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.     

Evaluation of the blood analyzer Beckman Coulter LH 750: analytic performance, decision rules

We evaluated the new analyzer Beckman Coulter, an instrument dedicated to the cell blood count (CBC) and to the white blood cell (WBC) differential (including nucleated red blood cells (NRBCs)) over a global one month period, with three purposes: 1) evaluation of the analytical performance (precision, reproducibility, contamination, linearity); 2) accuracy of numerical results, by comparison to the laboratory instrument (CBC and WBC diff) or to the blood smear (NRBCs, low platelets); 3) evaluation, in terms of sensitivity and specificity, a set of abnormality messages built from the suspect flags and a few quantitative abnormalities. The analytical performances were found satisfactory. The WBC and platelet ranges of linearity were wider than in the GEN.S, as stated in the system specifications. However, the lack of adequate biological material made impossible the study of the whole mentioned linearity range. The accuracy of the CBC and differential parameters, as well as of reticulocytes, was studied with the Coulter GEN.S as reference instrument. The coefficients of correlation and the regression lines showed that the LH 750 results were similar to the GEN.S results. Furthermore, samples with thrombocytopenia and circulating NRBCs were evaluated and compared to the result obtained with microscopic lecture. The results showed a good relationship between platelets results given by the GEN.S and manual count leading to appropriate decision of transfusion. The correlation between GEN.S and manual count of NRBC was estimated as satisfactory. We used the LH 750 software to create conditional rules on the basis of qualitative and quantitative criteria, in order to define and enter a message system for detection of abnormalities. Our study showed that such a system flagged 95.7% of morphological abnormalities with a rate of unnecessary slide review (absence of any morphological abnormality on the blood smear) estimated at 8.3%. Furthermore, in 86% of the abnormalities studied, the relevant message was triggered. The Beckman Coulter LH 750 thus appeared as suitable in terms of validation efficiency.     

化学发光酶免疫分析法在乙肝病毒及核心抗体定性检测中的应用分析

目的 通过对比,探讨了化学发光酶免疫分析法在乙肝病毒及核心抗体定性检测中的应用价值.方法 选取2013年5月至2015年5月在我院的疑似乙型肝炎患者80例,抽取空腹血液样本后都分别进行乙肝病毒以及核心抗体的化学发光酶免疫分析法及ELISA法检测,并对其检测结果进行分析.结果 化学发光酶免疫分析法检出乙型肝炎病毒阳性78例,检出率为97.5％;而ELISA检出乙型肝炎病毒阳性76例,检出率为95.0％,两种方法的检出率对比差异无统计学意义(P＞0.05).化学发光酶免疫分析法对于乙肝病毒核心抗体IgM与IgG的检测阳性率分别为80.0％和70.0％,而ELISA法检测两种抗体的阳性率则分别为18.8％和20.0％,化学发光酶免疫分析法对乙肝核心抗体IgM与IgG的检测阳性率明显高于ELISA法(P＜0.05).ELISA法检出HBc-IgM的最低限为0.135 IU/ml,检出HBc-IgM最低限为0.143 IU/ml;化学发光酶免疫分析法检出HBc-IgM最低限为0.032 IU/ml,检出HBc-IgG最低限为0.038 IU/ml.结论 化学发光酶免疫分析法在乙型肝炎检测中具有高的检出率,尤其对乙肝病毒的核心抗体的检测敏感度较高,值得在临床推广应用.     

Simultaneous use of poly- and monoclonal antibodies as enzyme tracers in a one-step enzyme immunoassay for the detection of hepatitis B surface antigen.

A one-step third generation enzyme immunoassay (EIA) was developed for the detection of hepatitis B surface antigen (HBsAg) in human serum or plasma using a polyclonal (Pab-HBsAg) and two monoclonal antibodies to HBsAg (Mab1-HBsAg and Mab2-HBsAg). In this assay, the solid phase is coated with Mab1-HBsAg and the specimen is incubated simultaneously with peroxidase labelled Pab-HBsAg and Mab2-HBsAg. If HBsAg is present in a specimen it will form sandwich complexes with capture and tracer antibodies. The hook effect, observed in some HBsAg detection tests when a high concentration of HBsAg is present, was minimized in this assay by increasing the concentration of the peroxidase-labelled Mab2-HBsAg. The sensitivity of this assay for HBsAg/ay and HBsAg/ad subtypes in a standard (2 h incubation) procedure was 0.6 and 0.3 ng/ml and in an overnight (16-22 h incubation) procedure 0.2 and 0.15 ng/ml, respectively. Strong elimination of the hook effect was observed with specimens containing high levels of HBsAg compared with test results using peroxidase-labelled Pab-HBsAg alone as enzyme tracer. This EIA offers a procedure, with a high specificity and wide range of sensitivity for the detection of HBsAg in human sera or plasma.     

