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.     

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.     

Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg

A second generation radioimmunoassay (RIA) and enzyme-linked immunoassay (EIA) for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) was developed which utilizes recombinant DNA-derived HBsAg (rHBsAg) in place of human plasma derived HBsAg. In these sandwich assays, rHBsAg immobilized on a solid phase was used to capture anti-HBs from the specimen and rHBsAg conjugated to horseradish peroxidase or radiolabeled with 125I was used as a detecting reagent. These rHBsAg-based assays were compared to a commercial radioimmunoassay for anti-HBs detection (AUSAB RIA). For a population of 1711 sera and plasma specimens, 99.2% overall agreement was demonstrated between the recombinant RIA and EIA and 98.6% agreement was observed between the recombinant assays and AUSAB-RIA. The recombinant assays demonstrated equivalent sensitivity and detectability to AUSAB RIA. Most discrepant samples were low-level reactive by AUSAB-RIA, generally less than 10 mIU/ml, and likely represent nonspecific reactivity since no other marker for hepatitis B infection was detected in these samples.     

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.     

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.     

Improved economics of HBsAg screening with commercial radioimmunoassay reagents.

Commercial 125I anti-HBs was processed to yield a sixfold improvement in economy without significant loss of sensitivity or specificity. Additional polystyrene beads were coupled with commercially supplied anti-HBs. The modified assay (Mod-RIA) was compared with commercial RIA, EIA, and RPHA using established HBsAg panels. Mod-RIA was also compared with HEPATEST (RPHA) for screening 71 200 fresh blood donations during an 11.5-month period.     

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.     

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.     

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.     

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.     

Detection of an antigen (AN6520), possibly related to non-A, non-B hepatitis, by monoclonal antibodies. I

The antibody to AN6520 antigen, which was isolated from the liver of a patient with non-A, non-B hepatitis (NANBH), has been detected frequently in convalescent sera from patients with NANBH by the passive hemagglutination (PHA) test. In a further study, we established hybridoma cells secreting antibodies against AN6520 antigen and obtained ascitic fluids with PHA titers ranging from 1:10(5) to 1:10(7). In immunodiffusion with AN6520 antigen, all monoclonal antibodies were found to form an identical precipitin line. These lines were also identical to those formed by rabbit antiserum against AN6520 antigen and by convalescent sera from patients with NANBH. With one of the monoclonal antibodies, 1-F12, solid-phase radioimmunoassay (SP-RIA) for detecting AN6520 antigen was developed as well as blocking RIA for anti-AN6520 antibody detection. The antigen assay was 50 times more sensitive than the reverse passive hemagglutination (R-PHA) test, with a sensitivity threshold of the 1 ng/ml of antigen solution; the antibody assay was 10 times more sensitive than PHA. The results with this blocking RIA were mostly in agreement with the data obtained by PHA. Furthermore, the antigen in human sera, which had never been detected by R-PHA test, could be detected by SP-RIA.     

Detection and quantitation of hepatitis-B surface antigen immune complexes (HBsAg-ICs) by an antigen-specific method. I. Detection and quantitation of in vitro prepared HBsAg-ICs

An antigen-specific method has been developed for direct detection and quantitation of HBsAg-ICs. The method involves (1) precipitation of HBsAg-ICs with 3.5% PEG; (2) dissociation of the PEG precipitated ICs by treatment with NaSCN, NaI, KBr, low or high pH buffers, trypsin or papain; and (3) detection and titration of HBsAg and/or anti-HBs liberated from ICs. Treatment with 2 M and 3 M NaSCN, papain or trypsin liberates HBsAg, while following treatment with 3 M and NaI free anti-HBs is detectable. Trypsin digestion (2 mg/ml, 30 min at 37 degrees C) proved to be most effective for disrupting HBsAg-ICs formed at equivalence as well as in excess of antigen or antibody. After trypsin digestion of the sample RPHA, RIA and ELISA may be used as a third step. Th     

'Aspira': a new sensitive assay for the detection of hepatitis B surface antigen.

A sensitive method for the detection of hepatitis B surface antigen (HBsAg) is described whereby the separation of specific antibody by affinity chromatography is incorporated into one of the assay reagents. HBsAg, which may be obtained from tissue culture fluids of a human hepatoma cell line, is first reacted with polystyrene beads. Unoccupied spaces on the plastic are next covered with a non-specific protein (e.g. albumin). The plastic is then reacted with either whole antiserum or an IgG fraction of anti-HBs. Care must be taken to cover all of the reacting sites of the first layer of HBsAg with sufficient anti-HBs. This results in a reagent of detecting antigen. No affinity purified antibody was necessary because the reagent, at this stage, contains only specific antibody on the surface. Antigen reacting with this reagent can be detected by the addition of radioactively labeled IgG fraction of anti-HBs, which will complete the 'sandwich'. This assay has been evaluated and found to be as sensitive as those commerically available.     

