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.     

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.     

'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.     

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.     

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.     

Sites that bind polymerized albumin on hepatitis B surface antigen particles: detection by radioimmunoassay.

Antibodies to polymerized human albumin (poly-HSA) could not be detected by using sensitive methods (enzyme-linked immunosorbent assay and radioimmunoprecipitation) in sera from chronic carriers of hepatitis B surface antigen (HBsAg) or in serial bleedings from one chimpanzee infected with type A hepatitis virus and one infected with non-A, non-B hepatitis virus. By a solid-phase radioimmunoassay, receptor sites for poly-HSA could be detected on HBsAg particles from sera containing either hepatitis B "e" antigen (HBeAg) or anti-HBe. Blocking experiments showed that monomeric HSA did not bind to this receptor. In general, the HBsAg particles from sera with HBeAg had more poly-HSA receptor sites or relatively more particles carrying this receptor compared with HBsAg from sera with anti-HBe. Microtiter plates coated with poly-HSA bound HBsAg from sera containing HBeAg with greater efficiency than did anti-HBs coupled to a solid phase (Ausria II beads), whereas with sera positive for anti-HBe, the two assays were equally sensitive. Decreased ability of HBsAg to bind to poly-HSA was seen in some sera which had been stored for a few years at 4 degrees C, whereas the binding to anti-HBs was unaffected. It is possible that polymers of albumin on the surface of hepatocytes could function as receptors for hepatitis B virus.     

Analysis of reagent lot-to-lot comparability tests in five immunoassay items

We investigated the degree of lot-to-lot reagent variation for 5 common immunoassay items. We measured the commercial as well as in-house controls for α-fetoprotein (AFP), ferritin, CA19-9, quantitative hepatitis B surface antigen (HBsAg), and hepatitis B surface antibody (anti-HBs) 10 times each by using both the old and the new lot of reagents whenever a reagent lot was changed, over a period of 10 months. The differences in the mean control values, the percent difference (% difference), and the difference to between-run standard deviation ratio (D:SD ratio) between successive lots were calculated. The % difference in mean control values between 2 reagent lots ranged from 0.1 to 17.5% for AFP, 1.0 to 18.6% for ferritin, 0.6 to 14.3% for CA19-9, 0.6 to 16.2% for HBsAg, and 0.1 to 17.7% for anti-HBs except negative controls of HBsAg and anti-HBs. The maximum D:SD ratios between 2 lots were 4.37 for AFP, 4.39 for ferritin, 2.43 for CA19-9, 1.64 for HBsAg, and 4.16 for anti-HBs. Thus, we have experienced extensive variability in lot-to-lot reagent variation for 5 immunoassay items, indicating that reagent lot-to-lot comparability tests should be continuously performed and that laboratories should determine their own acceptance criteria for each item.     

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.     

Highly Sensitive Detection of Hepatitis B Virus Surface Antigen by Use of a Semiautomated Immune Complex Transfer Chemiluminescence Enzyme Immunoassay

The performance of hepatitis B surface antigen (HBsAg) screening assays is continuously improved to reduce the risk of transfusion-associated hepatitis B. In this study, a semiautomated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of HBsAg, which is as sensitive as hepatitis B virus (HBV) DNA PCR, was developed; the ICT-CLEIA assay performance was compared with the performance of the Architect HBsAg QT assay and HBV DNA PCR. The specificities in the initial assay and after retesting were 99.50% (1,988/1,998 samples) and 99.95% (1,997/1,998 samples), respectively. The analytical detection limit was determined to be 0.2 mIU/ml using the 2nd International WHO HBsAg standard, and the cutoff value (0.5 mIU/ml) of the ICT-CLEIA assay was 8.0 standard deviations (SD) above the mean of the HBsAg-negative specimens. The ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs antibodies and demonstrated a 23.6-day-shorter window period using commercially available HBsAg seroconversion panels than the Architect HBsAg QT assay. Furthermore, the monitoring of the viral kinetics by the ICT-CLEIA assay and the HBV DNA PCR produced very similarly shaped curves during both the HBsAg seroconversion and reverse seroconversion periods. Therefore, the ICT-CLEIA assay may be useful not only for an earlier detection of HBV reactivation but also for the monitoring of hepatitis B patients.     

