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The Detection of Hepatitis B Surface Antigen by Radioimmunoassay Ian D. Gust & Noreen I. Lehmann To cite this article: Ian D. Gust & Noreen I. Lehmann (1975) The Detection of Hepatitis B Surface Antigen by Radioimmunoassay, Pathology, 7:4, 285-292 To link to this article:

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Date: 07 December 2016, At: 08:00

Pathology (1975), 7,pp. 285-92

THE DETECTION OF HEPATITIS B SURFACE ANTIGEN BY RADlOlM MUNOASSAY IAN D. GUSTand NOREEN I. LEHMANN Virus Laboratory Fairfield Hospital for Communicable Diseases, Fairjield, Victoria

Summary A comparative study w a s performed to assess the sensitivity and specificity of counterirnmunoelectrophoresis (CIEP) and radioirnmunoassay (RIA) for the detection of hepatitis B surface antigen. The 8,823 sera examined included selected reference panels and sera collected from populations w i t h low, moderate and high rates of chronic antigen carriage. Overall, hepatitis B surface antigen was detected i n 265 sera by CIEP and i n 376 by RIA. As well as detecting 46.4% additional positives. the RIA test detected all CIEP-positive sera; i.e., there were no false negative results. However, 1 5 0 sera (1.8% of the total tested) gave a positive result by RIA w h i c h was not repeatable on retesting. The explanation for this phenomenon appeared t o lie i n inadequate washing of the antibody-coated tubes.

The serological demonstration of hepatitis B antigen (HBsAg) in the blood is the most widely used method of demonstrating infection with hepatitis B virus (HBV). Many tests are available for detecting the antigen and these can be classified according to their ability to detect HBsAg at different titres in reference serum panels. The original method of detecting the antigen, double diffusion in agar gel (Blumberg et al., 1965) lacked sensitivity and was rapidly replaced by counterimmunoelectrophoresis (CIEP) (Gocke & Howe, 1970).However, the recognition that some units of blood in which antigen could not be detected by CIEP, were capable of transmitting hepatitis B, highlighted the need for more sensitive techniques. This was followed by the development of the passive haemagglutination (Vyas & Shulman, 1970), immune adherence (Mayumi et a f . , 1971), radioimmunoassay (RIA) (Ling & Overby, 1972) and radioelectrocomplexing (Simons, 1973) tests. Both the passive haemagglutination and RIA tests are produced commercially and are available locally. As most screening for HBsAg in this country has been performed by CIEP, the advent of a commercial RIA, which is claimed to be of high sensitivity, provided an opportunity for a comparative study of the two techniques.



Pathology (1975), 7, October

Tests Counterimmunoelectrophoresis CIEP was performed as previously described (Nastasi rt al., 1972). This technique is of proven sensitivity and comparable to the method used in the World Health Organization International Reference Centres in Houston and London (W.H.O. collaborative study, unpublished). Radioimmunoassay A solid phase RIA was performed using a commercially available kit (Ausria 1251,Abbott Laboratories). Test sera were added to polypropylene tubes coated with guinea-pig antibody (anti-HBsAg) to the surface antigen of the hepatitis B virus (HBV). After primary incubation to allow the formation of antigen-antibody complexes, the tubes were washed with buffer to remove unbound material. Following this, 12SI-labelledanti-HBsAg was added to each tube and incubated to allow the formation of an antibody-antigen-antibody sandwich. After further washing to remove unbound labelled antibody, the tubes were placed in an automatic gamma counter and the amount of radioactivity emitted by each tube was measured. Within limits, the greater the amount of antigen present in the test sample, the higher the final count obtained. To fit in with the work routine of the laboratory, the primary incubation was performed overnight (approximately 16 h at room temp.) and the secondary incubation for 1 h at 45°C. Previous studies had shown that this combination did not alter the sensitivity of the test. The tubes were washed by means of an electrically-operated repeating dispenser, with an attachment designed to deliver buffer two-thirds of the way down the tube and aspirate it from the bottom. The buffer is delivered via a thin pipe with multiple holes in the wall which ensures an even distribution of washing fluid around the tube. In each test, the first 7 tubes were inoculated with the negative control serum and the succeeding 3 tubes with the positive control serum supplied with the kit. The remaining tubes were inoculated with test sera: After counting, the mean value of the negative control tubes was calculated: those tubes inoculated with test sera, in which the count was equal to or greater than 2.1 times this value (the cutoff) were rewashed and recounted. This extra step ensured that tubes in which unbound labelled-antibody had been incompletely removed during the initial wash were recognized. Any serum which continued to produce a count greater than the cutoff mark was regarded as provisionally containing HBsAg and confirmation of this result was sought by other methods. Specificity tests The serum was first tested by CIEP and if positive, reported as containing hepatitis B antigen. If negative, the serum was retested by RIA and in addition a neutralization test was performed, using specific anti-HBsAg. The anti-HBsAg chosen was a high titre human serum which reacts by CIEP and is known to have antibody to both the 'd' and 'y' specificities of HBsAg. The dilution chosen for use in the test was determined by prior block titrations against a panel of antigens of both major subtypes. In the neutralization test, 100 pl of serum was added to an equal volume of antibody and incubated for 1 h at 3 7 T . The RIA test was then performed as described above except that an inoculum of 200 pl was used. Confirmation that the test serum contained hepatitis B antigen was obtained when the addition of the specific antibody reduced the count obtained below the cutoff level. Because a prozone phenomenon was encountered in some