Whole-blood counting immunoassay as a short-turnaround test for detection of hepatitis B surface antigen, anti-hepatitis C virus antibodies, and anti-Treponema pallidum antibodies

Whole-blood samples were used for a counting immunoassay (CIA) with the aim of developing a short- turnaround test. After optimization of the CIA, hepatitis B surface antigen (HBsAg), anti-hepatitis C virus antibodies (anti-HCV), and anti-Treponema pallidum antibodies (anti-TP) were detected as efficiently as by an enzyme immunoassay (EIA) with serum samples. The correlations between whole-blood CIA and serum EIA were 99.8, 97.1, and 99.4% for HBsAg, anti-HCV, and anti-TP, respectively. Whole-blood CIA may be of value when rapid screening of many samples is required.     

Evaluation of the Beckman Coulter HmX hematology analyser at a general hospital

The Beckman Coulter HmX is an hematological analyzer designed to provide a complete hematological profile including CBC, WBC differential (diff) and reticulocyte parameters. It has been evaluated in our laboratory over a two weeks period with three purposes: (1) a technical evaluation of the HmX performance, in regard to repeatability, linearity, carry over; (2) a comparison of numerical results (CBC, WBC diff) and analytical performance (flag sensitivity and specificity) with those obtained with the Coulter MaxM in use in our laboratory; (3) an analysis of the flagging algorithms using the blood smear as the reference method. The first part of the evaluation showed that the Beckman Coulter HmX is reproducible, and linear. The comparison between MaxM and HmX showed that the results given by the two instruments are similar and suggested that the Beckman Coulter HmX could replace the current hematology analyzer in use in our laboratory. The comparison of the flag system performance, between the Beckman Coulter HmX and the Coulter MaxM, has been performed with samples from three subgroups of patients (general departments, surgery and intensive care, hematological unit), and showed that the HmX is significantly more sensitive than the MaxM, with an higher global efficiency. The comparison of predictive values also showed a better performance of the HmX. In conclusion, the Beckman Coulter HmX is suited for an use in medium sized hospital laboratories (80 to 150 CBC diff/day), with good technical and analytical performance, a throughput of 75 samples/hr and a workstation allowing data management in accord with quality assurance guidelines in France.     

Evaluation of the performance of four rapid tests for detection of hepatitis B surface antigen in Antananarivo, Madagascar

Four rapid immunochromatographic assays--Determine HBsAg, Virucheck HBsAg, Cypress HBsAg Dipstick and Hexagon HBsAg--for human hepatitis B surface antigen (HBsAg) detection in human serum were evaluated. A collection of reference serum samples (91 HBsAg positive and 109 HBsAg negative) stored at -80 degrees C was used. Sensitivity and positive predictive value (PPV) exceeded 95%, and specificity and negative predictive value (NPV) exceeded 96% for all tests. The Determine HBsAg test performed best in this study, with a sensitivity of 97.8%, a specificity and PPV of 100%, a NPV of 98.2% and an accuracy rate of 99.0%. However, the differences between the tests were not significant. Other factors should therefore also be taken into account by the Ministry of Health in its decision to recommend a particular test: price, availability, delivery time and feasibility of whole-blood testing. The Determine test appears to be the most suitable for Madagascar, based on all these criteria. The use of this test, despite its lower sensitivity, could prevent blood-borne transmission of hepatitis B virus (HBV) in areas with limited resources.     

A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies

Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype.     

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.     

Immunofluorescent technique for the detection of monoclonal internal image anti-idiotypic antibodies of hepatitis B surface antigen

Monoclonal anti-idiotypic antibodies to HBsAg were screened by immunofluorescence for the presence of a subset behaving as the internal image of the original antigen. We describe the technique and the criteria fulfilled to establish that 2/6 monoclonals studied act as the internal images of the a determinant of hepatitis B surface antigen.     

