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Treponema pallidum subsp. pallidum is a fastidious spirochete and the etiologic agent of syphilis, a sexually transmitted infection (STI). Syphilis diagnoses and disease staging are based on clinical findings and serologic testing. Moreover, according to most international guidelines, PCR analysis of swab samples from genital ulcers is included in the screening algorithm where possible. It has been suggested that PCR might be omitted from the screening algorithm due to low added value. As an alternative to PCR, IgM serology might be used. In this study, we wanted to establish the added value of PCR and IgM serology for diagnosing primary syphilis. Added value was defined as finding more cases of syphilis, preventing overtreatment, or limiting the extent of partner notification to more recent partners. We found that both PCR and IgM immunoblotting could aid the timely diagnosis of early syphilis in ~24% to 27% of patients. PCR has the greatest sensitivity and can be applied to cases with an ulcer with suspected reinfection or primary infection. In the absence of lesions, the IgM immunoblot could be used. However, the IgM immunoblot has better performance in cases with suspected primary infection than in reinfections. The target population, testing algorithm, time pressures, and costs should determine whether either test provides sufficient value to be implemented in clinical practice., Competing Interests: The authors declare no conflict of interest.

Cutaneous leishmaniasis (CL) is firmly established in South America. We aimed to assess the detection of IgG antibodies against 14 and/or 16 kDa antigens by immunoblot (IB) for CL serological diagnosis in French Guiana, an area where many endemic pathogens could interfere with it. This study was performed retrospectively on sera from 141 patients at the Cayenne tertiary hospital: 30 were patients with confirmed CL, 71 were diagnosed with various other endemic pathogens, 11 were diagnosed with an autoimmune disease, and 29 controls had no history of CL. Antibodies bound to the 14 and/or 16 kDa antigens in 27 of the 30 CL patients' sera and in 39 of the 111 non-CL patients' sera (26 from the infectious diseases group, four from the autoimmune diseases group, and nine from the dermatology department). The method tested showed a high sensitivity (90%) and a low specificity (66%), and a diagnosis odds ratio of 17.5 (95% CI [4.6-78.0]). This IB may be helpful to exclude the diagnosis of CL, prompting physicians to look for another diagnosis in the case of a negative IB.

Strongyloides stercoralis is a soil-transmitted nematode that can cause life-threatening conditions in immunocompromised persons. In the United States, strongyloidiasis should be considered mainly in immigrants, refugees, or travelers. The confirmatory laboratory diagnosis is usually performed by detecting larvae from the stool, duodenal material, and sputum. In persons who are immunocompromised with severe strongyloidiasis, adult worms and eggs can be detected from duodenal material. For serological diagnosis, most assays use crude antigens to detect anti-S. stercoralis IgG. Recently, recombinant proteins such as rSs-NIE-1 and rSs-IR have been used to detect IgG antibodies. We used rSs-NIE-1 and rSs-IR recombinant antigens to develop a biplex Western blot assay to detect the IgG4 antibody in individuals with strongyloidiasis. The sensitivities of rSs-NIE-1 and rSs-IR were 97.4% and 90.8%, respectively, whereas the specificities were 97.6% and 98%, respectively. In conclusion, the biplex rSs-NIE-1 and rSs-IR immunoblot performs well in detecting IgG4 antibody in S. stercoralis-infected persons.

The use of high-quality antigen-specific immunoassays for detecting anti-neutrophil cytoplasmic antibodies (ANCA) and anti-glomerular basement membrane (GBM) autoantibodies is recommended in patients with suspected ANCA vasculitis and/or anti-GBM disease. We analysed the diagnostic performance of a semi-quantitative and rapid immunoblot (EUROIMMUN AG, Lübeck, Germany) in two settings. Patient sera from different cohorts (ANCA vasculitis n  = 187, anti-GBM disease n  = 19, and disease controls n  = 51) were used. The diagnostic performance of the immunoblot was assessed when used as a confirmatory test for the presence of ANCA in suspected ANCA vasculitis and when evaluating the presence of ANCA and/or anti-GBM antibodies in AAV and/or anti-GBM disease patients with a rapidly progressive glomerulonephritis (RPGN). In a confirmatory test setting, the immunoblot had an optimal sensitivity and specificity of 97.4 and 98.1% for PR3-ANCA and 98.5 and 96.4% for MPO-ANCA, respectively. With increasing test result ranges, a higher interval likelihood ratio (LR) was found for both ANCA entities. When evaluating for ANCA in patients with RPGN, the highest diagnostic accuracy (sensitivity 92.9% and specificity 100%) was obtained by using different cut-off values of positivity for PR3- (>5) and MPO-ANCA (>10). Also, the diagnostic performance for detecting anti-GBM was good (sensitivity 100% and specificity 100%). There are advantages over other assays in terms of time, costs, and interpretation of results. The immunoblot is a useful addition to current guidelines, particularly when a rapid diagnosis is necessary.