sera containing high titres of hepatitis B antigen, it was occasionally necessary to perfom duplicate neutralization tests with the test serum diluted 1 : 10 and/or 1 :50. Sera tested Comparative panels Comparative testing was performed on a panel of sera distributed by the National Institutes of Health, and two panels of sera which had been compiled locally to assist with the evaluation of different methods of detecting HBsAg. Melbourne blood donors Sera were derived from voluntary blood donors attending the Melbourne Red Cross Blood Transfusion Service. This organization has employed routine donor screening by CIEP for more than 2 yr. Sera were also available from 2 donors in whom hepatitis B antigen had been detected by CIEP 6 mth previously but who were now negative by this test and from one donor who had been associated with 3 episodes of post-transfusion hepatitis but who had been consistently antigen-negative by CIEP. Children with Down’s syndrome Sera were obtained from children with Down’s syndrome living in a large Melbourne institution for the mentally retarded. Ante-natal patients Sera were obtained from routine ante-natal patients attending the Queen Victoria Hospital, Melbourne. Venereal diseases clinic Sera were collected from subjects attending the Government Venereal Diseases Clinic, Melbourne. Malaysian survey Sera collected from healthy volunteers living in different areas of Malaysia as part of a study of the distribution of antigenic subtypes in this region. Referredsera This batch consisted of sera submitted to our laboratory from other Melbourne hospitals for HBsAg testing. About 90% of these specimens were collected from staff and patients of renal units as part of regular screening programs.

RESULTS Comparative panels Comparative testing was performed on a panel of 61 sera (hepatitis B antigen panel number two) provided by the Division of Biologics Standards, National Institutes of Health, as part of a proficiency testing program. According to that body, 29 sera should be detectable by the combination of CIEP and complement fixation and 35 by RIA. The results obtained in this study are shown in Table 1. All 35 antigen positive sera were detected by RIA whereas only 22 were detectable by CIEP. Two locally-compiled comparative panels were tested, the first comprised 290 coded sera obtained from New Guinea natives, patients with acute viral hepatitis or other diseases, and mentally retarded children. HBsAg was detected by CIEP in ‘16 sera and by RIA in 24 (Table 1). The second local panel comprised 74 sera collected from selected blood donors attending the Port Moresby Red Cross Blood Transfusion Service and examined under code. HBsAg was detected by CIEP in 8 sera and by RIA in 11 (Table 1). Thus, in the 425 sera examined, HBsAg was detected in 46 (10.8%) by CIEP and in 70 (16.4%) by RIA. The RIA test was obviously superior, particularly in detecting low titre positives in the NIH test panel and in sera collected from patients with acute viral hepatitis.


Pathology (1975), 7, October


T A I I I .1~ Detection of HB,Ag by CIEP and RIA

’ Source of sera

HB,Ag detected CIEP


Grouv 1 N.i.H. panel




70 70 70 80

7 6 0 3

9 11 0 4






Local panel No. 1

(I) (2) (3) (4)

New Guinea Natives Hepatitis patients Non-hepatitis patients Mental patients

Local panel No. 2 Total ~~

70 ~

Group 2 839 3

0 0

0 3

Down’s syndrome Ante-natal patients V.D. clinic Malaysian survey

170 410 1000 961

63 2 17

68 6



Blood donors Special donors Group 3


22 60




Group 4 Other hospitals (8-mth period)




Melbourne blood donors A total of 839 sera were tested by RIA and no additional neutralizable positives detected. The sera of 2 CIEP-negative blood donors who had been CIEP-positive 3 mth earlier were found to be positive by RIA. In addition, serum from a donor who had been implicated in 3 episodes of post-transfusion hepatitis, but was CIEPnegative on all occasions, was found to be RIA-positive (Table 1). Children with Down’s syndrome Single sera were examined from 170 institutionalized children with Down’s syndrome; HBsAg was detected in 63 sera (37%) by CIEP and in 68 (40%)by RIA (Table 1).

