Clinical autoantibody detection by microarray

2017 ◽  
Vol 55 (4) ◽  
Author(s):  
Doreen Dillaerts ◽  
Heidi De Baere ◽  
Xavier Bossuyt
Keyword(s):  
Blood Donors ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Systemic Autoimmune Disease ◽  
Farr Assay ◽  
Nuclear Antigens ◽  
Antibody Assays ◽  
Low Sensitivity ◽  
Autoantibody Detection ◽  
Dsdna Antibodies

Abstract Background: AMiDot is a microdot array-based immunoassay that allows simultaneous detection of multiple autoantibodies on a single patient. We evaluated the AMiDot “Systemic Autoimmune Disease” (SAD) panel, which detects antibodies to 17 different antigens. Methods: AMiDot was performed on 184 samples from blood donors and on 280 randomly selected clinical samples containing antibodies to extractable nuclear antigens or to dsDNA. The results obtained by AMiDot on the clinical samples were compared to results obtained by EliA (Thermo Fisher) for anti-Ro60, anti-La, anti-RNP, anti-Scl-70, anti-CENPB, anti-Sm, and anti-Jo-1 and by Farr assay for anti-dsDNA. Discordant results were further analyzed by immunodot (D-tek). Results: Concordance between AMiDot and EliA was ≥87% and κ agreement ≥0.44. When compared to EliA and immunodot (in case of discordance between AMiDot and EliA), concordance improved to ≥91% and κ agreement to ≥0.77. The sensitivity of AMiDot (compared to EliA and immunodot, in case of discordance between AMiDot and EliA) was ≥93%, except for anti-Ro60 (84%). The concordance and κ agreement of AMiDot with the Farr assay (for dsDNA antibodies) was, respectively, 84% and 0.33. The sensitivity of AMiDot for dsDNA (compared to Farr assay) was 25%. The specificity was ≥97% (in blood donors as well as in clinical samples). The within-run imprecision was 9%–27% and the between-run imprecision 29%–39%. Conclusions: AMiDot offers an alternative to line immunodot assay. Individual antibody assays might suffer from low sensitivity.

scholarly journals Label-Free Plasmonic Biosensor for Rapid, Quantitative, and Highly Sensitive COVID-19 Serology: Implementation and Clinical Validation

2021 ◽  
Author(s):  
Olalla Calvo-Lozano ◽  
MIquel Sierra ◽  
Maria Soler ◽  
M.-Carmen Estevez ◽  
luis Chiscano-camon ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Clinical Validation ◽  
Label Free ◽  
Serological Tests ◽  
N Protein ◽  
Lateral Flow Assays ◽  
Low Sensitivity ◽  
Biosensor Device

Serological tests are essential for the control and management of COVID-19 pandemic, not only for current and historical diagnostics but especially for surveillance, epidemiological, and acquired immunity studies. Clinical COVID-19 serology is routinely performed by enzymatic or chemiluminescence immunoassays (i.e., ELISA or CLIA), which provide good sensitivities at the expense of relatively long turnaround times and specialized laboratory settings. Rapid serological tests, based on lateral flow assays, have also been developed and widely commercialized, but they suffer from limited reliability due to relatively low sensitivity and specificity. We have developed and validated a direct serological biosensor assay employing proprietary technology based on Surface Plasmon Resonance (SPR). The biosensor offers a rapid -less than 15 min- identification and quantification of SARS-CoV-2 antibodies directly in clinical samples, without the need of any signal amplification. The portable plasmonic biosensor device employs a custom-designed multi-antigen sensor biochip, combining the two main viral antigens (RBD peptide and N protein), for simultaneous detection of human antibodies targeting both antigens. The SPR serology assay reaches detection limits in the low ng mL-1 range employing polyclonal antibodies as standard, which are well below the commonly detected antibody levels in COVID-19 patients. The assay has also been implemented employing the first WHO approved anti-SARS-CoV-2 immunoglobulin standard. We have carried out a clinical validation with COVID-19 positive and negative samples (n=120) that demonstrates the excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor device as an accurate, robust, and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the management of COVID-19 patients and for the evaluation of immunological status during vaccination, treatment or in front of emerging variants.<br>

scholarly journals Label-Free Plasmonic Biosensor for Rapid, Quantitative, and Highly Sensitive COVID-19 Serology: Implementation and Clinical Validation

