Development and Technical and Clinical Validation of a Quantitative Enzyme-Linked Immunosorbent Assay for the Detection of Human Antibodies to Hepatitis B Surface Antigen in Recipients of Recombinant Hepatitis B Virus Vaccine

Pending removal from the market of a commercial assay (the AUSAB [Abbott Laboratories] enzyme immunoassay [EIA]) for the determination of antibodies to hepatitis B surface antigen (HBsAg), a new in-house quantitative enzyme-linked immunosorbent assay (ELISA) to measure antibodies against HBsAg (anti-HBs) was developed (anti-HBs in-house). Specific anti-HBs antibodies were sandwiched between the precoated HBsAg ad and ay subtypes purified from plasma from hepatitis B virus (HBV) human carriers and the recombinant HBsAg adw2 subtype tagged with horseradish peroxidase. The assay was calibrated against the 1st International Reference Preparation for anti-hepatitis B immunoglobulin (lot 1977-W1042). Analytical sensitivity and the limit of quantitation were estimated at 0.43 mIU/ml and 2.0 mIU/ml, respectively. Overall reproducibility was 11.86%, and accuracy was estimated to be 94.89%. More than 4,000 samples from seven clinical trials were tested with the anti-HBs in-house assay and compared to results generated with AUSAB EIA and AUSAB radioimmunoassay (RIA). During the technical validation, the anti-HBs in-house assay was compared to the AUSAB RIA as a reference (n = 919). Overall assessment of concordance and Deming''s regression analysis were performed. The coefficient of correlation between the AUSAB RIA and anti-HBs in-house assay was 0.9815 with a slope of 0.9187. The overall agreement between anti-HBs in-house and AUSAB RIA was 97.61%, considering the clinical cutoffs at 3.3 mIU/ml and 1.0 mIU/ml for the respective assays. From a clinical perspective, seroprotection rates and anti-HBs geometric mean antibody concentrations for individual studies calculated with either the in-house assay or the reference assays were similar. Conclusions of individual studies were confirmed. The performance characteristics of the in-house assay are acceptable. There is no evidence that use of the new assay would lead to different clinical conclusions from the reference method.Hepatitis B infection is a global health problem but most acutely affects developing countries (16). Currently there is no effective therapy against hepatitis B, whose disease spectrum ranges from asymptomatic disease to chronic liver diseases, including cirrhosis and hepatocellular carcinoma. Prevention of the illness through vaccination remains the method of choice for its control and eradication. Active immunization against hepatitis B infection can be achieved using vaccines containing either inactivated hepatitis B virus (HBV) surface protein (HBsAg) physicochemically purified from plasma from HBV human carriers or recombinant surface antigen produced by transfer of the S gene of HBV coding for HBsAg to an appropriate plasmid that is then inserted into the desired expression vector. The recombinant vaccine manufactured by GlaxoSmithKline Biologicals (GSK) is produced in yeast and is antigenically similar to plasma-derived HBsAg (4). Clinical studies of this recombinant vaccine either formulated as a single-component vaccine (Engerix-B; trademark of GSK) or formulated in combination with other antigens such as hepatitis A vaccine (Twinrix; trademark of GSK) or pediatric diphtheria-tetanus-pertussis-based vaccines (such as Infanrix hexa and Tritanrix HepB; trademarks of GSK) have proven its efficacy and immunogenicity (5).To date, commercial assays from Abbott Laboratories have been used at GSK to quantify the immune response to HBV vaccines in terms of antibodies against HBsAg (anti-HBs). However, since these assays are no longer commercially available in Europe, GSK has developed an in-house assay with adequate technical and clinical performance to ensure long-term supply of an assay with consistent quality. This paper describes the development, technical validation, and the clinical assessment of the new anti-HBs in-house assay.     

Quantitation of hepatitis B surface antibody by an automated microparticle enzyme immunoassay