Quantitative detection of hepatitis B surface antigen by chemiluminescent microparticle immunoassay during lamivudine treatment of chronic hepatitis B virus carriers

The usefulness of fully automated chemiluminescent microparticle immunoassay (Architect HBsAg QT) for monitoring serum levels of hepatitis B virus (HBV) during antiviral therapy remains unclear. Using this assay, hepatitis B surface antigen (HBsAg) was measured in 20 patients with chronic hepatitis B before and during lamivudine treatment. At the start of therapy, 12 patients had detectable hepatitis B e antigen (HBeAg) and 8 did not. The median serum HBV DNA level and HBsAg concentration (25th-75th centile) were 7.2 (6.1-7.8) log genome equivalents/ml and 3,932 (1,585-12,330) IU/ml, respectively. The HBsAg concentration was significantly higher in HBeAg positive than in HBeAg negative patients (P=0.031). There was a significant correlation between the HBsAg concentration and HBV DNA level (r=0.490, P=0.027). The HBsAg concentration negatively correlated with patient age (r=-0.395, P=0.085). After the start of lamivudine therapy, HBV DNA levels fell rapidly in all patients. Serum HBsAg concentrations also fell in most patients, but to a lesser extent. When drug-resistant variants emerged, serum HBsAg usually increased before biochemical breakthrough. Although HBV DNA was elevated persistently after the emergence of drug-resistant variants, the increase in HBsAg was transient. In some patients, the increase in HBsAg preceded the increase in HBV DNA. Monitoring of serum HBsAg concentrations with the use of Architect HBsAg QT, in addition to measurement of HBV DNA levels, is helpful for evaluating the response to lamivudine treatment and for the early detection of drug-resistant strains.     

Novel immunoassay for the detection of hepatitis B surface 'escape' mutants and its application in liver transplant recipients

Hepatitis B virus (HBV) strains with mutations in the surface gene are responsible for the failure of prophylaxis with hepatitis B immunoglobulin (HBIG) in a proportion of patients transplanted for HBsAg positive cirrhosis. So far, the emergence and evolution of these 'surface antibody escape' mutants have been studied by DNA sequencing. In this study the use of an immunoassay is described for diagnosis and characterisation of HBV recurrence after liver transplantation (OLT), based on a monoclonal antibody able to recognise both wild-type and mutant HBsAg. Pre- and post-transplant samples from 22 patients transplanted for HBsAg positive cirrhosis were studied: Group A: 12 patients who reinfected the graft despite receiving HBIG; Group B: 6 patients with no HBV recurrence with continuous HBIG; Group C: 4 patients with HBV recurrence without prophylaxis. By running the new assay in parallel with an immunoassay that is susceptible to HBsAg mutants, 4 of 12 cases were identified in Group A with HBV recurrence due to surface antibody escape mutants, whereas in 8 patients this was due to the wild-type HBV. The results from the immunoassays were confirmed in all cases by HBV DNA sequencing. The surface gene mutations in the 4 patients affected codons 144 and 145 and in one of these 4 patients HBV strains with mutations in both codons were detected before and after transplantation. The epitope recognised by the new monoclonal antibody that reacts with both wild-type and mutant HBsAg seems to remain stable in the HBIG-induced HBV mutants. This serological approach allows rapid and cost-effective screening for HBsAg escape mutants in the liver transplant setting and may be helpful in the selection of appropriate prophylaxis.     

Comparative evaluation of commercial enzyme immunoassay kits for detection of hepatitis B seromarkers.

The commercial hepatitis B enzyme immunoassay kits of Abbott Laboratories and Organon Teknika were compared for their sensitivity, specificity, and reproducibility in detecting the hepatitis B seromarkers hepatitis B surface and e antigens and antibodies to hepatitis B core, e, and surface antigens. With the exception of the Organon kit for antibodies to hepatitis B surface antigen, the specificity and reproducibility were about the same for both products, but the level of sensitivity was generally lower for the Organon kits; this, however, may not be critical in routine clinical application. The Organon kits have a longer shelf life and are cheaper.     

Enzyme-immunoassay in the detection of hepatitis B surface antigen.

A new enzyme-immunoassay (EIA, Hepanostika Microelisa System) for the detection of hepatitis B surface antigen was evaluated against other methods, namely complement fixation, Hepanosticon, AusRIA II and Finnish Red Cross Radioimmunoassay (FRC-RIA). EIA detected the greatest number of positive samples in a serum panel consisting of 142 sera from clinical hepatitis patients. FRC-RIA was the most sensitive method for subtype ad, while EIA detected the ay specimen at the highest dilution. None of the test systems gave the 'optimal' result in the screening test, and it is proposed that a separate procedure for each antigen subtype should be carried out to detect the greatest number of positive samples.     

