Evaluation of four commercially available Epstein-Barr virus enzyme immunoassays with an immunofluorescence assay as the reference method

Four commercially available enzyme immunoassays (EIAs) (Novitec, Biotest, Virotech, and DiaSorin) were evaluated, with an indirect immunofluorescence assay as the reference method, for Epstein-Barr virus (EBV) VCA (viral capsid antigen) immunoglobulin G (IgG), VCA IgM, or EBNA (EBV nuclear antigen) IgG at three different locations (Homburg, Stuttgart, and Dresden). Serum samples from 66 immunocompetent patients with infectious mononucleosis, 73 patients without prior EBV infection, and 96 patients with past EBV infections and 29 serum samples with possible cross-reactions to other herpesviruses were included. In addition, 25 samples from an extensively pretested panel that is commercially available (Boston Biomedica) were tested. Each sample was tested at only one location. The four EIAs varied considerably in performance. When analyzing for EBV diagnosis, the Novitec assay performed the best, with 4.9% discrepant diagnoses, followed by the Biotest, Virotech, and DiaSorin assays, with 6.8, 11.7, and 14.0% discrepant diagnoses, respectively. On the basis of single-parameter analysis, the Novitec assay also showed the lowest number of discrepant results, with 3.5%, compared with the Virotech, Biotest, and DiaSorin assays, which produced 5.4, 6.4, and 8.6% discrepant results, respectively. VCA assays using affinity-purified native antigens performed better than assays with recombinant or synthetic antigens. The synthetic EBNA-1s showed the lowest concordance with the reference compared to recombinant p72. Commercially available EBV EIAs differed considerably in performance; however, some proved to be reliable and convenient alternatives to the indirect immunofluorescence assay for routine diagnostics. Native antigens, rather than synthetic peptides, are favored for EBV serology testing.     

Evaluation of an indirect immunofluorescence assay for strongyloidiasis as a tool for diagnosis and follow-up

The diagnostic accuracy of an indirect immunofluorescence antibody test (IFAT) for Strongyloides stercoralis at different serum antibody titers was evaluated. To assess diagnostic sensitivity, sera from 156 patients with known strongyloidiasis were collected. Negative control sera were obtained from a composite group of 427 subjects (blood donors and hospitalized patients). With an area under the receiver-operating characteristic plot of 0.98, the IFAT showed a high level of diagnostic accuracy for strongyloidiasis. An antibody titer of > or = 1:20, with 97% sensitivity and 98% specificity, was identified as the diagnostic threshold with the best overall performance. Cross-reactions were evaluated with 41 additional samples from patients with other known helminth infections, and the IFAT detected low-titer positivity in only one subject with filariasis. A positive IFAT result at an antibody dilution of > or = 1:80 was virtually 100% specific, with 71% sensitivity. To test the usefulness of the IFAT as a monitoring tool, the changes in specific-antibody titers after treatment in a group of 155 patients were evaluated. Seroreversion or a decrease in antibody titer of twofold or more was observed in 60% of the patients. Response to treatment was directly correlated to the initial antibody titer, and a baseline titer of > or = 1:80 was identified as the best predictor of response. In conclusion, a positive IFAT result at an antibody dilution of >/=1:20 is the optimal cutoff for screening. A titer of > or = 1:80, with virtually no false-positive result, is a reliable cutoff for a serological assessment of treatment efficacy and for inclusion in clinical trials.     

Evaluation of a recombinant antigen ELISA for the diagnosis of acute toxoplasmosis and comparison with traditional antigen ELISAs.

An evaluation of five ELISA methods for the diagnosis of acute toxoplasmosis was undertaken by comparing a laboratory-produced ELISA employing two recombinant Toxoplasma gondii polypeptides as antigen with a laboratory-produced ELISA and three commercially available ELISAs employing traditional parasite antigen preparations derived from whole tachyzoites. With a panel of 75 sera from patients who showed either serological and clinical evidence of acute toxoplasmosis, or of diseases caused by other infectious agents, or from patients who showed no evidence of infectious disease, the ELISAs gave overall positive predictive values of 81.6-100%, negative predictive values of 87.8-100%, sensitivities of 81.3-100%, and specificities of 83.7-100%. No two ELISAs gave identical results with all sera tested. In total, the ELISA based on the two recombinant T. gondii polypeptides appeared to be the most specific ELISA in this comparison, showing positive predictive values and specificities of 100% for all groups of patients tested. The overall negative predictive value for this ELISA was 87.8% and the sensitivity was 81.3%. Therefore, the ELISA based on recombinant antigen appears to be a promising advance in the serological diagnosis of acute toxoplasmosis.     