The commercial tests currently available as second-level tests to detect ANA sub-specificities are generally used independently from the ANA immunofluorescence (IIF) pattern. The aim of this study was to evaluate the efficacy of the use of a customizable pattern-oriented antigenic panel by immunoblot (IB) using the International Consensus on ANA Patterns (ICAP) classification scheme, in order to introduce a novel and updated autoimmune diagnostic flowchart. 710 sera referred for routine ANA testing were selected on the basis of the ANA pattern according to the ICAP nomenclature (nuclear speckled AC-2,4,5; nucleolar AC-8,9,10,29; cytoplasmic speckled AC-18,19,20) and on an IIF titer ≥1:320. They were then assayed by three experimental IB assays using a panel of selected antigens. ICAP-oriented IB detected 515 antibody reactivities vs. 457 of traditional anti-ENA in the nuclear speckled pattern group, 108 vs. 28 in the nucleolar pattern group, and 43 vs. 34 in the cytoplasmic speckled pattern. This pilot study may lead the way for a new approach introducing an ICAP pattern-oriented follow up testing as a valid alternative to the existing standard panels, thus enabling more patients with autoimmune rheumatic disease to be accurately diagnosed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: To investigate the diagnostic yield of commercial immunodots to detect onconeural antibodies associated with paraneoplastic neurologic syndromes (PNSs), we analyzed the proportion of confirmed positive results using alternative techniques., Methods: Sera (n = 5,300) of patients with suspected PNS were tested by PNS+2 blot (Ravo Diagnostika; January 2016-May 2017) or EUROLINE PNS 12 Ag (Euroimmun; July 2017-November 2018). Positive samples were further explored by in-house indirect immunofluorescence and a third in-house technique (Western blot or cell-based assay) using recombinant protein. Those found negative by these 2 techniques were considered as nonconfirmed. We analyzed the relationship between band intensity and final confirmation. Clinical data were collected for all confirmed results and nonconfirmed EUROLINE immunodots., Results: PNS+2 blot was positive in 128/1,658 (7.7%) sera and confirmed in 47/128 (36.7%). EUROLINE was positive in 186/3,626 (5.1%) and confirmed in 56/186 (30.1%). Confirmation was highly variable among the antibodies tested, from 7.2% (PNS+2 blot) and 5.8% (EUROLINE) for anti-Yo to 88.2% (PNS+2 blot) and 65.0% (EUROLINE) for anti-Hu. None of the 27 weak positive sera by EUROLINE was confirmed. Band intensity in confirmed cases was variable among the antibodies from strong positive for all anti-Yo (n = 3) and anti-Hu (n = 11) to positive (n = 19) or strong positive (n = 9) for anti-SOX1. Among patients with a nonconfirmed EUROLINE result and available clinical information, all had an alternative diagnosis, and only 6.7% had cancer., Conclusions: Immunodots may be useful for PNS screening, but a threshold should be established for each antibody, and clinical information and confirmation by other techniques are essential., Classification of Evidence: The study provides Class IV evidence that immunodot assays for onconeural antibodies accurately identify patients with paraneoplastic neurologic syndromes., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background: Porcine cysticercosis has a negative impact on human health and the meat industry, as it makes infected meat unaproprieted for consuption and it is the main etiology of epileptic seizures in developing countries. There are multiple serological assays that use crude antigens with high sensitivity and specificity for the diagnosis of both porcine and human cysticercosis. Nonetheless, antigen preparation is time-consuming, needs a well-equipped laboratory and trained personnel and places those manipulating the meat at great risk for infection. New serodiagnostic approaches to the diagnosis of porcine and human cysticercosis have been directed towards the development of recombinant deoxyribonucleic acid technology for the generation of synthetic proteins that can serve as simplified, low-cost and harmless substitutes for native antigens. The aim of the present study was to further evaluate the recombinant Tsol-p27 protein as a target molecule in immunoassays for the serodiagnosis of porcine cysticercosis. From these data, we hoped to develop recommendations regarding its use in the serodiagnosis of porcine cysticercosis., Results: We studied a panel of 83 naturally infected pig sera from Angónia District, Mozambique, an endemic area for porcine and human cysticercosis. These sera were previously tested by antigen enzyme-linked immunosorbent assay (Ag-ELISA) to detect antigens of T. solium. The serum panel was processed by enzyme-linked immunoelectrotransfer blot (EITB) assay against the recombinant Tsol-p27 protein and the Ag-ELISA assay results were used to compare and evaluate the performance of Tsol-p27 for the diagnosis of cysticercosis. Out of 83 sera, 24 (29.0%) were positive for Tsol-p27 and 59 (71%) were negative in the same assay. From the 37 sera that tested positive to Ag-ELISA, 11 (13.3%) were positive to Tsol-p27, while from 46 sera that tested negative to Ag-ELISA, 33 (39.7%) also tested negative to Tsol-p27. The sensitivity and specificity of Tsol-p27 was 29.7% and 71.7%, respectively, while the positive predictive value and negative predictive value were 45.8% and 55.9%, respectively, as calculated using Medcalc® version 15.0 software (MedCalc Software, Ostend, Belgium)., Conclusion: While Tsol-p27 recombinant protein might be suitable for testing human sera, its performance in pigs is not acceptable, so other recombinant proteins should be evaluated alone or multiplexed.

Acquired Immunodeficiency syndrome (AIDS) is an important global public health issue. Increasing HIV/AIDS cases reported each year has become a serious health problem for our country. The fourth generation enzyme immunoassay (EIA) test is the first step in the laboratory diagnosis of human immunodeficiency virus (HIV) infection. When the EIA test is repeatedly reactive, antibody-based tests such as immuno blot (IB), line immunoassay (LIA), HIV 1-2 antibody differentiation immunoassay, and HIV RNA tests for the early period of infection are used as confirmatory tests. The aim of this study was to evaluate the results of three different methods for the diagnosis of HIV infection. HIV 1-2 IB and quantitative HIV-1 RNA PCR tests were performed in 199 patient samples. These samples were detected as the reactive or gray zone with HIV 1-2 Ab+Ag EIA test between 2010 and 2015 at Akdeniz University Hospital, Microbiology Laboratory. HIV 1-2 Ab+Ag determination in serum samples was performed with the EIA method (Elecsys HIV combi PT test, Roche Diagnostics, Germany). A commercial kit (INNO-LIA HIV I-II Score, Innogenetics, Belgium) was used for HIV 1-2 IB method. The presence of HIV-1 RNA was investigated by automated nucleic acid extraction and real-time PCR method (Ampliprep/COBAS Tagman HIV-1 Test, Roche Diagnostics, Germany) in plasma samples. For statistical analysis, SPSS, Mann Whitney U test was used, ROC analysis was performed and p<0.05 value was considered statistically significant. HIV 1-2 Ab+Ag EIA COI (cut-off index) median value was higher with positive HIV 1-2 IB and HIV-1 RNA results than negative HIV 1-2 IB and HIV-1 RNA results. These values were 394 (range: 11.5-2272) and 1.79 (range: 1.01-83.3) respectively and this difference was statistically significant (p< 0.001). HIV-1 RNA test results were positive in one patient with gray zone and two patients with negative HIV 1-2 IB result (viral loads were > 10.000.000, > 10.000.000 and 5.040.000 copies/ml, respectively). For the kit that we used for HIV 1-2 Ab+Ag EIA COI ratio of >16.45 had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 97.6%, 98.1%, 97.6% and 98.1%, respectively for the detection of HIV infection (r= 0.994, p< 0.001). HIV 1-2 Ab+Ag EIA S/CO ratio of < 9.26 had a sensitivity, specificity, PPV and NPV of 100%, 92.5%, 91.1% and 100% (p< 0.001). HIV infection is diagnosed if HIV 1-2 Ab+Ag EIA test result is repeatedly reactive and HIV 1-2 IB test and HIV-1 RNA tests are positive. In our study, HIV 1-2 Ab+Ag EIA COI median value was 394 (range: 11.5-2272) in this group of patients (p< 0.001). HIV-1 RNA PCR test was positive in three patients with > 10.000.000, 5.040.000 and > 10.000.000 copies/ml whose EIA tests were repeatedly reactive. HIV IB test was detected as the gray zone in one of them and as negative in the remaining two (HIV EIA S/CO values were 265, 9.5 and 131.8, respectively). These patients were diagnosed as acute HIV infection with clinical and laboratory findings. In conclusion, HIV RNA should also be performed and included in the diagnostic algorithm for acute HIV infection.