Radioimmunoassay for detection of hepatitis B surface antigen

A modification of radioimmunoassay (RIA) for detection of HBsAg is described. The schedule for purification and the method of iodinization of purified HBsAg are presented. The sensitivity of RIA and other immunological methods for detection of HBsAg was analysed comparatively. RIA in the modification described detected HBsAg in a concentration of 50 mg/ml.     

Chemiluminescence-linked immunoassay for detection of mumps virus antibodies.

We have developed a simple and sensitive chemiluminescence-linked immunoassay (CLIA) for determining mumps virus antibodies. Luminol molecules were used as markers, and polystyrene balls were used as antigen carriers. The CLIA was compared with an enzyme-linked fluorescence assay and a hemagglutination inhibition test on a total of 40 serum specimens obtained from 29 donors with natural infection or vaccination. There was good correlation between the three methods, and the sensitivity of the CLIA was about 10 times higher than that of the hemagglutination inhibition test, although it was slightly inferior to that of the enzyme-linked fluorescence assay. Moreover, the time course of light emission from the labeled antibody was rapid, and therefore in the CLIA the quantitation of the marker takes only a short time.     

New radioimmunoadsorbent method for detection of hepatitis B surface antigen.

A radioimmunoadsorbent assay for the detection of hepatitis B surface antigen (HBsAG) is described. The method uses small DEAE-cellulose columns to adsorb HBsAg from serum or plasma samples, and it uses 125 I-labeled antibody to HBsAg to detect the adsorbed antigen. A single 2-h incubation at 45 degrees C is used in the procedure. The sensitivity of the method was determined with the Bureau of Biologics HBsAg reference panels and was shown to be equivalent to other third-generation test methods. The specificity of the method was evaluated by testing specimens from blood donors, dialysis patients, and hospital staff in parallel with commercial radioimmunoassay kits for HBsAg. In the tests performed on 2,868 blood donor specimens, 6 positive specimens were identified by both the radioimmunoadsorbent method and the commercial radioimmunoassay kits. These positive specimens were confirmed by a counterimmunoelectrophoresis procedure. One nonconfirmable, repeatably positive specimen was observed with the radioimmunoadsorbent method. In testing 1,250 specimens from dialysis patients and hospital staff, 120 confirmable positive specimens were identified by both the radioimmunoadsorbent method and a commercial test. Overall, the false positive rate for the radioimmunoadsorbent method was found to be less than 1%.     

An enzyme immunoassay for detection of IgA class antibodies against hepatitis delta virus

A sensitive and reproducible enzyme-linked immunoassay (ELISA) for IgA class antibodies against the Delta antigen (HDAg) is described. Specificity of the assay was demonstrated by the absence of binding to an unrelated antigen or to uncoated plates and the finding that binding to HDAg was independent of total IgA concentrations in sera. Positive results were obtained with sera from 11 of 14 patients with chronic Delta virus infection (seropositive for HBsAg and IgM anti-HDAg, negative for IgM anti-HBc) at serum dilutions of up to 1:10(6). Sera from four normal healthy individuals and from 25 patients with chronic hepatitis B or other liver disorders who had no evidence of exposure to HDV were all negative in the assay.     

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.     

False-positive results with third-generation monoclonal hepatitis B surface antigen enzyme immunoassay.

Nonspecific hepatitis B surface antigen reactions with a third-generation enzyme immunoassay (Auszyme Monoclonal; Abbott Laboratories, North Chicago, Ill.) were investigated with 9,577 serum specimens in a clinical laboratory setting. Of the 196 serum specimens found reactive in Auszyme screen by the overnight procedure, 103 turned out to be true-positives, 71 were nonrepeatably reactive, and 22 were repeatably reactive but actually falsely positive (false-positive rate, 22 of 196, or 11.2%). Verification of the 196 screen reactives by the Auszyme 3-h incubation assay detected all but 4 true-positives, with a false-negative rate of 3.9% (4 of 103), and was negative for the rest. These observations reinforce the need for retesting all reactive specimens and confirming repeatedly reactive samples when the Auszyme Monoclonal test is used to detect hepatitis B surface antigen.