Evaluation of a reverse line blot assay for genotyping common human rotaviruses

To allow high-throughput genotyping of group A rotaviruses (RVA) in a routine surveillance setting, we developed a reverse line blotting method for the determination of the most common human RVA G- and P-genotypes: G1–G4, G9, G12, P[4], P[6] and P[8]. Using the reverse line blotting method on 951 clinical RVA positive feces samples, in 905 (95%) of the samples the G-genotyping yielded a result while in 945 (99%) of the samples the P-genotyping was successful. Comparison of the reverse line blotting-method as it is used currently to a sequence based method for genotyping RVAs showed an agreement of 96% for single strain infections (75 out of 78) but only 48% for mixed infections (10 out of 21).     

An immunofluorescence method for the diagnosis of primary syphilis using an absorption technique

An immunofluorescence technique for demonstrating Treponema pallidum in fixed films made from suspected chancre exudate is described. The method used is basically a reversal of the fluorescent treponemal antibody absorption test. It makes use of the fact that by using a syphilitic serum from which group antibody has been absorbed by an extract of Reiter treponemes, leaving only specific antibody, Treponema pallidum can be indentified. Neither the non-pathogenic genital treponemes nor mouth treponemes showed any fluorescence when tested by the technique described.All the reagents used in the test are available commercially, thus placing it within the scope of any laboratory with suitable fluorescence equipment.In the present state of knowledge of the treponematoses, this technique would seen to be the most accurate means of diagnosing primary syphilis because it permits the specific identification of Treponema pallidum in chancre exudate. It has the advantage over the dark-ground method that fixed films can be sent to the laboratory.     

建立产生gfp报道基因重组EB病毒细胞株

目的 建立产生带有gfp报道基因的重组EB病毒株。方法 构建一个有EBV DNABamHⅠ的C和H片段，gfp报道基因和puromycin选择基因的真核细胞穿梭质粒（pGFPUROEB)；采用电穿孔法将其转染于B95-8细胞中，并用荧光显微镜和流式细胞仪检测gfp的表达，PCR及Southern blot检测重组gfp EB病毒基因。结果 经puromycin的长期筛选。建立80%以上表达gfp的B95-8细胞株（称gfpB95-8),PCR检测有gfp基因，经Southern blot检测证实gfpB95-8可产生重组gfp的EB病毒。结论 构建了gfp报道基因和puromycin选择基因的真核细胞穿梭质粒；并成功建立产生带有gfp报道基因的重组EB病毒株。     

Evaluation of culture, immunofluorescence, and serology for the diagnosis of pertussis.

Nasopharyngeal culture, direct immunofluorescence, and serology of acute-phase and paired serum specimens were compared for the laboratory diagnosis of infections due to Bordetella pertussis in a community-based pediatric population with both high vaccine usage and high pertussis incidence. In 77 (37%) of 210 patients evaluated, one or more tests were positive for pertussis. A clinical illness compatible with pertussis was present in 52 (71%) of 73 pertussis test-positive and 42 (35%) of 119 test-negative patients (P less than 0.001). Nasopharyngeal culture was of low sensitivity (20 [26%] of 77 positive tests) but was most commonly confirmed by another positive pertussis test (85%). Direct immunofluorescence was both insensitive and nonspecific; only 6 (30%) of 20 cases positive by culture were positive by immunofluorescence, and only 4 (33%) of 12 of the culture-negative, immunofluorescence-positive cases could be confirmed by another positive pertussis test. Although serology by enzyme immunoassay proved to be the most sensitive of the laboratory tests (87%), this sensitivity could be achieved only by assaying both acute-phase and paired serum specimens and measuring immunoglobulin G (IgG), IgA, and IgM antibodies to two pertussis antigens (pertussis toxin and filamentous hemagglutinin). Loss of sensitivity occurred with any reduction in the number of these serologic assays performed. Optimal laboratory diagnosis of endemic pertussis in a pediatric population requires both nasopharyngeal culture and serology by enzyme immunoassay.     

Evaluation of a modified reverse line blot assay for detection and typing of human papillomavirus

Detection and typing of human papillomaviruses (HPVs) are requested by clinicians with growing frequency as part of the overall management of patients. A new method using consensus L1 amplification and a reverse line blot hybridization detection method has been described for broad spectrum HPV genotype identification. This method involves hybridization at 53 degrees C in a shaking water bath, which many laboratories might find cumbersome. We describe a modified hybridization step, using a dry-air incubator, that simplifies the detection protocol. Overall, comparable results were obtained by the modified, more easily performed protocol, indicating that laboratories might validate and use this modified method.     