Equine infectious anaemia (EIA) is a blood borne disease that is listed among the notifiable diseases of the World Organisation for Animal Health (OIE). EIA is also regulated by the OIE for the international trading provisions and is generally subject to control programmes. Since 2011, Italy has been conducting a surveillance plan based on a three-tier diagnostic system, using a serological ELISA as screening test, an agar gel immunodiffusion test (AGIDT) as a confirmatory method, and an immunoblot (IB) as an alternative confirmatory assay for discordant results between the first two tests. As for the in-house competitive ELISA (c-ELISA) and the AGIDT, the Italian National Reference Laboratory for EIA (NRL) validated the IB according to the OIE guidelines, employing eight panels containing positive sera, including those from EIA virus (EIAV) proven infected horses, and negative horse, mule and donkey sera collected from different geographical areas. In addition, two international reference image panels were employed for the optimization and the validation of the digital image reading system adopted that allows an impartial measurement of the serum reactivity in the IB assay. The immunological reactivity to EIAV antigens, p26, gp45 and gp90 adsorbed on the IB membrane, determines the serological status of the animal and for EIA, a p26 positive band together with at least one of the other antigen defines a subject as serologically positive for EIAV. For validation, the parameters assessed were threshold values, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility. These parameters were evaluated for each antigen as well as in combination, according to the diagnostic algorithm established above. The validation data defined the IB as having a satisfactory sensitivity, specificity, repeatability and reproducibility for all antigens and species tested. An instrumental recording of the results improves the confidence in using IB as a confirmatory test for EIAV, differently from the AGIDT that is read by an operator. The advantages of using the IB are its higher sensitivity, to that of the AGIDT, which allows an earlier detection of infection that reduces the risk of transmission and therefore the incidence of the EIA, and its higher specificity to that of the ELISA which is based on the discrimination of subjects reacting only against the p26, the antigen used by all ELISAs available, which are not considered as infected by EIAV. In particular, when this assay is used in outbreaks it can detect new cases earlier than the AGIDT, and therefore reduce the restriction period with an economic benefit for the animal owners and the public veterinary sanitary system., (Copyright © 2019. Published by Elsevier B.V.)

An estimated 41,200 people were newly infected with hepatitis C virus (HCV) in 2016 in the United States. Screening tests for antibodies to HCV may generate up to 32% false positivity in low-risk populations. Current Centers for Disease Control and Prevention (CDC) screening recommendations do not require confirmatory testing of a screening anti-HCV-positive test; however, confirmation is valuable for surveillance in the absence of HCV RNA testing. A recombinant immunoblot assay (RIBA) was used as a confirmatory assay for anti-HCV-reactive samples but was discontinued in 2013. Another anti-HCV confirmatory assay, INNO-LIA, is commercially available in Europe but is not approved by the Food and Drug Administration (FDA) in the United States. We report the development of an anti-HCV assay that was performed on an automated immunoblot platform using a fourth-generation HCV recombinant fusion protein. Based on testing of 70 well-characterized samples, of which 40 were HCV RNA and anti-HCV positive, 15 were HCV RNA positive/anti-HCV negative, and 15 were HCV RNA and anti-HCV negative, the specificity and sensitivity of the HCV-WES assay were 100% and 95%, respectively. Concordance between INNO-LIA and HCV-WES was determined by testing 205 HCV RNA-negative/anti-HCV-positive samples, of which 149 (72.7%) were positive by HCV-WES, while 146 (71.2%) were positive by INNO-LIA. We have shown proof of concept for the use of this test for confirmation of screened anti-HCV results. The HCV-WES assay has advantages over manual Western blot assays and INNO-LIA, including ease of use, lower cost, and reduced hands-on time., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Background: Assessments of commercial assays in detecting herpes simplex virus type 2 (HSV-2) antibodies have shown variable sensitivity and specificity, and variation in performance by global population., Objective: To evaluate performance of four assays in detecting HSV-2 antibodies in a composite Middle Eastern and North African (MENA) population. The assays are two ELISA kits: HerpeSelect ® 2 ELISA IgG and Euroimmun Anti-HSV-2 (gG2) ELISA (IgG), and two immunoblot (IB)/Western blot (WB) assays: HerpeSelect ® 1 and 2 Immunoblot IgG and Euroimmun Anti-HSV-1/HSV-2 gG2 Euroline-WB (IgG/IgM)., Study Design: Blood specimens were drawn from blood donors between 2013-2016 in Doha, Qatar. Twenty specimens from ten nationalities (Egypt, Iran, Jordan, Lebanon, Pakistan, Palestine, Qatar, Sudan, Syria, and Yemen; total = 200) were randomly selected and tested for HSV-2 antibodies., Results: In the six possible assay comparisons, Cohen's kappa statistics indicated fair to good agreement, ranging between 0.57 (95% CI 0.28-0.86) and 0.69 (95% CI 0.44-0.95). Meanwhile, positive percent agreement ranged between 50.0 (95% CI 18.7-81.3%) and 63.6% (95% CI 30.8-89.1%); negative percent agreement ranged between 97.8% (95% CI 94.4-99.4%) and 99.5% (95% CI 97.0-100.0%); and overall percent agreement ranged between 95.8% (95% CI 91.9-97.9%) and 97.5% (95% CI 94.2-98.9%). The two ELISA kits demonstrated comparable sensitivities and specificities ≥50% and >98%, respectively, with respect to the IB/WB assays., Conclusion: The study provided, for the first time, primary data on performance of these assays in diagnosing HSV-2 infection in MENA populations. Findings support comparable performance and utility of these assays, and demonstrate challenges in establishing seropositivity (versus seronegativity)., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Homodimer formation is essential for the normal activity of endothelial nitric oxide synthase (eNOS). Structural uncoupling of eNOS, with generation of enzyme monomers, is thought to contribute to endothelial dysfunction in several vascular disorders, including aging. However, low-temperature SDS-PAGE of healthy arteries has revealed considerable variation between studies in the relative expression of eNOS dimers and monomers. While assessing structural uncoupling of eNOS in aging arteries, we identified methodological pitfalls that might contribute to such variation. Therefore, using human cultured aortic endothelial cells and aortas from young and aged Fischer-344 rats, we investigated optimal approaches for analyzing the expression of eNOS monomers and dimers. The results demonstrated that published differences in treatment of cell lysates can significantly impact the relative expression of several eNOS species, including denatured monomers, partially folded monomers, dimers, and higher-order oligomers. In aortas, experiments initially confirmed a large increase in eNOS monomers in aging arteries, consistent with structural uncoupling. However, these monomers were actually endogenous IgG, which, under these conditions, has mobility similar to eNOS monomers. Increased IgG levels in aged aortas likely reflect the aging-induced disruption of endothelial junctions and increased arterial penetration of IgG. After removal of the IgG signal, there were low levels of eNOS monomers in young arteries, which were not significantly different in aged arteries. Therefore, structural uncoupling of eNOS is not a prominent feature in young healthy arteries, and the process is not increased by aging. The study also identifies optimal approaches to analyze eNOS dimers and monomers. NEW & NOTEWORTHY Structural uncoupling of endothelial nitric oxide synthase (eNOS) is considered central to endothelial dysfunction. However, reported levels of eNOS dimers and monomers vary widely, even in healthy arteries. We demonstrate that sample processing can alter relative levels of eNOS species. Moreover, endothelial dysfunction in aging aortas results in IgG accumulation, which, because of similar mobility to eNOS monomers, could be misinterpreted as structural uncoupling. Indeed, enzyme monomerization is not prominent in young or aging arteries.