2021 ◽  
Author(s):  
Olalla Calvo-Lozano ◽  
MIquel Sierra ◽  
Maria Soler ◽  
M.-Carmen Estevez ◽  
luis Chiscano-camon ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Clinical Validation ◽  
Label Free ◽  
Serological Tests ◽  
N Protein ◽  
Lateral Flow Assays ◽  
Low Sensitivity ◽  
Biosensor Device

Serological tests are essential for the control and management of COVID-19 pandemic, not only for current and historical diagnostics but especially for surveillance, epidemiological, and acquired immunity studies. Clinical COVID-19 serology is routinely performed by enzymatic or chemiluminescence immunoassays (i.e., ELISA or CLIA), which provide good sensitivities at the expense of relatively long turnaround times and specialized laboratory settings. Rapid serological tests, based on lateral flow assays, have also been developed and widely commercialized, but they suffer from limited reliability due to relatively low sensitivity and specificity. We have developed and validated a direct serological biosensor assay employing proprietary technology based on Surface Plasmon Resonance (SPR). The biosensor offers a rapid -less than 15 min- identification and quantification of SARS-CoV-2 antibodies directly in clinical samples, without the need of any signal amplification. The portable plasmonic biosensor device employs a custom-designed multi-antigen sensor biochip, combining the two main viral antigens (RBD peptide and N protein), for simultaneous detection of human antibodies targeting both antigens. The SPR serology assay reaches detection limits in the low ng mL-1 range employing polyclonal antibodies as standard, which are well below the commonly detected antibody levels in COVID-19 patients. The assay has also been implemented employing the first WHO approved anti-SARS-CoV-2 immunoglobulin standard. We have carried out a clinical validation with COVID-19 positive and negative samples (n=120) that demonstrates the excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor device as an accurate, robust, and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the management of COVID-19 patients and for the evaluation of immunological status during vaccination, treatment or in front of emerging variants.<br>

scholarly journals Label-Free Plasmonic Biosensor for Rapid, Quantitative, and Highly Sensitive COVID-19 Serology: Implementation and Clinical Validation

2021 ◽  
Author(s):  
Olalla Calvo-Lozano ◽  
MIquel Sierra ◽  
Maria Soler ◽  
M.-Carmen Estevez ◽  
luis Chiscano-camon ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Clinical Validation ◽  
Label Free ◽  
Serological Tests ◽  
N Protein ◽  
Lateral Flow Assays ◽  
Low Sensitivity ◽  
Biosensor Device

Serological tests are essential for the control and management of COVID-19 pandemic, not only for current and historical diagnostics but especially for surveillance, epidemiological, and acquired immunity studies. Clinical COVID-19 serology is routinely performed by enzymatic or chemiluminescence immunoassays (i.e., ELISA or CLIA), which provide good sensitivities at the expense of relatively long turnaround times and specialized laboratory settings. Rapid serological tests, based on lateral flow assays, have also been developed and widely commercialized, but they suffer from limited reliability due to relatively low sensitivity and specificity. We have developed and validated a direct serological biosensor assay employing proprietary technology based on Surface Plasmon Resonance (SPR). The biosensor offers a rapid -less than 15 min- identification and quantification of SARS-CoV-2 antibodies directly in clinical samples, without the need of any signal amplification. The portable plasmonic biosensor device employs a custom-designed multi-antigen sensor biochip, combining the two main viral antigens (RBD peptide and N protein), for simultaneous detection of human antibodies targeting both antigens. The SPR serology assay reaches detection limits in the low ng mL-1 range employing polyclonal antibodies as standard, which are well below the commonly detected antibody levels in COVID-19 patients. The assay has also been implemented employing the first WHO approved anti-SARS-CoV-2 immunoglobulin standard. We have carried out a clinical validation with COVID-19 positive and negative samples (n=120) that demonstrates the excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor device as an accurate, robust, and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the management of COVID-19 patients and for the evaluation of immunological status during vaccination, treatment or in front of emerging variants.<br>

scholarly journals Automated Low Cost Method of Quantification of Hepatitis C Virus

2015 ◽  
Vol 14 (3) ◽  
pp. 247-253
Author(s):  
Faiz MMT Marikar ◽  
Dammika Senevirathna ◽  
Neil Fernandopulle
Keyword(s):  
Viral Load ◽  
Hepatitis C ◽  
Blood Donors ◽  
Low Cost ◽  
Medical Science ◽  
Simultaneous Detection ◽  
Plasma Samples ◽  
Rt Pcr ◽  
Viral Genomes ◽  
Post Therapy