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.     

Analysis and comparison of two methods to detect hepatitis B antigen. Counterelectrophoresis (CEP) and reversed passive latex-agglutination (RPLA) technic in relationship to radioimmunoassay.

The authors evaluated two methods for detection of hepatitis B surface antigen, reversed passive latex agglutination and counterelectrophoresis, using radioimmunoassay as a control. The RPLA test (Antigen I, Pfizer Inc., Lot no. 403-94301-1) was compared with radioimmunoassay (RIA, Austria) and counterelectrophoresis (CEP) using serum specimens obtained at the Mount Sinai Medical Center, Miami Beach, Florida. Of the 100 selected sera tested as unknowns, 64 were positive by RIA and 36 were negative. The RPLA test yielded results comparable to those of the RIA technic in that the same 64 positives and 36 negatives identified by RIA were obtained with the RPLA test. Of the 64 positive samples obtained with the RIA technic, five yielded negative results with the CEP method. The RPLA test proved to be more sensitive than CEP in this survey.     

Solid-phase enzyme-immunoassay for detection of hepatitis B surface antigen.

The preliminary results of a solid-phase enzyme-immunoassay (EIA) for the detection of hepatitis B surface antigen (HBsAg) are presented. This method has been compared with the solid-phase radioimmunoassay (RIA) for HBsAg in dilution series of four HBsAg positive sera four national reference panels (The Laboratory Panel of the Central Laboratory of the Blood Transfusion Service of the Netherlands Red Cross, USA BOB Reference Panels Nos 2 and 3, and the 1st Panel of the National Reference Centre for virus Hepatitis at the Institute of Hygiene of the University of Göttingen, West Germany). In addition, the two test methods were compared in a weekly (up to 16 weeks) follow-up of 14 patients with acute viral hepatitis B. It was seen that, both by reading EIA test results with the naked eye and by colorimetric reading, the sensitivity and specificity of this test method compared very favourably with those of the RIA. EIA may have a slightly lower sensitivity than RIA for the subtype ad, while its sensitivity for the subtype ay may be slightly higher than that of RIA. These minor sensitivity differences may be due to the specificity profiles of the antisera used.     

A sensitive single reverse passive haemagglutination test for detecting both HBsAg and anti-HBs.

A trial of a modified reverse passive haemagglutination test for HBsAg using a 0.1% cell suspension instead of the recommended 1% showed an approximately eight-fold increase in detection sensitivity. The test can be performed within 30 minutes and lends itself to mass screening techniques. Confirmation tests can be done using the 0.1% method. In addition, the same serological plates and cells used for HBsAg screening can then be used to screen for high-titre anti-HBs. This makes the overall screening for both HBsAg and high-titre anti-HBs donors cheap and convenient.     

Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemagglutination techniques for detecting cytomegalovirus IgG antibody.

The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems. Cytomegalovirus (CMV) infection is an important cause of congenital brain damage and is also a major complication of both prolonged immunosuppressive therapy, especially in patients with organ transplants, and multi-donor blood transfusions. For serological diagnosis of infection, as well as for screening for antibody in patients and in blood donors, the solid-phase indirect radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques offer distinct improvements in sensitivity over previous methods. Although the principle of both tests, based on the detection of antigen-antibody reactions by means of a labelled anti-antibody, is the same, each possesses its own particular technical advantages and disadvantages, and both require their own expensive equipment for the reading of the results. There is still a lack of data on how they compare in sensitivity and specificity. The present study was undertaken to compare the two methods for the detection of CMV IgG and to evaluate them against the older techniques of complement-fixation (CF), passive haemagglutination (PHA) and anticomplement immunofluorescence (ACIF).     

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.     

A biotin/avidin solid-phase sandwich enzyme immunoassay for the antibody to hepatitis B surface antigen (anti-HBs)

A biotin/avidin solid-phase enzyme 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 (HBsAg) as a solid-phase 'capture' reagent and a mixture of biotinylated HBsAg and avidin-conjugated horseradish peroxidase as a probe 'detector' reagent. The assay was compared to a commercial radioimmune assay for anti-HBs detection. The two assays were found to measure the same molecules and to correlate well regarding anti-HBs titers.