Anti-multiple nuclear dots (anti-MND) and anti-SP100 antibodies in hepatic and rheumatological disorders

Multiple nuclear dots pattern has been described in primary biliary cirrhosis and, less often, in rheumatological disorders. Sp100 is the major antigen of multiple nuclear dots. We evaluated prevalence and diagnostic significance of multiple nuclear dots and anti-Sp100 reactivity both in hepatic and rheumatological diseases. A series of 283 consecutive liver patients (89 primary biliary cirrhosis, 12 primary sclerosing cholangitis, 85 autoimmune hepatitis, 97 hepatitis C virus-related chronic liver disease) and of 89 consecutive rheumatological cases were evaluated. Presence of multiple nuclear dots was assessed by indirect immunofluorescence on HEp-2 cells, anti-Sp100 reactivity by ELISA with recombinant protein. Multiple nuclear dots were detected in 20 patients (7%) with liver disease (of whom 15 with primary biliary cirrhosis), and in eight patients (9%) with rheumatological disorders. Anti-Sp100 was detected in 45 liver patients (16%), of whom 30 with primary biliary cirrhosis, but in only two with rheumatological disorders (2%) (P =0.0004). The concordance between multiple nuclear dots and anti-Sp100 in liver and rheumatological patients was 90% and 25% (P=0.0018), respectively. Among 89 consecutive patients with primary biliary cirrhosis, multiple nuclear dots and anti-Sp100 were present in 17% and 34%, respectively (P=0.0152). Anti-Sp100 positivity was associated with older age and higher gamma-globulin levels. Multiple nuclear dots are similarly observed in liver and rheumatological patients. In contrast, anti-Sp100 is more frequent in liver patients and is significantly more often detected in primary biliary cirrhosis, of which it can be regarded as a highly specific serological marker. The antigenic target of multiple nuclear dots in most rheumatological patients is other than Sp100.     

Performance characteristics and clinical utility of a hybrid ELISA for detection of ANA.

To investigate the clinical utility of a newly developed hybrid ELISA for antinuclear antibodies (ANA), a cross-sectional study of patients admitted to the Section of Rheumatology was initiated. The ELISA was compared to indirect immunofluorescence (IIF) on HEp-2 cells. Accuracy of tests was analyzed using receiver-operating characteristic methodology (ROC). In addition, diagnostic sensitivity, specificity and predictive values were calculated for each assay. Results from the ROC analysis showed a slightly superior accuracy for IIF as compared to ELISA. Furthermore, IIF showed higher diagnostic sensitivity and positive predictive value for all combinations of patients and reference populations. This was due to enhanced detection by IIF, in contrast to ELISA, of diagnostically useful antibodies. IIF detected 87.4% and ELISA detected 84.2% of sera with antibodies against extractable nuclear antigens (ENA). In addition, IIF detected diagnostically important antibodies that are not included among the anti-ENA. The hybrid ELISA either lacks or does not contain the relevant antigens in sufficient amount. Inclusion of these antigens may further enhance the performance characteristics of the ELISA.     

Detection of airborne allergen (Ole e 1) in relation to Olea europaea pollen in S Spain

BACKGROUND: In recent years, it has been demonstrated that the air carries not only airborne pollen but also plant particles of smaller size that have allergenic activity, and, being within the respirable range, these particles can trigger rapid attacks in the lower respiratory tract. The study of particles according to size (0.7-40 micro m) could provide valuable information on the real allergenic activity in the atmosphere. OBJECTIVE: The purpose of this study was to analyse the dynamics of airborne Olea europaea pollen in contrast to the allergenic activity of Ole e 1 in the atmosphere. METHODS: The analyses were carried out with a Hirst-type volumetric collector and a cascade impactor simultaneously during the MPS of the olive. The indirect ELISA was used to detect the allergenic activity. The sampling was performed in Granada city centre (S Spain), in the Science Faculty building on the University of Granada from 30 April to 26 June 2005. RESULTS AND CONCLUSIONS: This research demonstrates that both the allergenic activity as well as the pollen particles follow in a similar curve, except in periods before or succeeding the main Olea pollen season. The study of the distribution of the allergenic particles according to their sizes reveals that the highest concentrations are between 3.3 and <0.7 micro m, thus indicating that allergenic activity primarily involves paucimicronic particles.     

