Diagnostic performance of commercial serological assays measuring Bordetella pertussis IgG antibodies

Due to their specificity to B. pertussis antigens, immunoglobulin G (IgG) antibodies should be measured primarily for diagnosing pertussis. We compared the diagnostic performance of commercially available enzyme-linked immunosorbent assays (ELISAs) and chemiluminescent immunoassays (CLIAs) measuring IgG to B. pertussis antigens. An in-house ELISA with purified pertussis toxin (PT) was used as reference system. Commercial assays using PT only as coating antigen showed better performance as compared to those using a mixture of different antigens. The best diagnostic performances were achieved by CLIAs. Results were analyzed using a dual cutoff of either ≥125 IU/mL anti-PT IgG or ≥62 IU/mL anti-PT IgG for the in-house ELISA and accordingly to package inserts for commercial assays. Using the in-house ELISA at a 62 IU/mL cutoff, as the gold standard for interpretation of results from the commercial kits, resulted in lower sensitivity and higher specificity as compared to 125 IU/mL, thus, it may be especially useful in outbreak situations when high specificity is required.     

Sample commutability in external quality assessment surveys (EQAS) for thyroid-related antibodies--state of the art

External quality assessment surveys for thyroid-related antibodies have been offered by INSTAND for 20 years. During this time, some problems have remained, especially those between the similarity of samples sent and routine patient samples. Here the questions of "matrix effects" and "commutability of results" are topics discussed at most EQA-meetings. This short communication deals with the effects of lyophilization on results for thyroid-associated antibodies in an EQA-survey carried out by over 230 participants in October 2005 by INSTAND in Düsseldorf, Germany. The results show that there are small but statistically significant differences (at the level p < 0.05) in results from liquid and lyophilized samples from the same source, the tendency to higher results in the lyophilized samples for anti-TPO and anti-Tg and to lower results for TRAb. The precision in measurement was significantly better for anti-TPO and TRAb in the lyophilized samples, there being no significant difference for anti-Tg. The differences in results between liquid and lyophilized samples were minimal when compared with the numerical results for anti-TPO and anti-Tg, despite the fact that all kits were calibrated with the NIBSC reference materials, although the latter are now both 40 years old.     

Severe neonatal alloimmune thrombocytopenia caused by antibodies to human platelet antigen 3a (Baka) detectable only in whole platelet assays

BACKGROUND: Maternal antibodies that cause neonatal alloimmune thrombocytopenia are commonly identified by solid-phase assays that detect the causative antibodies on the basis of their reactions with specific PLT glycoproteins. Two cases of severe neonatal alloimmune thrombocytopenia caused by maternal antibodies specific for human PLT antigen 3a (HPA-3a [Baka]) that failed to give the expected reactions in some solid-phase assays were recently encountered. STUDY DESIGN AND METHODS: PLT-reactive antibodies were characterized by three different solid-phase assays and by flow cytometry. RESULTS: The two maternal antibodies gave negative reactions in the antigen capture ELISA, modified antigen capture ELISA, and MoAb immobilization of PLT antigens tests but reacted strongly in flow cytometry with intact PLTs that were HPA-3a+. Other sera samples specific for HPA-3a reacted equally well in all assays. CONCLUSIONS: The two antibodies appear to recognize an epitope on the HPA-3a+ form of glycoprotein IIb that is lost when PLTs are solubilized in detergent, as required for solid-phase assays. The diagnosis was made in these cases because no HLA antibodies were present, allowing an HPA-3a-specific reaction to be identified with intact PLTs as targets. Such antibodies are likely to be overlooked when HLA antibodies are also present.     