Ante-natal patients A group of 410 sera collected from consecutive ante-natal patients was tested. Hepatitis B antigen was detected by CIEP in 2 patients (0.49%) and by RIA in 6 (1.46%). (Table I). Five of the 6 women in whom antigen was detected were Southern Europeans. Venereal diseases clinic Of the 1,000 sera obtained from consecutive subjects attending this clinic, 17 were demonstrated to contain HBsAg by CIEP (1.7%) and 22 (2.2%) by RIA (Table 1).



Malaysian survey Sera from 967 Malaysias subjects were studied and of these, 39 (4%) were positive by CIEP and 60 (6.2%) by RIA (Table 1). Referred sera Of 5,009 sera referred to this laboratory in the 8-mth period November 1973 to June 1974, HBsAg was detected in 98 (1.9%) sera from 79 patients by CIEP and in 147 (2.9%) sera from 114 patients by RIA (Table 1). There were 35 patients in whom HBsAg was detected by RIA alone. Of these, 10 were in the incubation period or convalescent phase of hepatitis B, 8 had other forms of liver disease, 8 were blood donors, 6 were patients (2) or staff (4) of dialysis units and one had acute myeloid leukemia. No clinical information was available on the remaining 2 patients. Titrations A group of 20 sera known to be hepatitis B antigen-positive by CIEP were titrated in paralled by this method and RIA. Ten of these sera were obtained from patients in the acute phase of hepatitis B and 10 from chronic antigen carriers detected at the Red Cross Blood Transfusion Service. The results are shown in Table 2. The geometrical mean titre by CIEP was 7.5 and by RIA 2,610, representing a 348-fold increase in sensitivity by RIA. Non-repeatable results During the period of this study, 8,823 sera were screened for HBsAg and 265 (3%) were positive by CIEP and 538 (6.1%) by RIA. However, in the latter group, only 376 were confirmed by neutralization with specific antiserum, the remaining 162 being negative on repeat testing. Thus, non-repeatable results were obtained in 1.8% of all sera tested and 29.9% sera found to be positive on screening (Table 3). No serum was encountered which was RIA-positive on 2 occasions but could not be neutralized with specific anti-HBsAg ; i.e., no false positives were detected. However, 4 sera were encountered which because of a prozone phenomenon could only be neutralized after dilution. No serum was found to be CIEP-positive but RIA-negative; i.e., no false negative results were encountered.

DISCUSSION In this study, the sensitivity of 2 serological tests for the detection of HBsAg has been compared using sera obtained from a variety of populations.

Sensitivity Comparative titrations of known HBsAg-containing sera have shown that the RIA test is approximately 350 times as sensitive as CIEP, results which are in accord with the findings of other authors (Hansson & Johnsson, 1974; Miller, 1974). The value of this increased sensitivity was demonstrated in the studies described. In addition to detecting HBsAg in higher dilutions, RIA detected antigen in all sera which were CIEP-positive and in an additional 46.4% of sera which were CIEP-negative. The increased detection rate varied according to the particular group of sera examined but was highest in those batches which included old, stored sera, or sera which had suffered bacterial contamination. SpeciJicity Several groups of workers (Sgouris, 1973; Prince et al., 1973; Hansson & Johnsson, 1974) have reported a high proportion of false (i.e., non-neutralizable) positive results with the RIA test. These results appeared to be due to the presence, in some sera, of substances which react with the guinea-pig component of the anti-HBsAg used in the