Validation of a recombinant based antibody ELISA for diagnosis of human and canine leishmaniasis

In this study, a recombinant chimeric antigen (CA) ELISA was validated as a single test for both human and dog leishmaniasis. Serum panels included 327 human and 339 canine IFAT-positive and 1113 human and 1078 canine IFAT-negative samples. CA-ELISA was carried out using the same serum dilution, and labelled protein A as secondary reagent. Test performances were calculated using ROC analysis. For the human panel, the test showed diagnostic accuracy (DA) 0.974, specificity (Sp) 97.12%, sensitivity (Se) 91.44%, and concordance (K) 0.88. The dog panel showed DA 0.998, Sp 99.54%, Se 98.54%, and K 0.98. The proposed method is the best recombinant antigen-based ELISA, and can be used as IFAT substitute for mass screening.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) with affinity-purified Em18 and an ELISA with recombinant Em18 for differential diagnosis of alveolar echinococcosis: results of a blind test

Alveolar echinococcosis (AE) is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE) is also endemic. Both AE and CE are highly endemic in China, and both serologic detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems. Evaluation of Western blot analysis (WB) and enzyme-linked immunosorbent assay (ELISA) for the Em18 antigen, using affinity-purified and recombinant Em18, was carried out "blindly" using 60 human sera from patients diagnosed in France. The results were compared with those obtained using a commercially available Echinococcus WB immunoglobulin G (IgG) kit developed in France. The Em18 WB and Echinococcus WB IgG showed very similar results for detection of AE. Both affinity-purified Em18 or a recombinant Em18 WB and Echinococcus WB IgG seem useful for identification of AE, and the latter seems appropriate for both AE and CE, whereas affinity-purified Em18 ELISA and the newly developed recombinant Em18 ELISA appear to be suitable for detection of AE, especially for epidemiological surveys.     

Evaluation of Four Commercially Available Epstein-Barr Virus Enzyme Immunoassays with an Immunofluorescence Assay as the Reference Method

Four commercially available enzyme immunoassays (EIAs) (Novitec, Biotest, Virotech, and DiaSorin) were evaluated, with an indirect immunofluorescence assay as the reference method, for Epstein-Barr virus (EBV) VCA (viral capsid antigen) immunoglobulin G (IgG), VCA IgM, or EBNA (EBV nuclear antigen) IgG at three different locations (Homburg, Stuttgart, and Dresden). Serum samples from 66 immunocompetent patients with infectious mononucleosis, 73 patients without prior EBV infection, and 96 patients with past EBV infections and 29 serum samples with possible cross-reactions to other herpesviruses were included. In addition, 25 samples from an extensively pretested panel that is commercially available (Boston Biomedica) were tested. Each sample was tested at only one location. The four EIAs varied considerably in performance. When analyzing for EBV diagnosis, the Novitec assay performed the best, with 4.9% discrepant diagnoses, followed by the Biotest, Virotech, and DiaSorin assays, with 6.8, 11.7, and 14.0% discrepant diagnoses, respectively. On the basis of single-parameter analysis, the Novitec assay also showed the lowest number of discrepant results, with 3.5%, compared with the Virotech, Biotest, and DiaSorin assays, which produced 5.4, 6.4, and 8.6% discrepant results, respectively. VCA assays using affinity-purified native antigens performed better than assays with recombinant or synthetic antigens. The synthetic EBNA-1s showed the lowest concordance with the reference compared to recombinant p72. Commercially available EBV EIAs differed considerably in performance; however, some proved to be reliable and convenient alternatives to the indirect immunofluorescence assay for routine diagnostics. Native antigens, rather than synthetic peptides, are favored for EBV serology testing.     

Rapid diagnosis of extensively drug-resistant tuberculosis by use of a reverse line blot hybridization assay

Drug resistance in tuberculosis (TB) is a matter of grave concern for TB control programs, as there is currently no cure for some extensively drug-resistant (XDR) strains. There is concern that this resistance could transmit, stressing the need for additional control measures, rapid diagnostic methods, and newer drugs for treatment. We developed an in-house assay that can rapidly detect resistance to drugs involved in the definition of XDR-TB directly from smear-positive specimens. Two hundred fifteen phenotypically XDR-TB isolates and 50 pansusceptible isolates were analyzed using a reverse line blot hybridization (RLBH) assay. The assay was also successfully applied to 73 smear-positive clinical specimens. The RLBH assay exhibited good sensitivity for the detection of resistance to isoniazid (99%), rifampin (99%), fluoroquinolones (95.3%), and second-line aminoglycosides (94.8%). The results from application of this assay on direct smear-positive clinical specimens revealed 93% concordance with the phenotypic drug susceptibility test (DST) results for the above-mentioned drugs. The time to accurate DST results was significantly reduced from weeks to 3 days. This molecular assay is a highly accurate tool for screening for XDR-TB, which achieves a substantial reduction in diagnostic delays.     