Comparison of six commercial tick-borne encephalitis IgM and IgG ELISA kits and the molecular characterization of their antigenic design

Tick-borne encephalitis virus (TBEV) diagnosis is mainly based on the detection of viral-specific antibodies in serum. Several commercial assays are available, but published data on their performance remain unclear. We assessed six IgM and six IgG commercial enzyme-linked immunosorbent assay (ELISA) kits (ELISA-1 through ELISA-6) using 94 samples, including precharacterized TBEV-positive samples (n=50) and -negative samples (n=44). The six manufacturers showed satisfactory sensitivity and specificity and high overall agreement for both IgM and IgG. Three manufacturers showed better reproducibility and were the most sensitive (100%) and specific (95.5–98.1%) for both IgM and IgG. Two of them were also in agreement with the clinical interpretation in more than 90% of the cases. All the assays use inactivated virus as antigen, with strains showing approximately 94% homology at the amino acid level. The antigenic format of the assays was discussed to further improve this TBEV diagnostic tool.     

Serological diagnosis of Helicobacter pylori infection

H. pylori gastritis elicits both a local and a systemic immune response. Although multiple invasive tests are available for the diagnosis of H. pylori infection, serum testing for anti-H. pylori antibodies is a minimally invasive test available to diagnose this infection. Latex agglutination is available, but most kits use ELISA. The sensitivities and specificities of many commercial kits were more than 90%. However, most of these serologic detection kits were made in Western countries. There might been the influence of geographical differences on test results. Recently, serological test has been reported about of usefulness in monitoring after eradication therapy at long-term period.     

New highly sensitive sandwich ELISA system for soluble endoglin quantification in different biological fluids

Soluble endoglin (sEng) is a fragment of a membrane-associated receptor (CD105) expressed on endothelial cells, mesenchymal stem cells and trophoblast cells. It is considered as a regulatory factor involved in the development of preeclampsia (PE) and cancer-associated neo-angiogenesis. The purpose of this study was to describe a new sandwich ELISA for sEng quantification. In contrast to three commercial kits, the new ELISA was able to quantify sEng not only in blood plasma and cell culture supernatants, but also in urine and cerebrospinal fluid. The assay detected up to two orders of magnitude higher antigen levels than did the commercial kits. Using Western blot assay followed by SDS-PAGE or Blue Native PAGE, we demonstrated a heterogeneous nature of sEng molecules. Antigen heterogeneity is considered as a factor contributing to the pronounced differences in its content estimations by different ELISAs. We obtained evidence indicating that the assay was capable of detecting heterogenious sEng molecules. The new ELISA was validated as a quick, specific and accurate method for sEng quantification. Despite the differences in antigen content estimates, the assay had similar diagnostic performance as widely used commercial kit for the detection of severe PE in pregnant women based on plasma sEng contents. Moreover, the new assay was able to delineate diseased patients based on antigen levels in urine. Therefore, the new ELISA is a potentially valuable tool for in vitro and clinical studies.     

Prevalence of non-A, non-B hepatitis/hepatitis C virus antibody in laboratory quality-assurance sera.

Quality-assurance sera (QAS) are prepared from pooled sera composed of thousands of individual donations. Previous studies documented that a substantial percentage of individual QAS test positive for viral disease markers, including antibodies to human immunodeficiency virus and to hepatitis B surface antigen. We tested 239 QAS from various proficiency programs and commercial sources to determine the prevalence of hepatitis C virus (HCV) antibody. We tested samples for anti-HCV by using an enzyme immunoassay (EIA; Abbott Labs.) and an enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostics). We observed an overall positive rate of 49% by one or both assays in all categories of sera tested. In addition, we found a greater rate of positivity (58%) in proficiency program samples than in commercial samples (43%). We found discrepant results between the two assays for 15 of 239 samples (6%). In the discrepant samples, the EIA result was positive, whereas the ELISA result was negative. Anti-HCV positivity in QAS has important implications for laboratory personnel handling these samples.     

An undetectable source of technical error that could lead to false negative results in enzyme linked immunosorbent assay of antibodies to HIV-1

Since the institution of routine testing for antibodies to Human Immunodeficiency Virus (HIV) using the enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of this assay system has received significant scrutiny. During previous use of this methodology, we have quantified rates of false biological positive results using commercial kit assays in a normal donor population. In this study, we have identified a potential source for false negative results. Using multiple lots of two different commercial ELISA kits, the absorbance readings at the test end point could not differentiate between normal non-reactive donor samples and blanks containing no sample. These results occur using normal donor samples, even though the assays could distinguish between blank wells and the manufacturers' "normal controls", provided with the assay. Our findings suggest that a technical pipetting error is presently undetectable, either visually or by statistical methods, and could permit an untested, potentially HIV-1 positive, unit to be released into the transfusable blood supply. A possible solution is suggested.     