Testing for antibodies to AIDS-associated retrovirus (HTLV-III/LAV) by indirect fixed cell immunofluorescence: specificity, sensitivity, and applications

Seropositivity to the AIDS-associated retrovirus, HTLV-III/LAV, has profound implications. Simple and reliable tests are needed to detect such antibodies. A rapid, sensitive indirect immunofluorescence assay (IFA) on acetone-fixed virus-producing CEM/LAV-N1 cells was adapted for detection of human antibodies to HTLV-III/LAV. Specific and nonspecific patterns of of immunofluorescent reactivity were easily distinguished, and results paralleled those obtained by Western blotting and radioimmunoprecipitation (RIP), indicating that there is no need to confirm IFA positivity. In contrast, the commercial enzyme-linked immunosorbent assay (ELISA) was less reliable. False positives occurred with sera from seven hemophiliacs that were negative on Western blots, and false-negative reactions were observed on two occasions. These involved low-titer AIDS-patients' sera that were positive on Western blots, and from one of which virus was successfully isolated. Our results emphasize the requirement for confirmatory assays when the ELISA test is used for primary screening of sera for antibodies to HTLV-III/LAV. The IFA method is especially well-suited to quantitative analysis of serum antibody levels. Our data suggest that serum antibody titers rise as disease progression occurs, ultimately falling as severe complications ensue. It is suggested that in laboratories where the demand for HTLV-III/LAV antibody testing is not excessive (1,000-2,000 sera/month), IFA could serve as the only serological assay for both screening and epidemiological purposes.     

PCR detection of HIV proviral DNA (gag) in the brains of patients with AIDS: comparison between results using fresh frozen and paraffin wax embedded specimens.

AIMS--To adapt the polymerase chain reaction (PCR) technique of HIV detection to paraffin wax embedded brain tissue and to compare the results with those obtained using frozen tissue. METHODS--HIV antigen and HIV proviral DNA were detected in specimens of frontal lobe using immunohistochemistry and PCR, respectively. DNA was extracted from fresh tissue using standard methods whereas the technique for extracting DNA from paraffin wax embedded tissue was partly modified. RESULTS--Twenty cases were examined. HIV DNA was detected in 16 cases in frozen specimens. Of these, 15 were also positive when paraffin wax embedded material was analysed. CONCLUSIONS--This study shows that HIV proviral DNA can be detected in formalin fixed, paraffin wax embedded brain tissue by PCR. The results obtained from paraffin wax embedded specimens showed a similar degree of reliability to those from fresh frozen brain. Factors such as fixative, fixation time, and delay in performing post mortem examinations did not seem to influence PCR amplification as positive results were obtained with specimens left in fixative for up to eight months, as well as in cases where post mortem examinations had been delayed for up to four days.     

Sandwich ELISA for detection of goats’ milk in ewes’ milk and cheese

A sandwich ELISA has been developed for the detection of defined amounts of goats’ milk (1–100%) in ewes’ milk and cheese. Polyclonal antibodies were raised in rabbits against goat caseins (GC). The resultant antibodies were affinity purified by immunoadsorption of the crude antiserum on to columns containing immobilized cow, ewe and goat caseins, followed by elution of the goats’ milk‐specific antibodies (anti‐GC) from the column containing the goat caseins. The anti‐GC antibodies bound to the wells of a microtiter plate were used to capture the goats’ caseins from milk or cheese mixtures. Further immunorecognition of the captured proteins was achieved with the same antibodies conjugated to biotin. ExtrAvidin‐peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of ewes’ milk and cheese containing variable amounts of goats’ milk.     

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus)

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.     

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) for the detection of picogram quantities of IgE

Existing methods of measuring IgE in in vitro peripheral blood lymphocyte (PBL) cultures are not sufficiently sensitive to detect IgE when it is present in small amounts. This paper describes a modification of a two-site ELISA which increases the sensitivity of the assay 10–20-fold. By using the Fab′ fragment of either rabbit or mouse monoclonal anti-IgE conjugated to alkaline phosphate (AP) as the detector, the background of the assay was reduced sufficiently to permit signal amplification, using a commercially available amplified AP substrate. With this assay as little as 10 pg/ml of IgE could be detected. The interassay coefficient of variation was 15–18% between 1200 and 100 pg/ml IgE (n = 14) and there was a good correlation with a commercial IgE radioimmunoassay (RIA) (r = 0.98, N = 38).     

Presence of tonofilaments and thymic serum factor (FTS) in thymic epithelial cells of a fresh water fish (carp: Cyprinus carpio) and a sea water fish (bass: Dicentrarchus labrax)

Using indirect immunofluorescence we demonstrated the presence of FTS in two fishes; the size of epithelial cells of the carp is smaller than of the bass.     