Diagnostic testing for Lyme disease (LD) remains dependent on detection of antibodies to LD Borrelia using serologic assays, in adherence to the standard two-tiered testing (STTT) algorithm. We present the first analytic evaluation of the automated Borrelia B31 ViraChip IgM and IgG microarray immunoblot (MIB) assays (Viramed Biotech AG, Planegg, Germany) in comparison to two different, semiautomated blot assays for LD, including the Borrelia B31 ViraStripe IgM and IgG line immunoassays (LIAs) (Viramed) and the MarDx Borrelia burgdorferi IgM and IgG Western blot (WB) assays (Trinity Biotech, Carlsbad, CA), using prospectively collected sera ( n = 411) and archived, clinically characterized samples ( n = 91). We show comparable overall agreement (>84%) of the ViraChip MIB assays against the two aforementioned LD blot methods. The ViraChip MIB assays were also compared to a consensus standard, whereby samples were classified as positive or negative for IgM or IgG to B. burgdorferi if the analyte-matched ViraStripe LIA or MarDx WB assay were positive or negative, respectively. The ViraChip IgM and IgG MIB assays showed >93% positive, negative, and overall agreement versus these consensus criteria. The ViraChip MIB assays were associated with a time savings of 28 min to process one full batch of samples compared to the time required for the ViraStripe LIAs. The ViraChip MIB assays can be programmed and performed on an open-system, automated enzyme-linked immunosorbent assay (ELISA) processor, negating the need for assay-specific equipment and enabling laboratories to consolidate LD testing onto a single platform. We conclude that the ViraChip IgM and IgG MIB assays may be added to the repertoire of supplemental, second-tier blot testing systems for diagnosis of LD., (Copyright © 2018 American Society for Microbiology.)

Antibody use is a critical component of cardiovascular physiology research, and antibodies are used to monitor protein abundance (immunoblot analysis) and protein expression and localization (in tissue by immunohistochemistry and in cells by immunocytochemistry). With ongoing discussions on how to improve reproducibility and rigor, the goal of this review is to provide best practice guidelines regarding how to optimize antibody use for increased rigor and reproducibility in both immunoblot analysis and immunohistochemistry approaches. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/guidelines-on-antibody-use-in-physiology-studies/ .

Experimental measurements require calibration to transform measured signals into physically meaningful values. The conventional approach has two steps: the experimenter deduces a conversion function using measurements on standards and then calibrates (or normalizes) measurements on unknown samples with this function. The deduction of the conversion function from only the standard measurements causes the results to be quite sensitive to experimental noise. It also implies that any data collected without reliable standards must be discarded. Here we show that a "1-step calibration method" reduces these problems for the common situation in which samples are measured in batches, where a batch could be an immunoblot (Western blot), an enzyme-linked immunosorbent assay (ELISA), a sequence of spectra, or a microarray, provided that some sample measurements are replicated across multiple batches. The 1-step method computes all calibration results iteratively from all measurements. It returns the most probable values for the sample compositions under the assumptions of a statistical model, making them the maximum likelihood predictors. It is less sensitive to measurement error on standards and enables use of some batches that do not include standards. In direct comparison of both real and simulated immunoblot data, the 1-step method consistently exhibited smaller errors than the conventional "2-step" method. These results suggest that the 1-step method is likely to be most useful for cases where experimenters want to analyze existing data that are missing some standard measurements and where experimenters want to extract the best results possible from their data. Open source software for both methods is available for download or on-line use.

CV2 antibodies (CV2-ab) associate with paraneoplastic neurological syndromes (PNS) and small-cell lung cancer. This study was designed to assess the sensitivity of two widely used anti-CV2 commercial kits. Fifty three sera with CV2-ab identified by immunohistochemistry on paraformaldehyde-perfused rat brain were tested with two commercial immunoblot kits (Euroimmun AG, and Ravo Diagnostika) and 4 (7.5%) of them were negative with the commercial kits. The 4 samples were positive by immunofluorescence on HEK293 cells transfected with CRMP5 and immunoblot of these cells lysate. A few CV2-ab-positive sera may be missed by commercial immunoblots. Negative samples from patients with high suspicion for PNS should be tested by alternative methods., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Quantitative analysis and modeling of signaling pathway components can reveal important information about the dynamics of that system, including the relative stoichiometries and affinities between the individual signaling components, as well as rate-limiting steps in the signaling pathway. In this chapter, we present a method that we developed to quantify the steady-state ratio of core Hedgehog (Hh) signaling components in both cultured cells and the Drosophila embryo, a physiologically relevant tissue.

The loading controls used for quantitative immunoblotting of mammalian proteins are not appropriate for use with Dictyostelium discoideum. Actin levels, for example, change greatly during Dictyostelium development. In addition, Dictyostelium-specific antibodies for other potential control proteins are not commercially available. Here we demonstrate the use of labeled streptavidin to detect biotinylated mitochondrial 3-methylcrotonyl-CoA carboxylase α (MCCC1), providing a robust and convenient tool for quantitative normalization of Dictyostelium Western blots, as well as fluorescently labeling mitochondria for microscopy of fixed cells.

A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th-16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.

Various serological tests were used for the diagnosis of glanders in the past but still complement fixation test (CFT) is the internationally prescribed test for trading equines. A new immunoblot (IB) technique has recently been introduced to overcome the well known shortcomings of CFT i. e. a considerable number of false positive and negative results and anticomplementary effects of sera. The objective of this study was the comparative evaluation of two glanders CFT antigens commercially available at Central Veterinary Institute ofWageningen UR, Lelystad, NL (CIDC) and at c.c.pro GmbH, Oberdorla, DE (c.c.pro) in a glanders endemic area regarding specificity and sensitivity. A total of 1678 serum samples from the endemic region (Province Punjab, Pakistan) and a non-endemic area (Germany) were analysed. All sera tested positive or suspicious with CFT were analysed by the confirmatory IB to exclude CFT false positive results. Both CFT antigens showed 100% sensitivity. The use of CIDC or c.c.pro antigen resulted in specificities of 77.45% or 75.71% for sera from endemic area and 93.75% or 94.79% for sera from non-endemic areas, respectively. The results demonstrate the different performances of identical tests in different epidemiologically settings. Based on these results, the combined use of CFT and IB is highly suggestive for the serodiagnosis of glanders. Good agreement was calculated between CFT (using either c.c.pro or CIDC antigen) and immunoblot.

The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and P-glycoprotein (Pgp) are important in the distribution and elimination of many drugs and endogenous metabolites. Due to their membrane location and hydrophobicity it is difficult to generate purified protein standards to quantify these transporters in human tissues. The present study generated transporter proteins fused with the S-peptide of ribonuclease for use as standards in immunoquantification in human liver and small intestine. Quantification of the S•tag™, a 15 amino acid peptide, is based on the formation of a functional ribonuclease activity upon its high affinity reconstitution with ribonuclease S-protein. S-tagged transporters were used as full-length protein standards in the immunoquantification of endogenous BCRP, MRP2, and Pgp levels in 14 duodenum and 13 liver human tissue samples. Expression levels in the duodenum were 305±248 (BCRP), 66±70 (MRP2), and 275±205 (Pgp) fmoles per cm(2). Hepatic levels were 2.6±0.9 (BCRP), 19.8±10.5 (MRP2), and 26.1±10.1 (total Pgp) pmoles per g of liver. The mean hepatic scaling factor was 35.8mg crude membrane per g of liver, and the mean duodenal scaling factor was 1.3mg crude membrane per cm(2) mucosal lining. Interindividual variability was greater in duodenal samples than liver samples. It is hoped that this innovative method of quantifying these transporters (and other membrane proteins) will improve in vivo-in vitro extrapolation and in silico prediction of drug absorption and elimination, thus supporting drug development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Elevated protein oxidation is a widely reported hallmark of most major diseases. Historically, this 'oxidative stress' has been considered causatively detrimental, as the protein oxidation events were interpreted simply as damage. However, recent advances have changed this antiquated view; sensitive methodology for detecting and identifying proteins susceptible to oxidation has revealed a fundamental role for this modification in physiological cell signalling during health. Reversible protein oxidation that is dynamically coupled with cellular reducing systems allows oxidative protein modifications to regulate protein function, analogous to phosphoregulation. However, the relatively labile nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. Consequently, specialized methods to stabilize protein oxidation in combination with techniques to detect specific types of modification have been developed. Here, these techniques are discussed, and their sensitivity, selectivity and ability to reliably identify reversibly oxidized proteins are critically assessed.

