Biological standards for hepatitis B virus assays

Journal of Hepatology, 1992; 25: 251-255 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 016%8278/92/$05.00

251

HEPAT 01144

6. Gerkena, W.

Gerlichb, C. B&hot”, .C. Thomasd, F. Boninoe, Carneiro de Mouraf and M.-H. Meyer zum

“1.Medizinische Klinik und Poliklinik, Johannes Gutenberg University,Mainz, Germany; binsritutfiir Virologie. University of Giessen, Giessen, Germany; ‘I.k5El? V U-75. CHU Necker and Insi?turPasteur, Paris, France; dSr. Maips Hospital Medical School, London, United Kingdom; ‘Ospedale Molinerte, Turin, hazy; and rMedicai School of Lisbon, Lisbon, Portugal

During the past few years, most serological assays for the diagnosis of HBV have been based on solid-phase radio- or enzyme-immunoassays. These tests are now commercially available and give a satisfactory level of nearly 100% correct results for qualitative results. However, most average laboratories cannot quantitate their findings accurately. Most test kits do not provide instructions for quantitation and reference sera with defined concentrations. This is unfortunate due to the medical problems of viral hepatitis, e.g. monitoring of therapeutic trials, where only quantitative or at least serniquantitative results provide the necessary information for making decisions. From a clinical point of view, therefore, the purpose of this paper is to focus on some essential points for the biological standardization of hepatitis I3 virus serology.

es for negative and positive control samples

In general, biological standardization of HBV serology depends on the quality of the reagent, the equipment of the laboratory and on the performance of rhe assays (1). Controls for qualitative tests

Good laboratory practice should include at least 3 negatives and 3 positives in every test run on an immunoassay. The positive control should have a concentration which is close to the assumed limit of detection. If, for instance, the detection limit of a HBsAg test kit is guaranteed by the producer to be 0.5 nglml, the positive control should not contain more than 1 @ml. Besides

clear definition of a cut-off value between positive and negative results, definition of a grey zone is advisable within results need to be repeated. Furthermore, criteria are required which help to decide on the final result after repetitive testing. These trivial minimal requirements are not always satisfied by :ome commercially produced test kits. In these cases, internal quality criteria have to be established.

Controls for quantitative tests

Proper selection of control samples for quantitation is very important. A sufficient number of negative blood samples (n = 8) from different healthy blood donors could be used as a negative control. Instead of one fixed positive control sample, we suggest using a geometric dilution series of a strongly positive sample. Dilution shonld be selected so that the highest dilution is clearly below the average detection limit, whereas the lowest dilution should produce a signal within the saturation zone. Thus, for immunoassay of HBsAg, geome;;;cal dilutions between 100 and 0.01 ng/ml in several 4-fold dilution steps can be used. This is not much more expensive or laborious than 3 replicates of one fixed positive control sample and allows accurate control of the detection limit and a semiquantitative estimate of HBsAg concentrations in weakly positive samples. A less expensive version of this approach is to use one weakly positive control serum with a concentration close to the detection limit and a strongly positive control which gives a signal at The upper range of the proportionality between signal and concentration.

Correspondence: Eurohep - Pathobiology Study Group, G. Gerken, M.D./K.-H. Meyer zum Biischenfelde, Poliklinik, Johannes Gutenberg Universitlt Mainz, Langenbeckstrasse 1, 6500 Mainz, Germany.

M.D., I. Medizinische Klinik und

G. GERKEN et al.

252

Standards for quantitation of HBV markers

log

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An internationally recognized reference sample has only been defined for anti-HBs (2). Subreference samples have been derived locally and by test kit producers. Thus, there is no reason to report ill-defined ratios in-

10

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I detection limit

I

HB5Ag

concentration of sample

Fig. 1. Calibration curve for quantitation [according to Gerlich and

Thomssen (l)].

DefGtion of cut-off value Another problem is the definition

of cut-off

values

which are best calculated from negative controls. Historically, they have been defined as 2.1 times the mean value of negative controls (NC); 2.1 is 7 times the standard deviation (SD) plus NC. It should always be verified however, that 2.1 times the negative controls is higher than NC plus 5 times the SD. Otherwise this result is better used as the cut-off itse!f. With inhibition assays, 50% of the negative control is often used as cutoff. For certain anti-HBc test kits 30% is advisable. This increases the sensitivity of these assays for detecting true low-level re’ctive samples. There is no general agreement, however, concerning the diagnostic usefulness of testing for IgM anti-HBc for differentiation of acute, chronic and past hepatitis B infection. Future developments based on IgM capture assays, recombinant antigens and new technologies, e.g. fully automated immunoassays using the IMX-instrument system, are required to establish an international standard.