A fully automated microparticle enzyme immunoassay, IMx AUSAB, was developed for the detection and quantitation of antibody against hepatitis B surface antigen (anti-HBs). The IMx AUSAB assay can complete 24 tests in less than 45 minutes. Anti-HBs concentrations in specimens are calculated automatically by comparison of the specimen rate to values determined from a stored standard curve. IMx AUSAB sensitivity is 2-3 mIU/ml, equivalent in sensitivity to AUSAB RIA or EIA. Specimens from blood donors, diagnostic and hospital patients, hepatitis B vaccinees, and individuals with a variety of infectious and autoimmune diseases tested in parallel by IMx AUSAB and AUSAB RIA or IMx AUSAB and EIA gave overall qualitative agreement of 97.8% (1265/1293) and 99.1% (1281/1293), respectively. The prevalence of anti-HBs ranged from 5.9% in volunteer blood donors to 47.0% of specimens from a sexually transmitted disease clinic. Most discordant specimens (18/34) were low level reactive (less than 10 mIU/ml) by AUSAB RIA, but negative by IMx AUSAB and AUSAB EIA. These specimens were also negative for antibodies to hepatitis B core antigen (anti-HBc). Six discordants were low level reactive by IMx but negative by RIA and EIA. Three of these six specimens were also reactive for anti-HBc suggesting that the IMx AUSAB reactivity resulted from the presence of low level anti-HBs. Quantitative agreement between IMx AUSAB and RIA or IMx and EIA for 106 specimens ranging in anti-HBs concentration from 1 to 30,000 mIU/ml gave linear correlation coefficients of 0.91 and 0.96, respectively. The IMx test was useful for monitoring hepatitis B vaccine response and seroconversion levels after hepatitis B infection.     

Comparison of two techniques for detecting antibody to HBsAg during a hepatitis B vaccine study

Two assays for the detection of antibody against hepatitis B surface antigen (anti-HBs) were compared. The first was a direct sandwich radioimmunoassay (RIA) which detects, in principle, antibody against any epitope of hepatitis B surface antigen (HBsAg). The second assay was an inhibition enzyme-linked immunosorbent assay (ELISA). In this assay a fixed amount of HBsAg which can be blocked by anti-HBs is measured in a direct sandwich test. Prevaccination screening sera (n = 191) and follow-up sera obtained from high risk groups (n₁ = 85; n₂ = 41) during two hepatitis B vaccine studies were compared in RIA and ELISA. In prevaccination sera either HBsAg or anti-HBs were detected by ELISA. Full agreement between the results of RIA and ELISA for anti-HBs was obtained in sera containing more than 10 IU/1 anti-HBs. Both tests showed variable results at low titres. Experiments with monoclonal anti-HBs indicated that ELISA is less sensitive for subtype specific antibodies (anti-d, anti-y), which may explain that there were consistent differences between RIA and ELISA in a minority of cases.     

The utilization of biotin-antibiotin interaction for the detection of antibody to hepatitis B surface antigen (anti-HBs)

A biotin-antibiotin solid-phase enzyme-linked immunoassay for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) is described. The assay utilizes hepatitis B surface antigen as a solid-phase 'capture' reagent and a mixture of biotinylated HBsAg and antibiotin-conjugated horseradish peroxidase as a detector reagent. The assay was compared to a commercial enzyme immunoassay (AUSAB EIA) which used the biotin-avidin system for anti-HBs detection. The two assays were found to measure the same molecules and to correlate well regarding anti-HBs titers.     

Evaluation of assay methods for hepatitis B surface (HBsAg) antigen and its antibody (anti-HBS) in viral hepatitis B (VHB)-HBsAg-positive

The sensitivity of methods for the detection of HBsAg and its anti-HBs was compared in serial 1200 sera samples from 30 patients with VHB-HBsAg-positive. HBsAg was tested by gel-diffusion (GD), counter-immunoelectrophoresis (CIE), reversed haemogglutination (rHA), radioimmunoassay (RIA), and enzyme immunoassay (EIA). RIA and EIA methods are statistically significantly more sensitive compared with the other methods (P<0.0005). By these methods the minimal concentrations of HBsAg in sera can be proved. Although there is no statistically significant difference in the sensitivity between RIA and EIA, the latter is more sensitive if the subtype ay-HBsAg is considered (12 sera samples). In 24 patients the subtype was ay, in two ad, and in four it could not be differentiated.In 70% of patients anti-HBs was proved by RIA and in 10% by CIE, i.e., in 73% and 9% of sera samples, respectively. In 117 sera samples of these patients the sensitivity of RIA and EIA was compared for determination of anti-HBs. No statistically significant difference between the methods for determination of anti-HBs was found (50.42% 40.17%). No immune response to HBsAg has been observed in 9 cases, but 6 of them have remained permanent carriers of this antigen.     

Significance of isolated hepatitis B core antibodies detected by enzyme immunoassay in a high risk population.