α-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinson's disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.

Objective: Serological testing for myositis-specific or associated autoantibodies [myositis-specific antibody (MSA) and myositis-associated antibody (MAA)] is useful for the diagnosis of idiopathic inflammatory myopathies (IIMs). However, available assays are neither standardized nor validated. The objective is to evaluate the accuracy of a commercial line blot assay for myositis diagnosis., Methods: IgG antibodies against Jo-1, PL-7, PL-12, PM/Scl, Ku, Mi-2 and Ro52 antigens were detected by a line blot and in-house RNA immunoprecipitation or immunoblot. We tested sera from 208 IIM patients, 50 healthy subjects and 180 control patients (11 non-autoimmune myopathy, 23 muscular dystrophy, 11 UCTD, 68 SLE, 36 SSc, 22 SS and 9 arthropathy)., Results: MSAs or MAAs were detected in 98 (47%) out of the 208 IIM patients by line blot: anti-Jo-1 in 43 (21%), anti-PL-7 or anti-PL-12 in 8 (4%), anti-Mi-2 in 9 (4%), anti-PM/Scl in 9 (4%), anti-Ku in 10 (5%) and anti-Ro52 in 49 (24%). Overall specificity was: 100% for anti-Jo-1, anti-PL-7 or PL-12 and anti-PM/Scl; 96% for anti-Ku; 98% for anti-Mi-2; and 76% for anti-Ro52. In-house testing confirmed line blot results regarding anti-Jo-1, anti-PM/Scl and anti-Ku, while it was more accurate than line blot in detecting anti-Mi-2 (7 vs 4% sensitivity, 100 vs 98% specificity), and anti-aminoacyl-tRNA synthetase (anti-ARS) non-Jo-1 antibodies (11 vs 4% sensitivity, 97 vs 99% specificity)., Conclusions: Line blot could be a suitable serological test in the diagnostic workup for myositis, and it represents a reliable alternative to more time-consuming procedures. Continuous effort is recommended in order to improve its accuracy.

We studied the diagnostic accuracy of a rapid immunochromatographic test (ICT) for detection of IgM against scrub typhus (ST ICT) and an immunoblot test for the detection of IgM against murine typhus (MT IBT) by using admission serum samples from 1,030 febrile patients in Laos. Sensitivity and specificity for the ST ICT determined by using the diagnostic criteria of a four-fold increase in IgM against Orientia tsutsugamushi between paired samples were 23.8% (95% confidence interval [CI] = 15.9-33.3%) and 86.2% (95% CI = 84.1-88.6%), respectively. Sensitivity and specificity for the ST ICT determined by using an admission IgM titer > or = 1:400 were 39.1% (95% CI = 34.1-44.2%) and 99.5% (95% CI = 98.7-99.9%), respectively. Sensitivity and specificity for the MT IBT determined by using the criteria of a four-fold increase in IgM against Rickettsia typhi between paired serum samples were 61.2% (95% CI = 53.7-68.3%) and 86.5% (95% CI = 84.1-88.8%), respectively. Sensitivity and specificity for the MT IBT determined by using an admission IgM titer > or = 1:400 were 54.6% (95% CI = 49.1-60.0%) and 94.1% (95% CI = 92.0-95.7%), respectively. Both assays had relatively good specificity but low sensitivity and thus have limited utility for admission diagnosis.

Various attempts have been made to improve Epstein-Barr virus (EBV) serodiagnosis by developing more practical and objective methods than immunofluorescence-based assays. In the present study, the performance of immunoblot-based assays were evaluated by comparing the results obtained by the gold standard immunofluorescence antibody (IFA) test for the detection of IgM and IgG antibodies against EBV viral capsid antigen (anti-VCA). Serum samples of 277 patients admitted to Ege University Hospital for routine EBV diagnosis were included in the study. The age range of the patients was 3 months-89 years (mean 28 years) and 104 of them were females and 173 were males. All the samples were assayed by commercial immunoblot (Euroline IgM and IgG; Euroimmun, Germany) and IFA (EBV-CA IgG and IgM, Euroimmun, Germany) methods. Crosstabulation, chi-square test and phi (phi) measures in SPSS 16.0 statistical package programme were used for data analysis. Of the 216 samples that were interpreted as positive with immunoblot-based IgM assay, only 34 (15.7%) were confirmed as positive with IFA, whereas 162 (75%) were negative, and 20 (9.3%) were equivocal (phi = 0.167; low correlation). Of the 85 samples that were anti-VCA IgG positive with immunoblot assay, 82 (96.5%) were positive, 2 (2.3%) were negative and 1 (1.2%) were equivocal with IFA (phi = 0.441; significant correlation). When the indeterminate results obtained by IFA test were excluded from the evaluation, the correlation between immunoblot VCA IgG and IFA IgG was 85.4% (88/103) and between immunoblot VCA IgM and IFA IgM was 27.3% (69/253). When the intensities of bands were evaluated for IgM testing, it was noted that as the intensity of the bands increased (1+ to 3+), IFA VCA IgM reactivity rates increased (from 9.9% to 29.5% for p19 band; from 24% to 85.7% for gp125 band). For immunoblot VCA IgM testing, 165 samples were found to be positive only for VCA p19 band. Of these samples, 135 (81.8%) were negative, 15 (9.1%) were positive and 15 (9.1%) were equivocal with IFA. It is observed that even though immunoblot assays with automated blotting and scanning systems can be a convenient alternative to immunofluorescence assay, the rate of false positivity obtained for VCA IgM was high (75%). It was concluded that in laboratories which apply immunoblotting as a primary screening test for EBV serodiagnosis, the positive VCA IgM results (particularly isolated p19 band positivity) and the presence of low intensity bands, should be confirmed by IFA testing.

Beef tenderness is a very complex and multifactorial sensorial meat quality trait, which depends partly on muscle characteristics. This tissue is very variable according to animal type (age, breed and sex) and rearing conditions. Consequently, beef tenderness exhibits a great variability. Different research programs have revealed several genes or proteins which could be good markers of beef tenderness. In order to validate the relation of these markers with beef tenderness on a large population of bovines, it is necessary to have a large-scale and trusty technique which can access different quantities of proteins related to tenderness. In this study we firstly compared Western-Blot and Dot-Blot. Secondly, we evaluated Dot-Blot technical and biological capabilities for the quantification of protein biomarkers. The results demonstrated that the Dot-Blot technique with fluorescence detection presents numerous interests. This technique allows a good reproducibility and permits the simultaneous analysis of a large number of samples. The Dot-Blot technique defined and validated in this study can be used for protein biomarkers analyses, notably to predict beef tenderness. Another major result of this study is that about 5 to 10 animals per group are required to detect large differences (>1.5) in biomarker expression between tender and tough beef, whereas much larger numbers of animals (10 to 30) are required to detect smaller differences (about 1.2 to 1.3) taking into account the biological variability of these markers.