水痘-带状疱疹病毒抗原的ELISA定量检测方法的建立

目的:建立水痘-带状疱疹病毒(VZV)抗原的双抗体夹心ELISA定量检测方法,用于质控VZV灭活疫苗研发和生产中抗原含量.方法:以VZV中和单抗5F6C8为包被抗体、8H5D1为酶标抗体,构建定量检测VZV抗原的双抗体夹心ELISA方法,并对本方法的特异性、灵敏度、准确性、线性和稳定性等性能进行分析.结果:建立的双抗体夹心定量检测VZV抗原的ELISA方法,线性范围为0.4 μg～13 μg/ml,相关系数为R2=0.994,定量限度为0.4.μg/ml;变异系数CV＜ 15％、准确性回收率介于87.5％ ～ 111.6％之间,稳定性37℃6天的回收率＞80％.与VZV以外的相关病毒样本没有交叉反应.结论:构建的VZV抗原ELISA定量检测方法的各项性能符合定量检测需要,可用于VZV灭活疫苗的研发和生产过程的抗原含量检测.     

Screening for anti-neutrophil cytoplasmic antibodies (ANCA): is indirect immunofluorescence the method of choice?

Detection of ANCA has become an important tool for the diagnosis and monitoring of disease activity in Wegener's granulomatosis (WG). Unfortunately, a group of sera positive by the standard method for ANCA detection, indirect immunofluorescence (IIF), are negative when more specific tests with purified proteins are used. In order to examine this discrepancy we examined groups of sera selected for being (i) C-ANCA-positive by IIF; (ii) positive in proteinase 3 (PR3)-ANCA ELISA; and (iii) from 24 patients with WG. The following assays were used: IIF, PR3-ANCA ELISA and capture PR3-ANCA ELISA using MoAbs against PR3. Furthermore, since granule enzymes are released during coagulation, we also measured ANCA in complex with PR3. To test if granule enzyme release had any influence on ANCA detection, both serum and EDTA-plasma were collected from a patient with active WG. No difference, however, was found. In the IIF-positive group (n = 60) 68% of the sera were positive in PR3-ANCA ELISA, 86% in capture PR3-ANCA ELISA and 80% were positive for the PR3/IgG-ANCA complex. In the PR3-ANCA ELISA group (n = 105) 88% of the sera were positive by IIF, 98% in capture PR3-ANCA ELISA and 53% in the PR3/IgG-ANCA assay. To evaluate the tests clinically sera from 24 patients with WG were examined. In the remission group (n = 10) two patients were positive by IIF, four in the PR3-ANCA ELISA, and five in the capture PR3-ANCA ELISA. Fourteen had active disease, and in this group 11/14 were positive by IIF, 10/14 in PR3-ANCA ELISA and 12/14 by capture-ELISA. The correlation between IIF and capture PR3-ANCA ELISA titre (r = 0.72, P = 0.0095) was better than between PR3-ANCA ELISA and IIF (r = 0.56, P = 0.043). It is concluded that the capture PR3-ANCA ELISA is more sensitive than PR3-ANCA ELISA, and that the capture ELISA can be used for screening of PR3-ANCA.     

Preparation and evaluation of a recombinant Rift Valley fever virus N protein for the detection of IgG and IgM antibodies in humans and animals by indirect ELISA

This paper describes the cloning, sequencing and bacterial expression of the N protein of the Rift Valley fever virus (RVFV) ZIM688/78 isolate and its evaluation in indirect ELISAs (I-ELISA) for the detection of IgM and IgG antibodies in human and sheep sera. Sera used for the evaluation were from 106 laboratory workers immunised with an inactivated RVF vaccine, 16 RVF patients, 168 serial bleeds from 8 sheep experimentally infected with wild type RVFV and 210 serial bleeds from 10 sheep vaccinated with the live attenuated Smithburn RVFV strain. All human and animal sera that tested positive in the virus neutralisation test were also positive in the IgG I-ELISA. There was a high correlation (R² = 0.8571) between virus neutralising titres and IgG I-ELISA readings in human vaccinees. In experimentally infected sheep IgG antibodies were detected from day 4 to 5 post-infection onwards and IgM antibodies from day 3 to 4. The IgG I-ELISA was more sensitive than virus neutralisation and haemagglutination-inhibition tests in detecting the early immune response in experimentally infected sheep. The I-ELISAs demonstrated that the IgG and IgM response to the Smithburn vaccine strain was slower and the levels of antibodies induced markedly lower than to wild type RVFV infection.     