Pathology (1975), 7, October


TABLE 2 Comparative titres of HB,Ag obtained by CIEP and RIA Titre Serum no RIA


1 :4

I :lo24


1 :8

1 :8192


1 :16



1 :8



I :8

1 :8192


1 :8

1 :8192


1 :8

1 :I024


1 :2

1 :I024


1 :8

1 :I024


1 :4



1 :4

1 :I024


1 :32



1 :8

1 :lo24


1 :16

1 :8192


1 :32

1 :8192


1 :4

1 :I024


1 :4

1 :lo24


1 :8

1 :I024


1 :8



1 :4

1 :8192

1 :2- 1.32

Range Geometrical mean titre


I :1024-1:8192


TABLE3 Overall results of RIA test on 8,823 sera No. of sera





No. oositive


No. 316

No. 376

1 1

% Non-repeatable Total 1.8

Positives 29.9



test. This problem was not encountered during the current study, probably because 20% normal guinea-pig serum was included with the labelled antibody supplied with the kit. When performing neutralization studies to assess the specificity of RIA results, it may be necessary to test the sera at more than one dilution because of the occasional occurrence of a prozone phenomenon. We encountered 4 sera which could not be neutralized by our reference anti-HBsAg serum unless they were first diluted 1 : 10 or 1 :50. Failure to recognize this phenomenon could lead to some sera being inadvertently labelled as false positives. Repeatability With any test for HB,Ag that is likely to be used for mass screening of sera, it is important that the number of non-repeatable results obtained be within acceptable limits, Of the 8,823 sera tested, 162 were found to be RIA-positive on the screening but negative when the test was repeated. This represents a non-repeatability rate of 1.8%. The proportion of non-repeatable sera encountered in any particular run varied considerably and was independent of the source of the test sera and the age or batch of test material. The major factor influencing the incidence of non-repeatable results was failure to perform the test exactly as directed, the main problems being inadequate washing of the tubes after addition of labelled antibody or excessive delays before rewashing. In the latter half of the study, when the operators had become more familiar with technique, the incidence of non-repeatable results fell to less than 1%. Detection in blood donors If the results obtained in this study are extrapolated to the blood donor population, we could predict that the application of RIA techniques would result in the detection of one additional positive donor for every 2,000 sera tested. In addition, we would expect 10 to 20 units would require retesting because they had been positive in the screening test. Although this is a small increase in yield considering the additional labour and expense of the test, the extra effort would be justified as there is now considerable evidence that RIA-positive-CIEP-negative blood is infectious. Thus, in this study, 9 patients who developed post-transfusion hepatitis B had received blood from a donor who was RIA-positive and CIEP-negative and one of these donors had been associated with three separate episodes of post-transfusion hepatitis. The RIA is a very sensitive technique for detecting HB,Ag but is expensive and if large numbers of sera are being tested, requires access to an automatic gamma counter. Whilst it is likely to remain the method of choice in research laboratories, it is probable that the recently developed haemagglutination techniques, which appear to be simple, rapid and sensitive (Reesink et al., 1973; Cayzer et al., 1974) will be more suited for mass screening procedures. ACKNOWLEDGEMENTS The authors wish to thank the following persons for providing serum specimens which were used in the study: Drs Royle Hawkes, Graeme Woodfield, John Morris, David Pitt, Greg Buist and Sumitra Kamath. Miss Maria Nastasi and Mrs Mary Dimitrakakis provided excellent technical assistance. This study was supported by a grant from the National Health and Medical Research Council. Address j o r reprint requests: I . D.G. Virus Laboratory, Fairfield Hospital, Yarra Bend Road, Fairfield, Victoria

3078, Australia



Pathology (1975), 7, October

References BLUMBERG, B. S., ALTER, H. J. & VISNICH, S. (1965): A ‘new’ antigen in leukemia sera. J . Amer. med. ASS.191, 541-546. CAYZER,I., DANE,D. S., CAMERON, C. H. & DENNING, J. V . (1974): A rapid haemagglutination test for hepatitis B antigen. Lancet. 1, 947-949. GOCKE, D. J . & HOWE,C. (1970): Rapid detection of Australia antigen by counterimmunoelectrophoresis. J . Immunol. 104, 1031. HANSSON, B. G . & JOHNSSON, T. (1974): Evaluation of a solid phase radioimrnunoassay (Ausria-125) for the detection of hepatitis B antigen (Australia antigen) in two clinical materials. J . din. Puth. 21,35-39. LING,C. M. & OVERBY, L. R. (1972): Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with lZ51-antibody.J . Immunol. 109, 834. MAYUMI, M., OKOCHI, K. & NISHIOKA, K. (1971): Detection of Australia antigen by means of immune adherence haemagglulination test. Vox Sung. 20, 178-181. MILLER,J . P. (1974): Third generation detection systems for hepatitis B antigen. Hepatitis Symposium, Melbourne (in press).

NASTASI, MARIAC., PRINGLE, R. C. & GUST, I. D. (1972): The detection of Australia antigen and antibody by cross-over imrnunoelectrophoresis. Ausr. J . med. Technol. 3, I 1 I-- 115. PKINCE, A. M., BKOTMAN, B., JASS, D. & IKKAM, H. (1973): Specificity of the direct solid phase radioimmunoassay for detection of hepatitis B antigen. Lancet. 1, 1346 1350. REESINK, H. W., DUIMEL, W . J. & BRUMMELHUIS, H. G . J . (1973): Evaluation of a new haemagglutination technique for the demonstration of hepatitis B antigen. Luncer. 2, 1351-1353. SGOURIS, J. T. (1973): Limitations of the radioimmunoassay for hepatitis B antigen. New Eng. J . Med. 288, 160-161. SIMONS,M. J . (1973): Detection of hepatitis B antigen by radioelectrocornplexing. Bull. Wld Hlth Org. 49, 107-109 VYAS,G . N . & SHULMAN, N . R. (1970): Haemagglutination assay for antigen and antibody associated with viral hepatitis. Science. 170, 332-333.

The detection of hepatitis B surface antigen by radioimmunoassay.

A comparative study was performed to assess the sensitivity and specificity of counterimmunoelectrophoresis (CIEP) and radioimmunoassay (RIA) for the ...
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