The serology of chronic hepatitis B infection revisited.

The serology of chronic hepatitis B infection has been established through the use of commercial immunoassays to measure the structural antigens of the hepatitis B virus and their respective antibodies in serum. However, the commercial assays have not been designed to detect serum antibodies in the presence of an excess of circulating antigens. A series of serum samples from 200 HBeAg-positive, chronically infected hepatitis B patients with varying degrees of liver disease were analyzed using novel immunoassays designed to detect antibodies in the presence of circulating viral antigens. All patients, regardless of their liver disease, were seronegative for antibodies specific for the envelope antigens or the secreted nucleoprotein antigen (HBeAg) when the commercial assays were used. In contrast, virtually all chronically infected patients with liver disease and approximately 50% of patients without liver disease demonstrated anti-HBe and anti-envelope antibodies when sera were tested in the more sensitive immunoassays. Furthermore, asymptomatic patients could be serologically distinguished from symptomatic patients based on antibody fine specificity, titer, and IgG subclass. This study revealed that the majority of chronically infected hepatitis B patients produce a variety of antibodies for many years, and are not immunologically unresponsive, as suggested by the current assays.     

Development of a recombinant hepatitis B immunoglobulin derived from B cells collected from healthy individuals administered with hepatitis B virus vaccines: A feasibility study

Background

In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg).

Study Design and Methods

B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein–Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG₁ expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry.

Results

Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells.

Discussion

We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful.     

Specificity of an assay for antibodies to hepatitis B surface antigen

Eight percent of blood donors were found to have antibodies to hepatitis B surface antigen when tested by radioimmunoassay using commercial test kits. Four percent of positive reactions proved nonspecific when tested by the neutralization test using pooled surface antigen. The risks in relying solely on the reactivity in commercial- kit suggested that the specificity of the assay must be confirmed.     

Clinical significance of ELISA positive and immunofluorescence negative anti-dsDNA antibody

BACKGROUND: Positivity for anti-dsDNA antibody is a diagnostic criterion of systemic lupus erythematosus (SLE). In the present study, the significance of ELISA positive and Crithidia luciliae immunofluorescence test (CLIFT) negative anti-dsDNA sera was evaluated. METHODS: There were 371 consecutive serum samples submitted to for anti-dsDNA testing that were assayed using anti-dsDNA ELISA and CLIFT. Sera showing discrepant results were collected and then examined using 3 commercial anti-dsDNA and anti-ssDNA ELISA kits and by Farr assay. Medical records were reviewed for those patients who were ELISA positive and CLIFT negative for anti-dsDNA. RESULTS: Fifty-two patients of 100 anti-dsDNA ELISA positive patients were negative by CLIFT. For ELISA positive and CLIFT negative sera, Farr assays showed the highest positive rate (72.7%) for the 4 different anti-dsDNA assays (3 commercial kits and the Farr assay). Nearly 80% of 44 ELISA positive and CLIFT negative patients met >or=3 of the SLE classification criteria (excluding the anti-dsDNA criterion). CONCLUSION: Some anti-dsDNA ELISA kits have diagnostic efficiencies that are similar to that of the Farr assay. Moreover, the study identifies a group of patients that are ELISA positive but CLIFT negative for anti-dsDNA, and indicates that the majority of these patients have clinically relevant SLE.     

A comparison of ELISA assays as routine diagnostic test for detection of autoantibodies against extractable nuclear antigens.