单克隆抗体ELISA法快速诊断疱疹性脑炎

本文用单纯疱疹病毒单克隆抗体（ＨＳＶ－ＭｃＡｂ）ＥＬＩＳＡ法检测了１２８例病毒性脑炎患儿脑脊液中ＨＳＶ特异性抗原（ＨＳＶ—Ａｇ），２０例其他神经系统疾患也作了测定，３２例脑炎患儿同时取脑脊液和血清作ＨＳＶ—ＩｇＧ抗体水平测定比较，病毒性脑炎患儿的ＨＳＶ－Ａｇ阳性检出率为１９．５％，与血清／脑脊液的ＨＳＶ－ＩｇＧ测定比较，敏感性８４．６％，特异性９４．７％。用ＥＬＩＳＡ法检测脑脊液中ＨＳＶ－Ａｇ是一种简便、快速、敏感、特异的方法，对疱疹性脑炎具有早期诊断价值。     

Identification of tuna species (Thunnini tribe) by PCR-RFLP analysis of mitochondrial DNA fragments

Tunas are highly priced and the Atlantic bluefin tuna is most endangered trade fish in the world. For effective fishery management and protection of consumers’ rights, it is important to develop a molecular method to identify the species of tuna products. In this study, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was developed to identify 10 tuna or tuna-like species. Primers were designed to amplify six mitochondrial DNA fragments from each specimen, and three restriction enzymes including AfaI, DdeI, and AluI were used to analyze the short length fragments produced from ATPase and control region (CR) genes. Besides these, the phylogenetic relationships among tested fish species were analyzed based on phylogenetic trees constructed using the amplified COI and CR sequences. The results indicated that the developed PCR-RFLP really provided a useful and academic technique to identify high-priced tuna species.     

Enzyme-antibody conjugation by a heterobifunctional reagent and its application in enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis B surface antigen

The heterobifunctional reagent 3-(2-pyridyl-dithio)propionate (SPDP) was used to prepare defined conjugates composed of horseradish peroxidase (HRP) and goat anti-HBs IgG. The modification of HRP and IgG with SPDP was dependent on both the SPDP: protein molar ratio and the pH of the buffer. Conjugates were separated by a single affinity chromatographic step using concanavalin A-Sepharose 4B equilibrated with 0.1 M Tris-HCl buffer, pH 7.4, containing 0.5 M KCl and 1 mM EDTA. The conjugate was eluted with 10 or 100 mM alpha-methyl-D-mannoside and appropriate pools were made reflecting various HRP/IgG molar ratios. Each pool was examined for performance in an enzyme-linked immunosorbent assay for HBsAg. Conjugate composed of an HRP/IgG molar ratio of 2.5-4 yielded the greatest sensitivity.     

Treatment of malignant ascites and pleurisy by a streptococcal preparation OK-432 with fresh frozen plasma--a mechanism of polymorphonuclear leukocyte (PMN) accumulation

A single injection of a streptococcal preparation, OK-432, with fresh frozen plasma (FFP) (or fresh human serum) into the peritoneal or pleural cavity for the treatment of malignant ascites or pleurisy resulted in a complete reduction of ascitic fluid or pleural effusion in 5 out of 11 patients. FFP was used a further source of complement for the effective accumulation of antitumor polymorphonuclear leukocytes (PMNs) by complement-derived chemotactic factors in the cavity. C5a increased in the fluids 3-9 h after the injection and preceded a massive increase in PMNs. C1 inhibitor (C1INH) and C3b inactivator (C3bINA) decreased in several cases 6 h after the treatment. Chemotactic arachidonic acid metabolites, thromboxane B2(TXB2) as a characteristics of TXA2, and leukotriene B4(LTB4) also increased at the same time even in cases where C5a changed only minimally, and may play a role in accumulating antitumor PMNs in the cavity.     

Rapid diagnosis of echovirus type 33 meningitis by specific IgM detection using an enzyme linked immunosorbent assay (ELISA)

During an outbreak of meningitis in France (in the Lyon area), from June to October 1982, serum and stool samples were collected from 227 patients. An enzyme-linked immunosorbent assay (ELISA) for titrating IgG and IgM antibodies anti-echovirus type 33 was developed and compared with the virus isolation technique, and with the titration of neutralizing antibodies. In 39 patients excreting echovirus 33 in faeces, the ELISA test allowed a positive serodiagnosis in 85% of the cases by detection of specific IgM (64% of the cases) and by seroconversion (21%). Compared with the neutralization (Nt) test, ELISA was found to be more sensitive. The antibody titres in ELISA were over 50 times higher and detected earlier than the neutralizing antibodies. This early immune response allowed a rapid diagnosis by specific IgM detection in the acute sera collected within 8 days after the appearance of the clinical symptoms in more than 50% of the 97 patients examined, whereas the Nt test allowed a positive serodiagnosis in only 32% of the patients. The use of a caesium chloride purified antigen insured the specificity of the reactions.     