Calculation of quantitative results With commercially available HBV immunoassays a completely computerized system can be used to calculate automatically the quantitative results. Otherwise, an individual control using a calibration curve which correlates signal strength with the concentration of the positive reference serum is better for establishing valid quantitation (Fig. 1). Care must be taken to ensure that deviating points at the lower and upper ranges are not forced into an arbitrary function. Moreover, signals within the saturation zone need to be identiiied as such.

activity

units. can be clearly

correlated

with a de-

fined amount of HBs protein (3). Today, most kits include control samples with the defined amount of HBsAg expressed as ng/ml. Unfortunately, the calibration is not always correct and, moreover, the HBsAg activity in a given immunoassay may depend on the HBsAg subtype. Methodologically, quantitative immunoelectrophoresis is suitable for quantitation. In practice, a plasma containing a defined amount of HBsAg can be diluted from 1% to 100% in negative plasma. From this, the calibration curve can be established and compared with test samples. In general, the correlation between HBsAg activity and the amount of HBs protein is lost after heating, repeated freezing and thawing, and extensive purification. Consequently, the National Bureau of Sera and Vaccine in the Federal Republic of Germany (Paul Ehrlich Institute) has defined a HBsAg unit (known as ‘PEI unit’) which corresponds to 1 ng of fully reactive HBs protein. Reference samples for subtypes ad and ay are available. The Paul Ehrlich Institute has developed further reference samples for anti-HAV, anti-HBc, IgM anti-HBc, HBeAg and anti-HBe which are available for producers of test kits and for reference laboratories. These reference samples are also used by the Paul Ehrlich Institute for control and licensing of serological tests for viral hepatitis which are brought on to the German market. Gross deviations in sensitivity or specificity are prevented by this licensing procedure.

High accuracy quantitation

For the highest accuracy of serological assays, the socalled paraliel line assay must be performed (Fig. 2). For example, the test sample and the reference sample ale assayed in geometrical dilutions. The signals are plotted in double logarithmic scale and regression lines are determined with the assumption that the two titration curves are parallel. The distance of the dilution scale between the two curves gives the ‘re! ative potency’ of the sample with respect to this reference. If the titration curve of reference and sample are not parallel, exact quantitation is not possible because the two preparations differ in their properties. In this case we suggest determining the potency from the highest positive dilution of the sample.

BIOLOGICAL

STANDARDS

FOR HBV ASSAYS

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Fig. 2. Parallel lin- assay for quantitation [according to Gerlich and Thomssen (l)].

Dot blot hybridizution assay Molecular hybridization techniques have been successfully established to analyze serum V-DNA and represent a direct marker of HBV replication (4,5). The best way to obtain quantitative signals from hybridization ) assays or immunoblots has not yet been determined. A geometrical dilution of positive controls may allow visual comparison of the signals from the samples and dilutions which produce similar signal strengths. A standardized detection of HBV-DNA in HBsAg-positive sera can be derived from duplicates of serial dilution of cloned purified HBV-DNA1 e.g. from 400-3.1 pg/ml (Fig. 3) (6). In the same experiment, duplicates of differently diluted serum samples allowed comparison of a signal at the end-point dilution with some negative serum samples from blood donors without HBV markers. Using the dot blot hybridization assay, HBV-DNA can be detected at approximately up to 1.5 pg/ml and 2.104 genomtsiml respectively. Molecular solution hybridization assay Recently a new assay for HBV-DNA was designed on

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Reciprocal Dilution of Phaqe Standard Fig. 4. Solution hybridization assay in comparison with dot blot assay [according to Kuhns et al. (7)].

the basis of solution hybridization using iodine-labelled probes (7). This assay was compared with a filter of dot blot hybridization using a Ml3 phage standard which contained the whole HBV genome serially diluted in human serum negative for HBV markers (Fig. 4). Serum diluent also serged as a negative control. In this regard, the solution hybridization assay was linear over more than 3 orders of magnitude and included HRV-DNA concentrations ranging from 0.015 to 15 times the positive control (7). In clinical practice, the solution hybridization assay (8) has been found to be a simp!? qjlantitative method providing important information for monitoring, e.g. antiviral trials. Polymerax chain reaction (PCR) assay Currently, PCR technology for HBV-DNA provides a more than 1000 times increase in the sensitivity of the routine hybridization assays (9) (Fig. 5a; Table 1). Quclity and validity of the assay are now under investigation to reduce the possibility of carry-over, and to prevent sample-to-sample cross-contamination and false-positive results (10). A detection limit equal to 30-300 HBV TABLE 1 Sensitivity methods

Fig. 3. Semiquantitation by performing dot blot hybridization cording to Zyzyk et al. (6)].