Antimicrobial peptides (AMP) are an integral component of innate immunity. One of the most widespread AMP in fish are the piscidins, which have potent, broad-spectrum activity against viruses, bacteria, fungi, and parasites. The widespread phylogenetic distribution of piscidins suggests that they might play an important host defense role in many fish. Quantifying their expression is important in understanding how and where they function. Using a novel piscidin (piscidin 4) that we recently isolated from commercially cultured hybrid striped bass (white bass, Morone chrysops Rafinesque, female symbolxstriped bass, Morone saxatilis Walbaum male symbol), we optimized the conditions for measuring this piscidin via sandwich ELISA. We used an antibody to the highly conserved amino terminus of all piscidins as the capture antibody and a peroxidase-labeled antibody specific for the carboxy terminus of piscidin 4 as the detecting antibody. Specificity of the detecting antibody was confirmed by lack of cross-reactivity with other piscidins in ELISA, as well as specificity for piscidin 4 in tissue extracts via Western blotting. The accuracy of the test, defined as piscidin 4 recovery, was 96-103%. Precision, measured by the coefficient of variation, was 13-19%, and parallelism, determined by linearity of the response, had an r(2)>0.99. The ELISA paralleled the results obtained via Western blotting. Piscidin 4 levels expressed in gill tissue of healthy hybrid striped bass were well within concentrations that are lethal to important fish pathogens. Mean gill piscidin 4 in healthy hybrid striped bass was significantly greater than in either nutritionally stressed fish or in diseased (ectoparasite-infested) fish, suggesting that piscidin 4 can be significantly downregulated with stress or disease. These data suggest that the piscidin 4 ELISA might be a useful indicator of disease susceptibility, providing a new, sensitive tool for rapid screening of population health.

We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T. cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, São Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T. cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi.

Immunoblot is currently the preferred laboratory test to assist the diagnosis of limb-girdle muscular dystrophy (LGMD) 2A (calpainopathy). To assess whether immunohistochemistry may offer a reliable alternative screening we used two antibodies, Calp3-2C4 (exon 1) and Calp3-12A2 (exon 8), to label blots and sections of skeletal muscle from controls and patients with LGMD2A and other muscle diseases. In LGMD2A muscle biopsies a high degree of concordance was found with Calp3-2C4: labelling on sections was absent in patients with no bands on immunoblot and detected in those where CAPN3 bands were seen. Calp3-12A2 results were less consistent, with most samples retaining labelling. Interestingly, CAPN3 was found in all muscle sections from disease control patients irrespective of its detection on immunoblot. Our results show that immunohistochemistry with Calp3-2C4 has a similar pickup rate of LGMD2A as immunoblot and it may therefore be useful for distinguishing the majority of genuine CAPN3 defects from secondary protein reduction. However immunoblot is still needed when CAPN3 is present on sections to show secondary CAPN3 reduction and to identify LGMD2A with variable reduction of CAPN3 bands.

Facile assays for ADAMTS13, the metalloprotease that is absent or impaired in thrombotic thrombocytopenic purpura, could allow for timely diagnosis and management of this potentially fatal hematologic disorder; unfortunately, available assays employ methodologies that restrict use to only a few reference laboratories, resulting in reporting delays. Measurement of ADAMTS13 on an automated analyzer could allow widespread accessibility by routine clinical laboratories. We adapted a previously published technique to demonstrate that ADAMTS13 activity could be measured on an Instrumentation Laboratory automated coagulation analyzer after plasma digestion of a commercial source of von Willebrand factor (VWF). Results were obtained using a commercially available immunoturbidimetric assay for residual VWF activity. Samples from 114 patients with suspected thrombotic microangiopathy were analyzed by both standard immunoblot and our new semiautomated method with excellent agreement, particularly at the clinically relevant low levels of ADAMTS13 activity. This new method is semiautomated and offers quantitative results within 2 hr; our modifications allow for widespread applicability on instrumentation already in use by routine clinical laboratories., (Copyright 2008 Wiley-Liss, Inc.)

Background: Due to the westernization of living environments in Korea, a number of allergy patients are greatly increasing. External quality control assessments are difficult for allergy tests due to the lack of reference methods. We surveyed the current status of allergy tests performed in medical laboratories in Korea to prepare for an external quality control assessment in the near future., Methods: We conducted a survey on internal and external quality control trials, calibrations and its intervals, medical staffs, analyzers in use, an average number of tests per months, and report formats., Results: Among the 85 laboratories surveyed, 61 were doing allergy tests including 6 reference laboratories. There were two different types of analyzers (1) qualitative or semi-quantitative and (2) quantitative. These analyzers use either chemiluminescent or immunoblot method. Fifty-five laboratories were using 'qualitative or semi-quantitative' analyzers as a screening test and 31 laboratories were using quantitative analyzers. Most of the laboratories were only doing an internal quality control assessment included in the test kits. Excepting a few laboratories, calibrations were not done. About 34 laboratories reported numerical values with interpretative reports prepared by laboratory medical doctors., Conclusions: The necessity of external quality control assessment has become an issue for improving the quality of allergy tests. But due to the lack of standardization, it is difficult to carry out external quality control assessments. By grouping the laboratories in terms of the type of analyzers, we could overcome the problem of analyzer variations and launch an external quality control assessment program in the near future.

Objective: To evaluate the detergent and aqueous phases of crude saline extract (crude) of Taenia solium metacestodes extracted with Triton X-114, for the detection of immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA) and immunoblot for the laboratory diagnosis of human neurocysticercosis., Method: Serum samples were collected from 138 individuals and tested; 40 were diagnosed with neurocysticercosis (NC; group 1), 58 presented other parasitic diseases (group 2) and 40 were apparently healthy (group 3)., Results: The detergent phase showed only one dominant component (70-50 kDa), while the aqueous phase showed four (116, 110, 97 and 77 kDa) by silver staining. ELISA sensitivity and specificity were 92.5% and 84.5% for crude phase, 92.5% and 93.3% for detergent phase and 72.5% and 72.6% for aqueous phase., Conclusions: The immunoblot of the antigens confirmed the results obtained by ELISA. Detergent phase purified from T. solium metacestodes was an important source of specific peptides and was efficient in detecting T. solium metacestodes antibodies in serum samples. This phase can be used for detecting IgG antibodies to T. solium metacestodes in seroepidemiological investigations and in the diagnostic screening of NC patients.

Human cystic echinococcosis is a severe zoonotic infection caused by the larval stage of the taeniid tapeworm Echinococcus granulosus. The infection may be fatal if proper treatment is not provided; hence, early diagnosis is very important. Currently, ELISA and immunoblotting are the most reliable tests for serodiagnostic purposes, although their accuracy is largely dependent on the quality of the antigenic source used. Hydatid cyst fluid has been the antigenic extract of choice for primary immunodiagnosis of the disease, which is mainly based on the detection of antigens B and 5. Several problems are associated with this extract, however, including a lack of sensitivity and specificity, and difficulties with standardization of its use. This paper reviews recent advances in the identification and characterization of novel antigens that may be useful for the immunodiagnosing of human cystic echinococcosis, with emphasis on progress in recombinant technologies and synthetic peptides. Novel approaches are discussed, such as the design of antigenic extracts from other developmental stages of the parasite, as well as the usefulness of serum cytokine detection in the clinical follow-up of affected patients after surgical or pharmacological treatment.