OBJECTIVE: In an analytical evaluation, commercially available ELISA test kits for detection of antibodies directed against extractable nuclear antigens (ENA) were compared with the currently used combination of counterimmunoelectrophoresis and immunoblotting. DESIGN: Three screening ELISAs and two typing ELISAs were tested. These methods were fairly simple, easy to perform and "user friendly," because most of the reagents were ready to use. RESULTS: The agreement with the current methods was good, but the screening as well as typing ELISAs proved to be more sensitive, especially with regard to detection of SS-A auto-antibodies. The cut-off range of one screening assay was not well established and one typing assay suffered from problems with inaccuracy of package insert, purity of antigen and standardisation of reactivity (possibly caused by differences in amount of coated antigen). The other three ELISAs were reliable and sensitive for detection of ENA auto-antibodies. CONCLUSIONS: The ELISA ENA screen assays ENA-LISA polyvalent and Milenia ENA screen and typing assays ENA-LISA are reliable and sensitive for detection of autoantibodies in clinical specimens without substantial false negatives.     

Simple histidine-rich protein 2 double-site sandwich enzyme-linked immunosorbent assay for use in malaria drug sensitivity testing

A simple double-site sandwich enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum in vitro drug sensitivity tests based on measuring histidine-rich protein 2 (HRP2) is presented. The ELISA uses two commercial monoclonal antibodies and provides a drastically cheaper alternative to the test kits previously used in the HRP2 drug sensitivity test. The assay is simple to establish and perform. The sensitivity is comparable and the drug sensitivity results very closely match those obtained with the commercial ELISA kits (R(2) = 0.979; P < 0.001; mean log difference at the 50% inhibitory concentration = 0.07).     

国产化学发光免疫试剂检测血清HBsAg的应用评价

目的考核国产化学发光免疫分析试剂检测血清HBsAg的效果,探讨其在血液筛查和临床检测应用中的可行性。方法采用国产化学发光免疫分析试剂筛查345份HBsAg确认阳性的血清标本,比较其与常用的进口和国产酶联免疫检测试剂盒的漏检率;并采用不同浓度的标准HBsAg血清对国产化学发光免疫分析试剂进行线性分析和精密性试验。结果国产化学发光免疫分析检测试剂的漏检率高于Murex和Organon酶联免疫试剂盒,差异有统计学意义（P〈0．01）,而与国产英科新创（厦门）科技公司和上海科华生物技术有限公司的HBsAg酶联免疫诊断试剂盒的差异无统计学意义;采用标准血清进行线性分析,标本浓度在（0．5～150）ng／ml范围时与化学发光单位量（RLUs）呈良好的线性正相关;分别对低、中、高浓度标准血清进行板内和板间的重复检测,其反应具有良好的重复稳定性。结论化学发光免疫分析法检测血清HBsAg具有快速、可定量、反应稳定的特点,可用于血液筛查和临床标本的检测。     

抗乙型肝炎病毒表面抗原抗体国产ELISA试剂评价

目的　评价 6种国产抗乙型肝炎病毒表面抗原抗体 (抗 HBs)酶联免疫吸附试验 (ELISA)试剂盒。方法　将RocheElecsys 2 0 10电化学发光免疫分析系统上检测余留的抗 HBs标本 ,用 6种国产抗 HBsELISA试剂盒进行检测 ,评价 6种试剂盒的敏感度、检测范围、精密度和特异性。结果　 6种国产试剂盒的敏感度相差较大 ,10～ 4 0IU/L不等。 5 0IU/L以下时 ,浓度与吸光度呈直线关系 ,但吸光度数值较低 ;未发现高浓度时的钩状效应。 5 0IU/L的标本 ,A、B 2种试剂的批内变异系数 (CV)分别为 10 .1%和 13.7% ;日间CV分别为 14 .1%和2 4 .8% ;阴性标本在 6种试剂盒中出现了不同程度的假阳性。结论　国产抗 HBsELISA试剂的部分性能应改进和提高。     