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Fig. 5. Polymerase chain reaction assay (PCR) for the detection of serum HBV-DNA

[according to Gerken et al. (lo)].

particles/ml can be determined (11). Many precautions must be taken to guarantee the validity of the results; e.g., pre-PCR and post-PCR steps must be separated physically. Water buffers, pipette tips and microcentrifuge tubes should be autoclaved. Aliquots of premixed reagents must be prepared for daily use in a PCRproduct-free environment. Positive displacement pipettes are preferable and aerosols must be carefully prevented. DNA samples should be added last to each tube. PCR analysis must be repeated at least twice for the samples and negative controls should include normal genomic DNA extracted from serum, liver and leukocytes. Furthermore, a reagent control, such as PCR mix without template DNA, must always be run. Positive controls should be performed in each experiment with a predefined amount of DNA extracted from HBV-positive liver rumor tissue containing one copy of HBV-DNA per cell. Hybridization should be performed under stringent conditions. To re-evaluate sensitivity and specificity of the PCR assay, serial dilutions of different sera and cloned HBV-DNA should be subjected to PCR assay along with a target serum sample to give a semiquantitative estimate of the amount of PCR product (10). After PCR analysis, the autoradiogram can be quantitatively calculated by videodensitometry (Fig. 6). In addition, specific spots on the hybridized membrane can be cut out and subjected to liquid scintillation counting. This provides a practical approach to give a semiquantitative estimate of HBV-DNA using PCR methodology.

Use of monoclonal antibodies for HBV assays Monoclonal antibodies with diagnostic potential have been prepared in research laboratories against a battery of biological HBV targets. In general, due to their clonal

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Fig. 6. Semiquantitation of HBV-DNA sequences from PCR products [according to Gerken et al. (lo)].

nature, these antibodies demonstrate defined specificity, react with uniform avidity, and can be highly purified to homogeneity, thus providing reagent-grade material for analysis without limitation (12). However, the clonal origin of the antibody may cause certain problems. Each monoclonal fmtibody has highly defined properties. Large, differezlces in important properties such as solubility, affinity and lang-term stability have been observed between individual monoclonal antibodies. One major problem is that the high specificity of these antibodies may be too limited, so that the selection of suitable antibodies for a specific diagnostic purpose is of the utmost importance. In some instances, a single antibody may have enough specificity against a highly conserved epitope to be used as a general diagnostic reagent. Otherwise, to obtain the desired broad range of specificity it may be necessary to mix antibodies. Essentially, the development of high-affinity monoclonals is dependent on pure preparations of screening reagents. Immunization schedules are critical for the production of monoclonal antibodies. The route and time of immunization and the antigen concentrations are determined by establishing optimal conditions for the production of stable secreting hybridomas. Once antibodies have been produced, their properties must be characterized, e.g. subtype, antibody class and subclass, ascitic fluid concentration, affinity for HBsAg, associated epitopes and recognition sites (13). In practice, monoclonal antibodies can be used to construct radioimmunoassays, enzyme immunoassays, and