Objective: To assess the reliability of a new measurement of prostate-specific antigen (PSA) using a blotting-paper assay (nanotest) compared to the standard PSA immunoassay., Subjects and Methods: The PSA level was measured in 205 men volunteers (median age 70 years, range 41-75) using a nanotest and a standard PSA immunoassay, collected at the same time; 30 microL of capillary blood placed on to a blotting paper were collected for the nanotest and sent by mail to the same laboratory for the two assays. The results were compared statistically using the Spearman test, the intraclass correlation coefficient and the Bland-Altman test., Results: The nanotest threshold for an abnormal PSA level was 78 pg/mL, which corresponded to a standard PSA value of 3 ng/mL, with a sensitivity of 100%. There was a significant correlation (r = 0.98, Spearman test; P < 0.001) between the nanotest and the standard PSA assay. The intraclass correlation coefficient was 0.87. The Bland-Altman test showed a good agreement between the nanotest and the standard PSA assay, but there was an increasing proportional difference with increasing PSA value., Conclusion: There was a very high correlation between the nanotest and the standard PSA assay, especially for standard PSA levels of <5 ng/mL. Economic and clinical studies are indicated to confirm the utility of the nanotest in organized mass screening of prostate cancer.

An ultrasensitive, simple, and fast immunoassay for biotin-peptide detection using gold nanoparticles conjugated with antibodies has been developed. Biotin was covalently attached to a peptide and the biotin-peptide bound on a nitrocellulose membrane. Antibody-coated gold nanoparticles bound to the biotin-peptide formed red dots. With this method, 100 amol of the biotin-peptide was detected and no immunogold was bound to the membrane in the absence of biotin. The relative intensity of each dot was scored using Quantity One, a quantitative analysis software program. The linear working range of this assay was between 1 pmol and 1 micromol. The assay sensitivity was increased by silver enhancement to 100 zmol, and the linear working range was between 100 zmol and 100 fmol. This assay can be extended to detect target molecules, such as dioxin, digoxin, mercury, and so on, with matched antibodies and has potential broad applications in immunoassay.

Diagnosis of the parasitic infection cysticercosis is usually confirmed by serological assays. The electroimmunotransfer blot (EITB) for cysticercosis is a sensitive and specific assay, which uses six glycoprotein antigens on a strip to detect antibodies to Taenia solium cysticerci. Although the appearance of bands at any of these six sites is considered to be a positive result, a growing body of evidence suggests that the presence of a single 50-kDa band in this assay may not indicate infection. An audit of 984 samples tested over a 3-year period showed that only two (15.4%) of 13 samples with a single 50-kDa band were associated with a diagnosis of cysticercosis. Possible reasons for this include technical problems, cross-reactivity with other parasites or other diseases, or the presence of a non-specific band. The results suggest that the finding of a single 50-kDa band should be interpreted with caution.

Background: The routine use of hepatitis C virus (HCV) nucleic acid amplification testing (NAT) donor screening assays has provided an opportunity for revision of the current HCV supplemental testing algorithm, which requires that recombinant immunoblot assay (RIBA) be performed on every HCV enzyme immunoassay (EIA)-repeat-reactive donation. The FDA has approved variance requests to use a new algorithm that eliminates the need to perform RIBA when HCV NAT results are reactive. Data are provided in support of this new algorithm., Study Design and Methods: HCV EIA (including signal-to-cutoff optical density ratio), RIBA, and NAT data were compiled from 33.2 million donations screened over an approximately 4-year period by the American Red Cross and Blood Systems Laboratories. Further, donations having specific combinations of HCV EIA, RIBA, and minipool (MP) NAT results were evaluated, with more sensitive individual-donation (ID) NAT, to construct improved counseling messages for donors., Results: Of 47,041 EIA-repeat-reactive donations, 49.3 percent were RIBA-positive, 17.1 percent RIBA-indeterminate, and 33.5 percent RIBA-negative. NAT-reactive rates were 79.2, 2.5, and 0.18 percent for RIBA-positive, -indeterminate, and -negative donations, respectively. The new algorithm classified an additional 1 percent of donations as HCV-infected while at the same time detecting all infections classified as HCV-infected under the current algorithm. An additional 2.4 percent of RIBA-positive, MP NAT-nonreactive donations were reactive when a frozen-thawed aliquot was retested by ID NAT., Conclusion: Integrating HCV NAT results with RIBA results for purposes of donor notification allows more appropriate counseling messages to be given to EIA-repeat-reactive donors. The new HCV supplemental algorithm is an acceptable alternative to the current algorithm because it provides equivalent or superior accuracy in formulating donor counseling messages and may also result in reduced costs and more timely notification of infected donors.

Antibodies that specifically recognize signaling proteins (or individual phosphorylation events at specific residues in proteins of interest) have become important tools in the study of signaling pathways. However, the recognition properties of many commercially available antibodies have not been fully characterized. In the course of studies exploring PKC-delta phosphorylation mechanisms in cardiomyocytes, we have demonstrated that a BD Transduction Laboratories anti-PKC-delta MAb (generally viewed as an anti-PKC-delta protein antibody) recognizes PKC-delta in resting, but not in PMA-treated, cardiomyocytes. The observations that PKC-delta immunoreactivity is preserved when cultures are treated with PMA in the presence of a the PKC inhibitor GF-109203X and that PKC-delta immunoreactivity is restored by in vitro acid phosphatase treatment indicate that the epitope recognized by the BD Transduction Laboratories anti-PKC-delta MAb is masked by phosphorylation. The BD Transduction Laboratories MAb is poorly suited for studies that compare PKC-delta expression in resting and agonist-activated samples (or in studies of the relationship between PKC-delta phosphorylation and PKC-delta downregulation) because it artifactually displays PKC-delta phosphorylation as a decline in total PKC-delta protein. Other studies have shown that two anti-PKC-delta-pY(311) antibodies (manufactured by Cell Signaling Technology, Beverly, MA, and BioSource International, Camarillo, CA, respectively) specifically recognize stimulus-induced changes in PKC-delta-Y(311) phosphorylation on the endogenous PKC-delta enzyme, but the Cell Signaling Technology anti-PKC-delta-pY(311) antibody provides a better measure of Y(311) phosphorylation in overexpressed PKC-delta. Collectively, these studies have identified features of anti-PKC-delta antibodies that affect the interpretation of immunoblot analysis experiments. These findings related to PKC-delta may be symptomatic of a more pervasive feature of immunoblot analysis studies of phosphoproteins in general.