Serum samples, which were found positive for anti-HBc and negative for HBsAg and anti-HBs during routine testing with Abbott enzyme immunoassays (EIA), were collected prospectively. The samples were obtained from patients with a high risk of hepatitis B. Further analysis was carried out using radioimmunoassays and tests for additional markers of hepatitis B. 84 of the 203 initially collected samples (41.4%) were found positive for anti-HBs by RIA. Of the 119 samples negative for anti-HBs by RIA, 103 were available for further investigation, and 35 (34.0%) of these were negative for anti-HBc by RIA. This indicates that low sensitivity of the anti-HBs EIA and non-specificity of the anti-HBc EIA may account for about 60% of the cases presenting with isolated anti-HBc at initial testing. Fifty-two samples positive for anti-HBc by RIA, with isolated anti-HBc confirmed after RIA testing for HBsAg and anti-HBs, were examined further: anti-HBe was detected in 16 sera, anti-HDV in one serum and HBV-DNA in 5 sera by PCR. Additional testing of serum samples with isolated anti-HBc is warranted.     

Status and significance of testing for hepatitis B surface antigen and surface antibody

Following Blumberg's discovery of hepatitis B surface antigen (HBsAg), many attempts have been made to develop several in vitro diagnostic techniques for the detection of this antigen and its homologous antibody. The two-dimensional micro-Ouchterlony immunodiffusion has been the first technique used, rapidly replaced by procedures of increasing sensitivity characterized as second-generation and the currently available third-phase tests which include radioimmunoassay (RIA), reverse passive haemagglutination (RPHA), reverse passive latex agglutination (RPLA) and enzyme immunoassay (EIA). Among these, RIA appears to be the most sensitive and specific, whereas EIA, RPHA and RPLA have the advantage of long shelf-life of stable reagents, no need for sophisticated and expensive equipment and no hazard associated with the handling of radioactive isotopes. Moreover, the sensitivity of EIA should increase by objective reading with a colorimeter.The most sensitive method for the detection of surface antibody (anti-HBs) is again RIA, whereas passive haemagglutination (PHA) had the advantage of providing titres. Finally EIA, based on inhibition of a known amount of HBsAg, has at least the same sensitivity as PHA, but has the advantage that reagents are more stable and that it permits screening for both HBsAg and anti-HBs with the same reagents at the same time. The application of these highly sensitive techniques for the detection of HBsAg and anti-HBs has resulted in a consistent reduction in the incidence of post-transfusion hepatitis type B and in a better understanding of the aetiology, epidemiology and natural history of this infection.     

Comparison of two testing methods to determine hepatitis B surface antibody response to hepatitis B vaccine among health-care workers

Because of variations in reported seroconversion rates and to compare enzyme-linked immunoassay (EIA) and radioimmunoassay (RIA) methods to assess hepatitis B vaccine response, hepatitis B surface antibody (anti-HBs) was tested in 116 of 174 high-risk hospital employees enrolled in a hepatitis B vaccine program. All individuals were vaccinated with three injections of Heptavax-B, 1.0 ml containing 20 micrograms of HBsAg intramuscularly in the deltoid. The same lot of appropriately stored vaccine was used. Of the 41 individuals tested within zero to six months postvaccination, 35 (85%) and 38 (93%) were positive by EIA and RIA, respectively. Of the 75 individuals tested 7-24 months postvaccination, 61 (81%) and 71 (95%) were positive by EIA and RIA, respectively. Results of EIA tests performed at two laboratories were similar. Of 109 individuals positive by RIA, 13 did not have protective antibody levels. In contrast, of 96 individuals positive by EIA, only one did not have a protective antibody level. RIA may be a more sensitive test for anti-HBs, but a positive EIA result correlates better with protective antibody levels.     

Detection of human antibodies to hepatitis B surface antigen (HBsAg) by an enzyme-immunoassay for HBsAg.

We studied the feasibility of detecting antibody to hepatitis B surface antigen (anti-HBs) by an inhibition assay using the reagents of an enzyme-immunoassay for HBsAg (Hepanostika). Several modifications of the basic assay were investigated. Sensitivity was greatest when the test sample was incubated with a predetermined amount of HBsAg before the usual procedure of HBsAg detection. The presence of anti-HBs in the test sample was shown by a reduction of the solid-phase bound enzyme label. Results were obtained with a dilution series of serum samples containing anti-HBs, the anti-HBs Reference Panel of the American Bureau of Biologics, sera of hepatitis B patients, and sera of two individuals passively immunised with anti-HBs. The enzyme-immunoassay method showed at least the same sensitivity as passive haemagglutination. It was less sensitive than a commercially available radioimmunoassay (Ausab). There are no indications that non-specific reactions occur frequently. This study also revealed that the antigenaemia of acute hepatitis-B patients can be interrupted by a transient seroconversion.     

