Refining Timely Diagnosis of Early Syphilis by Using Treponema pallidum PCR or IgM Immunoblotting Next to Conventional Serology for Syphilis.

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.

Immunoblot for the Diagnosis of Cutaneous Leishmaniasis in French Guiana.

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.

Development of rSs-NIE-1 and rSs-IR Recombinant Antigen-Based Immunoblot for Detection of Antibody to Strongyloides stercoralis.

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.

Evaluation of the diagnostic performance of an immunoblot for ANCA and anti-GBM antibody detection.

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.

A new diagnostic algorithm for pattern-oriented autoantibody testing according to the ICAP nomenclature: A pilot study.

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.

Diagnostic yield of commercial immunodots to diagnose paraneoplastic neurologic syndromes.

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.

Further evaluation of recombinant Tsol-p27 by enzyme-linked immunoelectrotransfer blot for the serodiagnosis of cysticercosis in pigs from Mozambique.

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.

[Evaluation of Enzyme Immunoassay (EIA), Immunoblot and HIV RNA Polymerase Chain Reaction Test Results in the Diagnosis of Human Immunodeficiency Virus (HIV) Infection]. / Insan Immün Yetmezlik Virüsü (HIV) Enfeksiyonu Tanisinda Enzim 'Immunoassay' (EIA), Immünoblot ve HIV RNA Polimeraz Zincir Reaksiyonu Test Sonuçlarinin Degerlendirilmesi.

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.

Performance of four diagnostic assays for detecting herpes simplex virus type 2 antibodies in the Middle East and North Africa.

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).

An Automated Immunoblot Method for Detection of IgG Antibodies to Hepatitis C Virus: a Potential Supplemental Antibody Confirmatory Assay.

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.

Validation of an immunoblot assay employing an objective reading system and used as a confirmatory test in equine infectious anaemia surveillance programs.

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.

Potential pitfalls in analyzing structural uncoupling of eNOS: aging is not associated with increased enzyme monomerization.

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.

Evaluation of a Novel Microarray Immunoblot Assay for Detection of IgM- and IgG-Class Antibodies to Borrelia burgdorferi.

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.

Guidelines for authors and reviewers on antibody use in physiology studies.

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/ .

A Method and On-Line Tool for Maximum Likelihood Calibration of Immunoblots and Other Measurements That Are Quantified in Batches.

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.

Pitfalls in the detection of CV2 (CRMP5) antibodies.

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.