Restless legs syndrome (RLS) is a neurological disorder that may be related to iron misregulation at the level of the central nervous system. Evidence that iron is involved in RLS comes from magnetic resonance imaging data, autopsy studies, analyses of cerebrospinal fluid (CSF), and correlations of symptoms with serum ferritin. To further examine the possibility that brain iron status is insufficient in RLS, we determined ferritin levels in the CSF. Specifically, we differentiated between the H- and L-subunits of ferritin, because these peptides are expressed from different chromosomes and have different functions. We measured H- and L-ferritin subunit levels in control and RLS human CSF using immunoblot analysis and found that both H- and L-ferritin are significantly decreased in early but not late-onset RLS. Additionally, we quantified total protein in each CSF sample to establish that the decrease in ferritin subunits in RLS did not reflect a decrease in total protein in CSF. Furthermore, we used equal amounts of total CSF protein in the immunoblot analyses, in contrast to previously published studies that provided only volumetric data, to determine which approach was more accurate for quantifying the amount of ferritin relative to other proteins in CSF. Our results establish a protein standard in RLS, provide a comparative analysis of protein-controlled versus volumetric immunoblot techniques, and argue for a profound loss of iron storage capacity in the brain in RLS, specifically in the early onset RLS phenotype. These data suggest that CSF ferritin levels may provide a biomarker for assisting in the diagnosis of RLS.

Immunohistochemical assessment of 5-bromo-2-deoxyuridine (BrdU) in tissue sections is a widely used method to evaluate cell proliferation in vivo. However, this method requires time-consuming preparation of paraffin sections and laborious counting of BrdU-labeled nuclei on multiple sections. Here, we report the development of a rapid and reliable method to quantitate BrdU incorporation in intestinal and liver tissues using a dot-blot method. In vivo models of colon or liver proliferation were used to analyze the usefulness and reliability of this new method. Mice were killed after BrdU injection, and genomic DNA was isolated from the tissues, denatured, and dot-blotted onto a nitrocellulose membrane. The incorporated BrdU was detected with a BrdU monoclonal antibody, and the signal intensity was densitometrically quantified. Results were compared with BrdU index determined by conventional immunohistochemistry on the same tissue samples. The patterns of colonic BrdU incorporation during fasting and refeeding, measured by the dot-blotting method and the immunohistochemical method, were similar. The BrdU incorporation in the regenerating liver after partial hepatectomy, evaluated by these two different methods, showed a strong correlation (R(2) = 0.91, P < 0.01). In addition, the inhibition of colon proliferation by the phosphoinositol 3-kinase inhibitor wortmannin was demonstrated by this dot-blotting method. In conclusion, the dot-blotting technique described in this report provides an accurate, more efficient, and possibly more reliable method for the assessment of in vivo proliferation compared with conventional immunohistochemical determination of BrdU-labeling index.

Immunotherapy to prevent recurrence of clinical signs of atopic dermatitis (AD) is based on intradermal or serological tests that assist in identifying allergen-specific immunoglobulin E hypersensitivities. Unfortunately, the results of such tests can be negatively influenced by several factors, which include the age of the patients, the season of testing and the administration of anti-allergic drugs. Screening to predict when these expensive tests will be useful would benefit owners of dogs with AD. The objectives of this study were to determine whether a point-of-care allergen-specific immunodot assay (Allercept E-Screen, Heska Corp., Ft Collins, CO, USA) could predict results of either intradermal or Allercept full panel serological tests in atopic dogs. Thirty dogs living in the south-eastern USA were diagnosed with AD in accordance with current standards. Allergen-specific intradermal, serological and E-Screen tests were performed in all subjects. For flea, house dust mite and pollen allergens altogether, results of the E-Screen assay agreed with those of intradermal and serological tests in 26/30 dogs (87%) and 25/30 dogs (83%), respectively. In this group of dogs, the probabilities of obtaining intradermal or serological tests positive for these allergens were 70 and 67%, respectively. If either skin or serum tests were performed only in dogs with positive E-Screen tests, the probability of obtaining positive results would be increased from 70 to 95% and from 67 to 90%, respectively. In this population of dogs with AD, results of the E-Screen point-of-care immunodot assay was found to often agree with those of allergen-specific intradermal or Allercept tests for selected allergen groups.

Methods for the measurement of autoantibodies frequently provide controversial results. The objective of the present study was to evaluate the performance of Spanish Clinical Laboratories in the measurement of anti-Sm antibodies. A total of 23 laboratories participated, analysing 30 serum samples from patients with systemic lupus erythematosus and other autoimmune and non-autoimmune diseases. The laboratories used four extractable nuclear antigen screens, eight enzyme-linked immunosorbent assays (ELISAs) specific for anti-Sm, one line-blot, one dot-blot and one double immunodiffusion assay, from 15 different manufacturers. A total of 871 results were obtained. In general, very good sensitivity was obtained (95-100%), but specificity was moderate (52-86%) and must be improved. Most ELISAs and the line-blot were valid assays for anti-Sm detection and could serve as tests both for analysis and/or confirmation. The likelihood ratios indicated that both methods can be considered very useful or useful for the determination of anti-Sm antibodies. Nevertheless, the analytical quality of the methods for the measurement of anti-Sm antibodies could probably be improved by standardisation of the methods and the participation of laboratories in external quality control programs.

A standard operating procedure has been developed for an immunoslotblot assay of sulfur mustard adducts to DNA in human blood and skin for use in a field laboratory. A minimum detectable level of exposure of human blood in vitro (> or = 50 nM) sulfur mustard is feasible with the assay. In the case of human skin, a 1 s exposure to saturated sulfur mustard vapor (830 mg/m(-3)) could still be detected.

Rapidly progressive glomerulonephritis (RPGN) is characterized by rapid and progressive loss of renal function and the presence of crescentic glomerulonephritis (CGN). Early diagnosis and appropriate treatment is mandatory to prevent death and/or renal failure. We have evaluated an ANCA-GBM dot-blot diagnostic test in terms of sensitivity, specificity, and inter-observer effect in consecutive patients with RPGN ( n = 82). Control sera ( n = 34) included healthy and relevant disease controls. Dot-blots were independently evaluated by nine observers. Proteinase 3 (PR3)-ANCA, myeloperoxidase (MPO)-ANCA, and both were detected by ELISA in 36, 32, and 3 samples of 71 patients with pauci-immune CGN, respectively. Two additional samples were ANCA negative. The dot-blot revealed a sensitivity of 92-95% for PR3-ANCA and 80-86% for MPO-ANCA. The specificity of the dot-blot for PR3- and MPO-ANCA was 100%. In the patients with anti-GBM nephritis ( n = 9) anti-GBM was detected by both ELISA and dot-blot (sensitivity: 100%). The specificity of the anti-GBM dot-blot was 91-94%. However, the inter-observer effect was relatively high for detection of anti-GBM antibodies (24%). In conclusion, the ANCA-GBM dot-blot is a useful screening tool in situations where conventional ANCA testing is not readily available with excellent performance for PR3-ANCA detection, but less optimal sensitivity for MPO-ANCA and specificity for anti-GBM detection. Therefore, it is recommended to include the following advises in the report to the physicians: 1) patients with a high clinical suspicion for MPO-ANCA-associated RPGN and negative dot-blot must have conventional analysis for MPO-ANCA, and 2) negative anti-GBM dot-blot makes anti-GBM disease very unlikely, but positive samples should be confirmed by conventional anti-GBM tests.