14株抗乙型肝炎表面抗原单克隆抗体与变异表面抗原的反应模式及应用

目的制备抗乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)的单克隆抗体,检测单克隆抗体与15种变异HBsAg的反应模式。用筛选出的单抗建立快速检测变异HBsAg的ELISA实验方法,并做初步评价。方法用中国乙型肝炎病毒感染者血清中分离的HBsAg免疫BALB/c小鼠,通过杂交瘤细胞融合技术制备抗-HBs单克隆抗体。检测不同单克隆抗体与野生及变异HBsAg的反应性。筛选出两种可以较好识别变异HBsAg的单克隆抗体McAb2和McAb3,建立两种抗体ELISA检测HBsAg的方法。结果制备了14株抗-HBs单抗。经过初筛,有4种可以较好识别包括G145R在内的大多数变异HBsAg。优化了McAb2和McAb3检测HBsAg的条件,检测HBsAg的灵敏度较好,检测变异HBsAg的能力优于2种现行国产HBsAg检测试剂盒。结论用本实验制备的单抗可以很好地识别包括G145R在内的大多数变异HBsAg。     

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.     

Immunoassay detection of hepatitis B surface antigen mutants.

The increasing use of hepatitis B vaccination has had an overwhelming positive impact on the prevention of hepatitis B viral infection. Mutations in the hepatitis B surface antigen (HBsAg) gene occur as a result of vaccine escape mutants, anti-hepatitis B surface antigen immunotherapy, or in chronic hepatitis B viral infection. These mutants may present a challenge to immunoassay detection. Evaluation of the immunodetection of various HBsAg mutants has been sporadic, as the occurrence of these mutants is not common, and sufficient volume of serum samples is difficult to obtain. To investigate mutant detection, recombinant antigens were constructed to reflect mutations described in the literature occurring throughout the S gene. A limited number of serum samples exhibiting discordant immunoassay reactivity were also used to construct recombinant antigens. The evaluation of 25 HBsAg mutants across nine commercial assays of differing formats is described. Mutations affecting immunoassay performance were characterized as occurring mainly in loop 2 of the "a" determinant of HBsAg. It was determined that reagent epitope recognition was more significant for mutant detection than assay format.     

Immunoregulation of the in vitro anti-HBs antibody synthesis in chronic HBsAg carriers and in recently boosted anti-hepatitis B vaccine recipients.

We report a study on immunoregulation of in vitro antibody to hepatitis B surface antigen (anti-HBs) synthesis induced by pokeweed mitogen (PWM) from peripheral blood mononuclear cells (PBMC) in chronic hepatitis B surface antigen (HBsAg) carriers and in 'high responders', (anti-HBs RIA ratio greater than or equal to 20 in serum), recently boosted with anti-hepatitis B vaccine. Anti-HBs was detected in 11 days PBMC supernatants (SN) from 24 out of 36 'high responders', but in none from 31 chronic HBsAg carriers, despite detectable amounts of polyclonal IgG and antibody to hepatitis B core antigen (anti-HBc) were produced. The lack of anti-HBs production by chronic HBsAg carriers did not seem to be determined by suppressor influences because T lymphocytes from the majority of chronic HBsAg carriers, co-cultured with 'high responders' PBMC did not suppress anti-HBs production. Co-cultures between HBsAg carriers T4 positive (helper/inducer) cells and allogenic 'high responder' non-T cells produced anti-HBs antibody, indicating that HBsAg carrier T cells are not deficient in this allogenic helper function under PWM stimulation. Allogenic cocultures between HBsAg carrier non-T cells and 'high responder' T4 positive cells failed in anti-HBs production: a specific B lymphocyte defect might be involved in the lacking anti-HBs synthesis in chronic HBV patients. Antigen-induced specific anti-HBs synthesis experiments indicate that B cells themselves seem to be the target for HBsAg-induced suppression of anti-HBs antibody response.     