Mutation of the "a" determinant of HBsAg with discordant HBsAg diagnostic kits

Sera from 1003 in- and out-patients were investigated for hepatitis B surface antigen (HBsAg) mutation at Siriraj Hospital, Bangkok, Thailand. Individual samples were screened using two commercial HBsAg kits on automatic machine-based assays set up in parallel. The first kit was a sandwich ELISA kit that used monoclonal capture/monoclonal conjugate and color detection whereas the second was a sandwich MEIA, using monoclonal capture/polyclonal indicator and fluorochrome determination. Six specimens were found discordant by negative EIA and positive MEIA; two specimens of which were detectable of HBV DNA. Three out of four discordant results were confirmed by an anti-HBs neutralization assay. Based on direct sequencing, one HBsAg/anti-HBs sample with multiple mutations in the S gene was found. The mutation included the common glycine to arginine escape mutation at amino acid position 145 of the "a" determinant. The observation should alert the blood bank to the necessity of using screening kits capable of detecting HBV mutant carriers as well as providing verification for the phenomenon of vaccine-escape mutation in Thailand.     

Evaluation of immunochromatographic assay systems for rapid detection of hepatitis B surface antigen and antibody, Dainascreen HBsAg and Dainascreen Ausab.

We evaluated two immunochromatographic assays (ICAs), Dainascreen HBsAg for detecting human hepatitis hepatitis B surface antigen (HBsAg) and Dainascreen Ausab for detecting human hepatitis B surface antibody (anti-HBs) in human serum. The ICA systems are composed of a comb-shaped device that contains nitrocellulose strips on which complexes of HBsAg and anti-HBs can be visualized. The results can be read within 15 min of incubation. The limit of detection for HBsAg was 3.1 ng/ml and that for anti-HBs was 42 mIU/ml. Results of HBsAg detection agreed completely with those of conventional enzyme immunoassays (EIAs) and showed a 100% sensitivity (158 of 158 samples) and a 100% specificity (304 of 304 samples). The Dainascreen Ausab detected 184 of the 199 EIA-positive samples (sensitivity, 92.5%) and yielded 6 positive results among the 281 EIA-negative samples (specificity, 97.9%). The ICA systems are rapid and sensitive methods for detecting HBsAg and anti-HBs. They are low-cost systems that need no complex instrumentation for analysis and can be recommended for routine use in clinical microbiology laboratories.     

Long-term follow-up of Hepatitis B Surface antibody levels in subjects receiving universal Hepatitis B vaccination in infancy in an area of hyperendemicity: correlation between radioimmunoassay and enzyme immunoassay

The aims of the present study were to determine (i) the long-term immunogenicity and the decay rate of hepatitis B virus (HBV) surface antibody (anti-HBs) from universal hepatitis B vaccination at infancy for a healthy population in an area of hyperendemicity and (ii) whether the anti-HBs levels measured by enzyme immunoassay (EIA) were closely correlated with those assayed by radioimmunoassay (RIA) methods during long-term monitoring. A total of 1,337 apparently healthy children (696 boys and 641 girls) who were vaccinated against HBV at infancy and monitored for anti-HBs annually from 7 to 16 years of age entered the study. Serum samples were analyzed for anti-HBs by RIA at 7 to 15 years of age and were also analyzed by EIA at 13 to 16 years of age. Antibody titers were quantified in mIU/ml by EIA as well as by the ratio of the count in the sample to the count for a negative control (S/N) by RIA. In non-boosted children, the average decay of anti-HBs from 7 to 16 years of ages indicated that approximately 20% of the geometric mean titer decays per year. There was a good correlation between serum anti-HBs levels measured by the RIA and the EIA methods (r=0.91; P<0.0001). An equation for RIA to EIA level conversion was established: log EIA titer=-0.12+ (1.31 . log RIA S/N). The anti-HBs titers measured by EIA correlate well with the S/N assayed by RIA. The annual decay rate of the log anti-HBs level may help in planning booster immunizations for hypo-responders or individuals at risk in adolescence.     