This paper describes the development of a Dig-dUTP based multiplex real time RT-PCR for the simultaneous detection of HCV viral amount in plasma samples. Viral genomes were identified in the same sample by Dig-dUTP PCR 216 bp region. Analysis of known scalar concentrations of reference plasma indicated that the multiplex procedure detects at least 500 copies/ml of HCV. In addition, we also assayed HCV viral load in eighty co-infected patients and in fifteen blood donors, confirming the sensitivity and specificity of the assay. This method may represent a useful alternative method for the detection of HCV co-infection, reliable for a rapid and relatively inexpensive screening of blood donors. The assay may be used to determine post-therapy viral clearance.Bangladesh Journal of Medical Science Vol.14(3) 2015 p.247-253

scholarly journals International multicenter examination of MOG antibody assays

2020 ◽  
Vol 7 (2) ◽  
pp. e674 ◽  
Author(s):  
Markus Reindl ◽  
Kathrin Schanda ◽  
Mark Woodhall ◽  
Fiona Tea ◽  
Sudarshini Ramanathan ◽  
...  
Keyword(s):  
Excellent Agreement ◽  
Blood Donors ◽  
Clinical Care ◽  
Myelin Oligodendrocyte Glycoprotein ◽  
Antibody Assays ◽  
National Testing ◽  
International Testing ◽  
Ig G ◽  
International Multicenter ◽  
Aquaporin 4 Antibody

ObjectiveTo compare the reproducibility of 11 antibody assays for immunoglobulin (Ig) G and IgM myelin oligodendrocyte glycoprotein antibodies (MOG-IgG and MOG-IgM) from 5 international centers.MethodsThe following samples were analyzed: MOG-IgG clearly positive sera (n = 39), MOG-IgG low positive sera (n = 39), borderline negative sera (n = 13), clearly negative sera (n = 40), and healthy blood donors (n = 30). As technical controls, 18 replicates (9 MOG-IgG positive and 9 negative) were included. All samples and controls were recoded, aliquoted, and distributed to the 5 testing centers, which performed the following antibody assays: 5 live and 1 fixed immunofluorescence cell-based assays (CBA-IF, 5 MOG-IgG, and 1 MOG-IgM), 3 live flow cytometry cell-based assays (CBA-FACS, all MOG-IgG), and 2 ELISAs (both MOG-IgG).ResultsWe found excellent agreement (96%) between the live CBAs for MOG-IgG for samples previously identified as clearly positive or negative from 4 different national testing centers. The agreement was lower with fixed CBA-IF (90%), and the ELISA showed no concordance with CBAs for detection of human MOG-IgG. All CBAs showed excellent interassay reproducibility. The agreement of MOG-IgG CBAs for borderline negative (77%) and particularly low positive (33%) samples was less good. Finally, most samples from healthy blood donors (97%) were negative for MOG-IgG in all CBAs.ConclusionsLive MOG-IgG CBAs showed excellent agreement for high positive and negative samples at 3 international testing centers. Low positive samples were more frequently discordant than in a similar comparison of aquaporin-4 antibody assays. Further research is needed to improve international standardization for clinical care.

scholarly journals A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

2020 ◽  
Vol 59 (1) ◽  
pp. e01986-20
Author(s):  
Ibne Karim M. Ali ◽  
Shantanu Roy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Rdna Sequences ◽  
Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

scholarly journals Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

2020 ◽  
Vol 8 (12) ◽  
pp. 1950
Author(s):  
Laura Cerqueira ◽  
Sara Moura ◽  
Carina Almeida ◽  
Maria João Vieira ◽  
Nuno Filipe Azevedo
Keyword(s):  
Aspergillus Fumigatus ◽  
False Negative ◽  
Clinical Samples ◽  
Suitable Alternative ◽  
Fish Method ◽  
Negative Results ◽  
Specificity And Sensitivity ◽  
Pure Cultures ◽  
Low Sensitivity ◽  
Pna Fish

Aspergillus fumigatus is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatussensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL−1–1 × 103 conidia·mL−1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL−1 in sputum; 1 × 103 conidia·mL−1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield “fungal ball“ clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatussensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.

scholarly journals Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1358
Author(s):  
Leonard Schuele ◽  
Hayley Cassidy ◽  
Erley Lizarazo ◽  
Katrin Strutzberg-Minder ◽  
Sabine Schuetze ◽  
...  
Keyword(s):  
Influenza A ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Genome Coverage ◽  
Sequence Capture ◽  
Shotgun Metagenomic Sequencing ◽  
Oxford Nanopore ◽  
The One ◽  
Targeted Sequence Capture

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.

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