Prognostic value of HBsAg/IgM complexes in hepatitis B patients: nature of the proteins involved

Hepatitis B surface antigen (HBsAg)/IgM complexes were measured using a solid phase radioimmunoassay in sera of patients with acute or chronic hepatitis B. These complexes were found in 6 out of 18 patients four weeks after the onset of the disease and only one of them developed chronic hepatitis. HBsAg/IgM complexes correlated with HBsAg, hepatitis B e antigen (HBeAg), and pre-S2 concentration. The precipitation of HBsAg/IgM reactivity by polyethylene-glycol (PEG) and the binding of this activity to the surface of certain uncoated enzyme linked immunosorbent assay (ELISA) plates indicates that HBsAg/IgM positivity may reflect the presence of circulating complexes in serum. HBsAg and pre-S2 were found as components of the complexes but anti-HBs, anti-pre-S2, and polymerized human serum albumin (pHSA) were not. An immune binding between HBsAg and IgM is still questionable. Whatever the nature of the HBsAg/IgM complexes, their detection does not seem to be an earlier indicator of prognosis than HBeAg and/or pre-S2.     

Association of graft survival with host response to hepatitis B infection in patients with kidney transplants.

We studied the relation of host response to hepatitis B infection before transplantation with survival of kidney grafts in 79 patients receiving 87 transplants. Antibody to hepatitis B surface antigen (anti-HBs) signaled early graft rejection (median survival congruent to two months), whereas hepatitis B surface antigen (HBsAg) signaled delayed rejection (greater than 22 months). Patients with neither HBsAg nor anti-HBs had graft survival times (median congruent to 16 months) similar to the HBsAg carriers but significantly longer than the anti-HBs-positive patients (p less than 0.01). Similar results were observed when patients who received HLA-identical kidneys or had anti-HLA antibodies before transplantation were excluded. The highest probability of graft rejection was in patients with anti-HBs who received kidneys from male donors. The probability that such grafts would survive for four months was less than 20 per cent. HLA-nonidentical kidneys transplanted into patients with anti-HBs have a poor prognosis, whereas such grafts in HBsAg carriers have as good a prognosis as grafts in uninfected recipients.     

Detection of antibody to hepatitis B core antigen (anti-HBc) using a direct (antiglobulin) format and development of a confirmatory assay for anti-HBc

A direct (antiglobulin) solid-phase enzyme immunoassay for the detection of antibody to hepatitis B core antigen (anti-HBc) is described. The assay utilizes recombinant hepatitis B core antigen as the solid-phase 'capture' reagent and a mixture of monoclonal antibodies specific for human IgG and IgM conjugated to horseradish peroxidase as the 'detector' reagent. The direct assay demonstrated excellent sensitivity and specificity when compared with a commercially available competitive enzyme immunoassay. The direct assay format lends itself to a confirmatory assay for anti-HBc by addition of monoclonal anti-HBc to the reaction mixture. Feasibility of the confirmatory assay for anti-HBc was demonstrated using specimens reactive for anti-HBc as documented by both the direct and competitive assays.     

Acute hepatitis B viral infection in a patient with anti-HBs.

We report a patient with antibody to hepatitis B surface antigen (anti-HBs) but no antibodies to other hepatitis B virus components, who developed acute symptomatic type B hepatitis. The possible explanations for this unusual serological pattern are 1) the antibody-positive status, which developed against only a subdeterminant of hepatitis B surface antigen (HBsAg), arose naturally or as the result of cross-reaction with a variety of antigens; and 2) seroconversion to anti-HBs occurred in response to surface antigen of a mutant strain of hepatitis B virus (HBV). This anti-HBs positivity, in the absence of antibody to hepatitis B core antigen, does not provide natural immunization against HBV infection, and so is not protective. Individuals who are positive to anti-HBs antibody alone which is not elicited by HBV vaccine, should be vaccinated against possible HBV infection.     