接种甲型H1N1流感疫苗献血者血清中血凝素IgG抗体的检测

目的研制甲型H1N1流感(甲型流感)血凝素(HA)IgG抗体(抗-HAIgG)检测试剂,并用于对献血人群中甲型流感疫苗(甲流疫苗)接种者的抗-HAIgG检测。方法克隆表达甲型流感HA表位抗原(18-243aa),制备出酶联免疫吸附法(ELISA)抗-HA试剂,并对106份甲型流感流行前的献血者血清(甲型流感前组)、97份接种甲流疫苗后的献血者血清(接种甲流疫苗组)做抗-HAIgG检测。结果甲型流感前组:4例抗-HAIgG为阳性,102例阴性,阳性率为3.77%(4/106);甲流疫苗组:78例抗-HAIgG为阳性,19例阴性,阳性率为80.41%(78/97)。结论ELISA法检测甲型流感抗-HAIgG试剂具有较高的特异性、灵敏度和安全性,其在献血人群中的甲型H1N1病毒感染的流行病学调查、疫苗免疫血浆的筛选方面,具有一定的应用价值。     

Serologic testing for human immunodeficiency virus antibodies

Familiarity with available serologic tests for antibodies to human immunodeficiency virus (HIV) has become increasingly important in a wide variety of clinical settings. Enzyme-linked immunosorbent assay (ELISA) commercial kits are most often used as Enzyme-linked immunosorbent assay (ELISA) commercial kits are most often used as screening tests, and Western blot techniques are used for confirmation of positive results. ELISA specificity and sensitivity exceed 98%; the predictive value of a positive test varies from 2% for a weakly positive test in a low-prevalence population to 99% for a strongly positive test in a high-risk group. Confirmatory Western blot testing identifies antibodies with affinity for specific HIV antigens. Indeterminate Western blot antibody patterns necessitate subsequent testing or alternative methods for interpretation. A "window" period of up to 3 or more months follows acute HIV infection before seropositivity occurs.     

乙型肝炎血清学标志间非特异性交叉反应的研究

为证实乙型肝炎（乙肝）血清学标志酶联免疫吸附试验（ＥＬＩＳＡ）试剂中五种抗原抗体反应的特异性，采用排列组合的交叉反应方式，对Ａｂｂｏｔｔ和部分国产试剂盒中五种抗原抗体进行交叉反应研究，结果表明：所有生产商的乙肝血清学标志ＥＬＩＳＡ试剂盒中，酶标记抗－ＨＢｅ和包被的ＨＢｃＡｇ都存在交叉反应，大部分生产商的抗－ＨＢｃ与ＨＢｅＡｇ也存在交叉反应。实验证实抗－ＨＢｅ和抗－ＨＢｃ之间存在非恃异性交叉反应。考虑临床上抗－ＨＢｃ和抗－ＨＢｅ检测阳性率较高，非特异性交叉反应可能是重要原因。     

Influence of the macromolecular form of a B cell epitope on the expression of antibody variable and constant region structure

We investigated the influence of the macromolecular form of an epitope on the structure of antibody variable and constant regions expressed by the B cell population participating in an immune response to that epitope. Hybridomas were constructed from strain A/J mice undergoing either primary or secondary immune responses to p-azophenylarsonate conjugated to Brucella abortus (Ars-Bruc). We determined the sequences of the V genes expressed by hybridomas selected on the basis of expression of a single VH gene segment known to encode a large family of anti-Ars antibodies. These sequences were compared with the sequences of V genes expressed by a previously characterized panel of hybridomas isolated in the same way during the primary and secondary responses of A/J mice to Ars-KLH. The repertoire of Ars-specific V domains expressed among primary and secondary hybridomas elicited with these two forms of Ars were similar, as were the differences between primary and secondary V region somatic mutational alteration and affinity for Ars. In contrast, predominant expression of IgG2 anti-Ars antibodies was elicited in the secondary Ars-Bruc response, whereas secondary anti-Ars antibodies elicited with Ars-KLH are predominantly IgG1. Thus, differences in the macromolecular form of Ars clearly influence the isotypic profile of the anti-Ars response, but the expression, diversification, and selection of V domains elicited with this hapten are not greatly affected by such differences. Our results suggest that while isotype regulation is highly perceptive of the macromolecular form of a B cell epitope, V region regulation is primarily influenced by the molecular structure of that epitope.