Culture, ELISA and immunofluorescence tests for the diagnosis of conjunctivitis caused by Chlamydia trachomatis in neonates and adults

The relative value of culture, direct specimen antigen detection tests, i.e., enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) tests in the diagnosis of Chlamydia trachomatis infection was studied in 125 newborns and 121 adults with signs of conjunctivitis. Eye and nasopharyngeal samples were tested by culture using cycloheximide-treated or irradiated McCoy cells, ELISA (i.e., Chlamydiazyme, Abbott) and IF tests (i.e., Chlamyset, Orion and MicroTrak, Syva). Of the neonates, 70 (35 boys and 35 girls) and 54 (33 males and 21 females) of the adults were positive in one or both eyes in one or more tests: 191 (39%) in cultures, 173 (35%) in ELISA and 160 (33%), 176 (36%) in each of the IF tests. Using culture as standard reference, the sensitivities of ELISA and the IF tests were 88%, 81% and 87%, while the corresponding specificities were 99%, 98% and 97%, respectively. The predictive values for a negative test (PVN) were 93%, 89% and 92% and for a positive test (PVP) 98%, 96% and 94%. Of the 124 cases chlamydia-positive in the eyes, 67 (54%), 76 (61%), 64 (52%) and 70 (57%) were positive in nasopharyngeal samples in one or more of culture, ELISA and the two IF tests, respectively. The sensitivities of ELISA and the IF tests in nasopharyngeal samples were 87%, 78% and 81%, while the corresponding specificities were 90%, 93% and 91%, respectively. The predictive values for a negative (PVN) test were 95%, 92% and 93%, and for a positive test (PVP) 76%, 81%, and 77%. Nasopharyngeal swabs were more often positive in cases with 2 or more weeks' duration of symptoms than in those with shorter duration.     

建立一种用重组蛋白检测抗口蹄疫病毒抗体的间接ELISA方法

目的研究以重组蛋白作为包被抗原检测口蹄疫病毒（FMDV）感染后的动物血清中特异性抗体的可能性,为建立一种非病毒颗粒的ELISA检测试剂盒提供实验依据。方法利用自行构建表达的O型口蹄疫病毒VP1表位肽重组蛋白（VP1epi）作为包被抗原,采用间接ELISA方法确定抗原的最佳工作浓度和包被方法,优化各项实验条件,并以FMDV感染后的豚鼠血清作为标准阳性血清确定ELISA方法的特异性和灵敏度。结果FMDV感染后的阳性豚鼠血清可以很好地识别VP1epi重组蛋白,用此蛋白包被检测抗FMDV抗体的灵敏度可达1∶3200,并证明所检测的抗体是FMDV特异性的。结论VP1epi重组蛋白可以替代FMDV颗粒用于建立检测抗FMDV抗体的ELISA试剂盒。     

Evaluation of commercial enzyme immunoassays compared to immunofluorescence and double diffusion for autoantibodies associated with autoimmune diseases.

A commercially available enzyme immunoassay system for detecting autoantibodies to double-stranded DNA, deoxyribonucleoprotein, Smith, ribonuclearprotein, Sj?gren's syndrome-associated antigens A and B, and scleroderma-associated antigen 70 was compared to the conventional immunofluorescence assay for double-stranded DNA and double diffusion assays for extractable nuclear antigens. There was excellent correlation between methods, but it appears that the enzyme immunoassays are more sensitive. Based on the results of this study, the authors recommend performing anti-nuclear antibody screening at two dilutions, with enzyme immunoassay follow-up of appropriate patient sera that are positive on anti-nuclear antibody testing. Nucleolar and centromere pattern anti-nuclear antibodies are diagnostic for variants of scleroderma and need no further evaluation. Negative anti-nuclear antibody tests performed using HEp-2 tissue culture cells require no further evaluation.     

A method for the determination of antibody affinity using a direct ELISA

Antibody affinity is of major significance in immunoassays. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the conditions of the assay used. Here a method is described for the determination of binding constants (K) in a direct ELISA with the use of the computer program LIGAND. Five of the antibodies studied bound to their antigen with two classes of antigen binding site, while all the other antibodies studied reacted with only a single class of antigen binding site. The accuracy of the method and the implications for antigen-antibody reactions are discussed.