Sensitivities of four new commercial hepatitis B virus surface antigen (HBsAg) assays in detection of HBsAg mutant forms

Mutations in hepatitis B virus surface antigen (HBsAg) involving amino acid substitution within the immunodominant "a" determinant may affect the performance of commercial HBsAg assays. The performances of four HBsAg assays that recently received Conformité Européene marking, Advia Centaur HBsAg (Bayer), Monolisa HBsAg Ultra (Bio-Rad), Liaison HBsAg (Dia Sorin), and Vidas HBsAg Ultra (bioMérieux), were compared with that of the routinely used HBsAg assay AxSYM HBsAg V2 (Abbott). Assays were evaluated for (i) analytical sensitivity performance with a national reference HBsAg panel (including 10 samples with calibrated HBsAg concentrations from 0.04 to 2.24 ng/ml) and (ii) the detection of HBsAg mutants by studying a panel of 35 HBsAg mutants (23 collected from patients and 12 recombinant mutants). The limits of detection of these assays were <0.15 ng/ml (from 0.089 to 0.121 ng/ml). The sensitivity performances for mutant virus detection varied, ranging from 37.1% to 91.4%. The lack of detection of these mutants by commercial assays was probably due to the epitope recognition of the anti-HBs assay reagents in the capture phase and in the conjugates. The prevalence and clinical impact of HBsAg mutants are under investigation. However, the manufacturers must be vigilant in the design of the assays in order to reduce the risk of missing a broad range of described S gene mutants.     

Immunogenicity of low doses of hepatitis B vaccine in children: a study in 650 New Zealand children

Six hundred and fifty New Zealand children from 2-12 years of age were vaccinated three times with 2 mcg intramuscular (IM) doses of Merck Sharp and Dohme plasma-derived hepatitis B vaccine (H-B-Vax), at 0, 1, and 6 months, and tested 2-3 months later for antibody to hepatitis B surface antigen (anti-HBs) by radioimmunoassay (RIA). Overall, 96.5% of the children seroconverted for anti-HBs by RIA, having levels greater than 2.1 RIA S/N units, with 91.2% having values greater than 10 S/N units. Anti-HBs levels were also determined by enzyme immunoassay (EIA), by which method a significantly better response was demonstrated in 2-4-year-olds than in older children. This study demonstrated that a satisfactory anti-HBs response was obtained using one-fifth of the recommended doses of hepatitis B vaccine.     

Rapid detection of HBsAg and anti-HBs by enzyme-immunoassay

Enzyme-immunoassay (EIA) for HBsAg is a reliable method with third generation sensitivity. Using a mechanized system for washing and reading and computerized data processing, subnanogramme amounts of subtypes ad and ay could be detected in approx. 5.5 h.Using EIA-kit reagents and an HBsAg ‘standard’, anti-HBs could conveniently be detected. Sensitivity comparisons with dilution series of the WHO anti-HBs standard showed RIA to be 8–128 times more sensitive than EIA for anti-HBs. However, similar percentages of positives were found in a patient population, and discrepancies were found between RIA and EIA results which cannot be explained on the basis of sensitivity differences only. A careful examination of the specificity of EIA and RIA is indicated.     

Low-dose vaccination against hepatitis B in children: one-year follow-up

Six hundred forty-three children, negative for markers of hepatitis B virus (HBV) infections, were given three X 2-micrograms doses of Merck, Sharp and Dohme (MSD) plasma derived hepatitis B vaccine (H-B-Vax) at monthly intervals. Twelve months after the first dose of vaccine, antibody to hepatitis B surface antigen (anti-HBs) was detected in 89% of children by radioimmunoassay (RIA) and in 83% by enzyme immunoassay (EIA). Seroconversion rates and anti-HBs titres were significantly greater in 1-4-year-olds than in older children (p less than 0.01). Eighteen children with no anti-HBs or other markers of HBV at this time were given 10 micrograms of vaccine and tested one month later. Seventeen developed anti-HBs, 12 at levels consistent with an anamnestic response. Forty-nine HBV-marker-negative children seroconverted for antibody to hepatitis B core antigen (anti-HBc) in the 8-month period before or the 12-month period following vaccination. Forty-six of these children were positive for anti-HBs, and one has been confirmed as a chronic carrier of hepatitis B surface antigen (HBsAg). Three cases of clinical hepatitis B in children have been seen in the community since the vaccination programme began. Two of these were amongst the estimated 5% of children who were not vaccinated. The third was in a vaccinee and occurred 4 1/2 months after the last dose of vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the technical and clinical performance of the Elecsys® HBsAg II assay with the Architect®, AxSym®, and Advia® Centaur HBsAg screening assays