Sensitive and rapid detection of Clonorchis sinensis infection in fish by loop-mediated isothermal amplification (LAMP)

The fish-borne clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is endemic in a number of countries with over 35 million people being infected globally. Rapid and accurate detection of C. sinensis in its intermediate host fish is important for the control and prevention of clonorchiasis in areas where the disease is endemic. In the present study, we established a loop-mediated isothermal amplification (LAMP) approach for the sensitive and rapid detection of C. sinensis metacercariae in fish. The specificity and sensitivity of primers designed from the C. sinensis cathepsins B3 gene were evaluated, and specific amplification products were obtained with C. sinensis, while no amplification products were detected with DNA of related trematodes, demonstrating the specificity of the assay. The LAMP assay was proved to be 100 times more sensitive than a conventional polymerase chain reaction for detection of C. sinensis. The established LAMP assay provides a useful tool for the rapid and sensitive detection of C. sinensis in fish, which has important implications for the effective control of human clonorchiasis.     

Genetic detection and multilocus sequence typing of vanA-containing Enterococcus strains from mullets fish (Liza ramada)

Enterococci have emerged as important nosocomial and community-acquired pathogens in humans. The presence of vanA-enterococci was investigated in 103 fecal samples recovered from mullets fish (Liza ramada). All fecal samples were inoculated in Slanetz-Bartley agar plates supplemented with 4 mg/L of vancomycin for vancomycin-resistant enterococci (VRE) recovery and two isolates/sample were characterized. Antibiotic susceptibility was tested for 11 antibiotics by disk diffusion and agar dilution methods. VRE identification was performed by biochemical and molecular methods. Additionally, the mechanisms of resistance to glycopeptides (vanA, vanB, vanC1, vanC2, and vanD) and other antibiotics [erm(A), erm(B), tet(L), tet(M), aph(2')-aac(6'), aph(3')-IIIa, ant(6'), vat(D), vat(E)] as well as the presence of enterococcal surface protein (esp) and hyl virulence factors were investigated. vanA-Enterococcus faecium isolates were recovered from 4 of 103 tested samples, and they showed glycopeptide and erythromycin resistances. Three of them were also ampicillin resistant, two showed resistance to tetracycline, ciprofloxacin, and kanamycin, and one showed resistance to gentamicin. The tet(M) and erm(B) genes were found in all tetracycline- and erythromycin-resistant strains, respectively. The aph(3')-III and aph(2')-aac(6') genes were identified in the kanamycin- and gentamicin-resistant isolates, respectively. The IS1216 element was identified within vanX-vanY region of Tn1546 in two vanA isolates. The hyl and esp virulence genes were found in four and two isolates, respectively. vanA-strains were ascribed to sequence types ST280 (two isolates) and ST273 (two isolates), including both lineages into the clonal complex CC17. Mullets fish can excrete VRE in their feces and may be a reservoir for such resistant bacteria that can be transmitted to other animals including humans.     

Evaluation of a total hepatitis C virus (HCV) core antigen assay for the detection of antigenaemia in anti-HCV positive individuals

A new, sensitive enzyme immunoassay has been developed for detecting and quantifying total hepatitis C virus (HCV) core antigen in anti-HCV positive or negative sera ("trak-C", Ortho Clinical Diagnostics, Raritan, NJ). The purpose of this study was to evaluate the performance of trak-C as an additional laboratory diagnostic marker of viraemia. The performance was compared to HCV-RNA detection in the "screening" of sera from a large heterogeneous population of hospitalised patients and outpatients. Six hundred and eighteen anti-HCV negative sera, 405 anti-HCV positive/HCV-RNA negative sera, 604 anti-HCV positive/HCV-RNA positive sera and 67 anti-HCV negative sera containing antigens or antibodies potentially interfering with the performance of the assay were analysed. Supplemental HCV antibody testing was performed using a commercial strip immunoblot assay. HCV-RNA was investigated using a qualitative commercial assay. A quantitative commercial RT-PCR was used for the analysis of selected samples. Sensitivity and specificity values were 94.7 and 100%, respectively. The latter was also confirmed when anti-HCV negative samples containing potentially interfering antigens/antibodies were examined. Sensitivity below 100% was probably due to an antigenaemia below the detection limit of trak-C. Besides, because 65.6% of HCV-RNA positive/trak-C negative samples presented specific antibodies against all four RIBA antigens, the hypothesis was raised that, in some cases, the dissociation step efficiency could be sub-optimal. In conclusion, trak-C seems suitable for identifying HCV infection on large based populations. It is a rapid to perform, reliable and specific assay that can be adapted to any laboratory setting.