Co-occurrence of HBsAg and anti-HBs: Two consecutive infections or a sign of advanced chronic liver disease?

Simultaneous presence of hepatitis B surface antigen (HBsAg) and antibodies to the surface antigen (anti-HBs) was detected in 32 out of 89 Dutch chronic hepatitis patients of Caucasian race. HBsAg was subtyped ad in 28 and ay in four cases. Anti-HBs could be subtyped in 25 cases using reference antigens discriminating between d, y, and w1-4 subdeterminants. In 20 patients HBsAg subtype ad (HBsAg/ ad) was accompanied by antibody to subdeterminant y (anti-y), whereas HBsAg/ ay and anti-d were simultaneously detected in the serum of one patient. The antibody pattern in sera from the remaining patients was complex. Eighteen anti-HBs-positive patients were matched for age, histology, and hepatitis B e antigen (HBeAg) status with 18 anti-HBs-negative patients. Differences in risk factors for acquiring a hepatitis B infection were not found. These results do not support the hypothesis that co-occurrence of HBsAg and anti-HBs is due to two consecutive infections with hepatitis B virus. The frequency of the co-occurrence of HBsAg and anti-HBs was found to be related to the degree of progressive liver disease, since anti-HBs was found in three out of 23 asymptomatic carriers, in four out of 20 chronic persistent hepatitis patients, in 20 out of 41 chronic active hepatitis patients, and in all five patients with chronic active hepatitis and cirrhosis. The high frequency of anti-HBs in advanced liver disease may be the result of a disturbed immunologic response mechanism.     

Hepatitis B virus infection in upper and lower Egypt

The relative prevalence of hepatitis B surface antigen (HBsAg), anti-HBs, and anti-hepatitis B virus core antigen (HBc), as markers of hepatitis B virus infection, among 1,866 apparently healthy residents of two Egyptian provinces representing Upper and Lower Egypt populations was determined using solid-phase radioimmunoassay (SPRIA). The prevalence rate of HBsAg in the Egyptian population was moderately high (10.1%); it was higher in the Upper Egypt (11.7%) than the Lower Egypt (8.0%) population and more frequent in young adults--especially those of Upper Egypt--and males than females in both populations. The prevalence of anti-HBs gradually increased with age; it was higher in the Lower Egypt (51.1%) than the Upper Egypt (41.7%) population, and it was higher in females than males. A remarkably high infection rate, as shown by the prevalence of anti-HBc, was found in both populations (88.0%), with minor variations depending on age, sex, and geographic area.     

筛选出一株可识别多种变异HBsAg的单克隆抗体及其应用

目的 制备筛选可识别变异表面抗原(hepatitis B surface antigen,HBsAg)的单克隆抗体(monoclonal antibody, mAb).用筛选出的单克隆抗体建立检测变异HBsAg的ELISA实验方法.方法 用血源HBsAg免疫Balb/c小鼠,通过杂交瘤细胞融合技术制备抗-HBs单克隆抗体.不同单克隆抗体包被酶标反应孔,检测真核细胞表达的野生及变异HBsAg,了解各种单克隆抗体的反应模式 .筛选出可以较好识别变异HBsAg的单克隆抗体Hb1,优化该抗体ELISA检测HBsAg的方法,与 8种HBs Ag检测试剂比较检测变异HBsAg的能力.结果 经过筛选,得到一种可以较好识别包括G145R在内大多数变异HBsAg的单克隆抗体.检测变异HBsAg的能力优于市售HBsAg 诊断试剂.结论 用本实验制备的单克隆抗体可以用于ELISA检测变异HBsAg,减少HBsAg变异株的漏检率.