HBsAg at a level less than I ng per ml. It is, therefore, unlikely that low prevalence of HBsAg (0.03%) found in this study compared to nationwide prevalence of 0.07 percent is due to differences in sensitivity of test methods. We are, however, unable to explain the difference.

In our experience, Auszyme I I is more demanding, in terms of attention to detail and precision, than most RIA tests. This might be one of the reasons why regions had high false positive rates on screening tests. Washing of beads must be thorough yet not excessive. Beads must not be allowed to overdry as this may result in inactivation of horseradish peroxidase causing lower net absorbance values. A parallel situation in RIA would be the removal of radioisotope 1 ) by excessive washing, resulting in lower counts. The Auszyme I1 technique is vulnerable on this account and, theoretically, it is possible for excessive drying to cause a weakly reactive sample to give a change in adsorbance value below the cutoff.

Auszyme 11 requires an extra 30 minutes of incubation time to complete the test; however, unlike radioimmunoassays, samples in this test do not have to be counted for I minute, thus allowinga considerable time saving. Using a special filter photometer (Quantum,@ Abbott Laboratories), Auszyme I 1 could provide the test results earlier than most RIA tests. The solution of enzyme substrate, o-phenylenedi- amine, is instable. Therefore, once prepared, it must be used within 1 hour. Due to the instability of color developed as a result of an enzyme reaction, the test must be read within 2 hours. The test must be repeated if the time of reading exceeds 2 hours.

On balance, we found Auszyme I1 to be a sensitive, specific, and reliable test that is suitable for routine screening of blood for HBsAg. Tests based on ELISA technology have the potential for improvement in sensitivity since even minute amounts of active enzymes may amplify the production of color used to define the positivity of a sample; such amplification is not possible with assays based on radioisotopes.

TRANSFUSION Vol. 23. No. I - 1983

AL.

Acknowledgments We thank the Medical and Technical Directors of the American

Red Cross Blood Services Regions of Birmingham. AL; Columbus. OH; Johnstown. PA; and Portland. OR. for their participation in this study. We also express our appreciation to the technical staff of these regions for their assistance.

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References Goldfield M. Bill J. Colosimo F. The control of transfusion associated hepatitis. In: Vyas GN, Cohen SN. Schmid R. eds. Viral hepatitis. Philadelphia: The Franklin Institute Press, 1978:405. Engvall E. Perlmann P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G . Imrnuno- chem 1971;8:871-4. Wei R, Knight GJ. Zimmerman DH. Bond HE. Solid-phase enzyme immunoassay for hepatitis B surface antigen. Clin Chem 1977;23:813-5. Wolters G . Kuijpers L. Kacaki J. Schuurs A. Solid-phase enzyme-immunoassay for detection of hepatitis B surface antigen. J Clin Pathol 1976;29:873-9. Dimitrakis M. Gust ID. Comparison of three solid phase radioimmunoassay techniques for the detection of hepatitis B surface antigen. Aust J Med Lab Sci 1980;1:59-63. Nath N , Dodd R Y . Ledman R, Fang CT. Barker LF. Comparative evaluation of radioimmunoassay kits used for testing blood for hepatitis B surface antigen. Am J Clin Pathol 198 1:75:2 14-8. Reesink H W. Lafeber-Schut LJ, Aaij C. Reerink-Brongers EE. Comparison of six “third-generation”tests for the detection of HBsAg. Vox Sang 1980;39:61-72. Vincent SR and Whitson KJ. Anevaluation of three commercial R I A systems for the detection of hepatitis B surface antigen. Aus J Med Lab Sci 1980;1:13-22. Adachi H. Fukada I-, Funahashi S. Kurahori 7’. lshikawd E. Sandwich enzymo immunoassay of hepatitis B surface antigen (HBsAg). Vox Sang 1978;35:219-23. Caldwell CW, Barrett JT. Enzyme irnmunoassay for hepatitis B and its comparison to other methods. Clin Chirn Acta

Hyland CA. Mason EC, Harden PA. Shaw AE. Mayrer D, Hope SL.. Sensitivities of radioimmunoassay and enzyme- linked immunosorbent assay for detection of hepatitis B surface antigen. Vox Sang 1979;36:137-41. Wolters G, Kuijpers L, Kacaki J. Schuurs A. Enzyme- linked immunosorbent assay for hepatitis B surface antigen. J Inf Dis 1977;136:S311-7. Kacaki J. Wolters G, Kuijpers L. Stulemeyer S. Results of a multicentre clinical trial of the solid-phase enzyme immunoassay for hepatitis B surface antigen. Vox Sang 1978;35:65-74. Bastiaans MJS, Dodd RY, Nath N. Pineda-Tamondong G, Sandler SG, Barker LF. Hepatitis-associated markers in the American Red Cross volunteer blood donor population. I. Trends in HBsAg detection, 1975-78. Vox Sang 1980;39:1-8. Nath N. Fang CT, Berberian H, et al. Hepatitis-associated markers in the American Red Cross volunteer blood donor population. 11. Distribution of level of HBsAg reactivity by radioimmunoassay and occurrence of nonspecific reactivity. Vox Sang 1980;39:73-8.

1977;81:305-9.

Nrapendra Nath. Ph.D., Head, Hepatitis Section American Red Cross Blood Services Laboratories.93 12 Old Georgetown Rd., Bethesda, MD 20314. (Reprint requests)

Roger Y, Dodd, Ph.D.. Head, Transmissible Diseases and Immunology Laboratory, American Red Cross Blood Services Laboratories.

Chyang T. Fang, M.S., Hepatitis Section, Transmissible Diseases and Immunology Laboratory, American Red Cross Blood Services.