South East Asia has some of the highest prevalence rates of hepatitis B virus (HBV) infection (≥8%) in the world, and the emergence of hepatitis B surface antigen (HBsAg) mutant strains is a growing problem. Assays with the highest levels of sensitivity, including mutant detection, should be used for routine HBsAg screening. In this large multicenter study, the clinical and technical performance of the fully automated Elecsys HBsAg II assay was compared with the Architect, AxSYM, and Advia Centaur HBsAg assays for HBsAg screening. Nine laboratories (three each from Thailand, Korea, and Singapore) compared the Elecsys HBsAg II assay with their routine HBsAg screening assay against a range of stored and routine clinical samples, including recombinant mutants. The Elecsys HBsAg II assay demonstrated equivalent sensitivity and specificity to the Architect HBsAg assay. However, the Elecsys HBsAg II assay recognized a native mutant sample (L94S, L97V, L98V, T123A) that the Architect HBsAg assay failed to detect. The AxSYM and Advia Centaur HBsAg assays appeared less sensitive for the detection of early HBV infection and also failed to detect some of the recombinant mutant strains. There was almost complete agreement between the Elecsys HBsAg II assay and comparator assays with respect to routine serum samples. The results of this study demonstrate that the Elecsys HBsAg II assay is a highly sensitive and specific screening assay for HBsAg and detects reliably the most important and clinically relevant HBV mutants and genotypes. It is suitable for routine HBsAg screening in Asia. J. Med. Virol. 82: 755–762, 2010. © 2010 Wiley‐Liss, Inc.     

Long-Term Follow-Up of Hepatitis B Surface Antibody Levels in Subjects Receiving Universal Hepatitis B Vaccination in Infancy in an Area of Hyperendemicity: Correlation between Radioimmunoassay and Enzyme Immunoassay

The aims of the present study were to determine (i) the long-term immunogenicity and the decay rate of hepatitis B virus (HBV) surface antibody (anti-HBs) from universal hepatitis B vaccination at infancy for a healthy population in an area of hyperendemicity and (ii) whether the anti-HBs levels measured by enzyme immunoassay (EIA) were closely correlated with those assayed by radioimmunoassay (RIA) methods during long-term monitoring. A total of 1,337 apparently healthy children (696 boys and 641 girls) who were vaccinated against HBV at infancy and monitored for anti-HBs annually from 7 to 16 years of age entered the study. Serum samples were analyzed for anti-HBs by RIA at 7 to 15 years of age and were also analyzed by EIA at 13 to 16 years of age. Antibody titers were quantified in mIU/ml by EIA as well as by the ratio of the count in the sample to the count for a negative control (S/N) by RIA. In nonboosted children, the average decay of anti-HBs from 7 to 16 years of ages indicated that approximately 20% of the geometric mean titer decays per year. There was a good correlation between serum anti-HBs levels measured by the RIA and the EIA methods (r = 0.91; P < 0.0001). An equation for RIA to EIA level conversion was established: log EIA titer = −0.12 + (1.31 · log RIA S/N). The anti-HBs titers measured by EIA correlate well with the S/N assayed by RIA. The annual decay rate of the log anti-HBs level may help in planning booster immunizations for hyporesponders or individuals at risk in adolescence.     

Comparison of enzyme immunoassay with radioimmunoassay for the detection of hepatitis B surface antigen

A direct solid-phase enzyme immunoassay (Auszyme I) and a direct solid-phase radioimmunoassay (Austria II) for detection of hepatitis B surface antigen (HBsAg) were compared in tests with a panel of 347 human sera. Compared with RIA, EIA showed a sensitivity of 98% with 153 HBsAg-positive sera and a specificity of 99% with 194 HBsAg-negative sera. Sera that gave false negative and false positive results by EIA were re-examined by both RIA and EIA to confirm the initial result. Use of less than the recommended volume of serum for EIA produced results inconsistent with RIA in four of 27 sera examined. Quantitative correlation between RIA and EIA was low (r = 0.691). Positive controls used for EIA showed considerable variation from day to day, although intra-assay variation was much less. The sensitivity of the EIA method examined compares favourably with previously published EIA studies and with the RIA used in this study. Auszyme I EIA is a sensitive and specific third generation test for HBsAg that offers several advantages over currently used RIA techniques.     

A method for coupling the hepatitis B surface antigen to aldehyde-fixed erythrocytes for use in passive hemagglutination

A method for coupling the hepatitis B surface antigen (HBsAg) to aldehyde-fixed erythrocytes for use in passive hemagglutination assays for anti-HBs is described. This provides a stable and inexpensive reagent having a sensitivity for anti-HBs detection which is only slightly less than solid phase radioimmunoassay.