BIOLOGICAL

ST4NDARDS

FOR HBV ASSAYS

immunoblotting techniques. Furthermore, monoclona~s can be used in immunohistopathology (14). With the same antibody, sensitivity of an immunoassay also depends on the methodology of the assay. For example, the enzyme immunoassay technique seems to be more sensitive than immunoblotting and easier to perform for routine use (15-17). Recently, synthetic peptides derived from the HBV sequences have been used to characterize the specificities of both monoclonal antibodies and target preparations (18). in addition, peptides can be used to establish HBV diagnostic assays in the detection of pre-Sl and pre-S2 antibodies during HBV infection, for example (19). However, conformational epitopes of the S- or C-region caneferences 1 Gerlich WH, Thomssen R. Terminology, structure and laboratory diagnosis of hepatitis viruses. In: McIntyre N et al. (Eds.). Oxford Textbook of Clinical Hepatology, Lond#on: Oxford LJniversity Press, 1991. 2 Stamm P. Gerlich WH, Thomssen R. Quantitative determination of antibody against hepatitis B surface antigen: measurement of its binding capacity. J Biol Standard 1.980; 8: 59-68. 3 Gerlich WH, Thomssen R. Standardized detection of hepatitis I3 surface antigen: determination of its serum concentration in weight units per volume. Dev Biol Standard 1975: 30: 78. 4 Weller IVD, Fowler IF, Monjardino J. Thomas HC. The detection of hepatitis B virus DNA in serum by molecular hybridization: a more sensitive method for the detecrion of complete hepatitis B virus particles. J Med Virol 1982; 9: 273-80. 5 Scotto J. Hadchouel M. Hery C et al. Detection of HBV-DNA in serum by a simple spot hybridization technique: comparison with results for other viral markers. Hepatology 1983: 3: 27984.

6 Zyzyk E, Gerlich HW. Uy A et a!. Assay of hepatitis B virus genome titers in sera of infected subjects. Eur 3 Clin Microbial i986; 5: 330. 7 Kuhns MC, McNamara AL, Cabal CM et al. A new assay for the quantitative detection of hepatitis B viral DNA in human serum. In* Zuckerman AJ (Ed.), Viral Hepatitis and Liver Disease. New York: A.R. Liss Inc., 1988; 258-62. 8 Kuhns MC, McNamara AL, Perillo RP et al. Quantitation of hepatitis B viral DNA by solution hybridization. J Med Virol 1989: 27: 274-81. 9 Kaneko S. Finestone SM. Miller RH et al. Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J Clin Microbial 1989: 27: 1930-3. 10 Gerken G, Paterlini P, Manns M et al. Assay of hepatitis B virus DNA by polymerase chain reaction and its relationship to PreS-

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not be mimicked by short peptides.

Although the reliability of hepatitis virus serology performed by clinical laboratories is generally good or even excellent, internal and external quality controls would be useful. Some principal guidelines for clinical practice have been discussed. Quantitative standardization would provide clinically relevant information, not only for HEN-diagnosis, but also for therapeutic trials, -which are often performed in large international multicenter studies .

and S-encoded viral surface antigens. Hepatology 1991: 13: l58166. 11 Larzul D, Gigne F, Sninsky J et al. Detection of HBV-sequences in serum by using in-vitro amplification. J Virol Methods 1988: 20: 227-37. 12 Ben-Porath E, Fujita YK, Wands JK. Hepatitis B monoclonal antibody testing. In: Popper H, Schaffer F (Eds.j, Progress in Liver Diseases. Vor; VIII. New York: Grnne & Stratton, 1988: 347-66. 13 Petit MA, Cape1 E. Rio?tot M et al. Antigenic mapping of the surface proteins of infectious hepatitis B virus particles. J Gen Viral 1987: 68: 2759-67. 14 Dienes HP. Gerlich WH. Wdrsdiirfer M et al. Hepatic expression patterns of the large and middle hepatitis B virus surface protein in viremic and non-viremic chronic hepatitis B. Gastroenterology 1990: 98: 1017-23. 15 Gerken G, Manns M, Gerlich WH et al. Immune blot analysis of viral surface proteins in serum and liver of patients with chronic hepatitis B virus infection. J Med Viral 1989: 29: 261-5. 16 Gerken G, Manns M, Gerlich WH et al. PreS encoded viral surface protein in relation to the major surface antigen in acute hepatitis B virus infection. Gastroenterology 1987; 92: 18s-8. 17 Gerken G, Manns M. Gerlich WI-I et al. Sensitivity of immunoblotting compared with enzyme immunoassays in the det’ection of preS protein in acute hepatitis B. In: Zuckerman AJ (Ed.). Viral Hepatitis and Liver Disease. New York: Alan R. Liss, Inc., 1988; 277-9. 18 Neurath AR, Kent SBH, Strick N, Parker K et al. Delineation of contiguous determinants essential for biological functions of the preS sequence of the hepatitis B virus envelope protein: its autogenicity, immunogenicity and cell-receptor recognition. Ann Inst PasteurNirol 1988: 139: 13-38. 19 Alberti A. Cavaletto D, Pontisso P et al. Antibody response to preS2 and hepatitis B virus induced liver damage. Lancet 1988: i: 1421-4.