A novel enzyme-linked immunosorbent assay using a mixture of human native and recombinant proteinase-3 significantly improves the diagnostic potential for antineutrophil cytoplasmic antibody-associated vasculitis

2008 ◽  
Vol 68 (2) ◽  
pp. 228-233 ◽  
Author(s):  
J Damoiseaux ◽  
C Dähnrich ◽  
A Rosemann ◽  
C Probst ◽  
L Komorowski ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Odds Ratio ◽  
High Sensitivity ◽  
Antineutrophil Cytoplasmic Antibody ◽  
Antineutrophil Cytoplasmic Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Proteinase 3 ◽  
The Novel ◽  
Capture Elisa ◽  
Diagnostic Potential

Background:Antineutrophil cytoplasmic antibodies (ANCA) with a C-ANCA or P-ANCA pattern are detected in ANCA-associated vasculitis (AAV). While in most patients with AAV a C-ANCA pattern is due to reactivity with proteinase-3 (PR3)-ANCA, some C-ANCA-positive sera do not react with PR3.Objective:The development and evaluation of a direct enzyme-linked immunosorbent assay (ELISA) for PR3-ANCA with increased sensitivity.Methods:A mixture of human native (hn) and human recombinant (hr) PR3 was used as antigen coating. The resulting ELISA (anti-PR3-hn-hr) was compared with ELISAs using directly coated hn-PR3 or hr-PR3, as well as with a hn-PR3 capture ELISA. Assay characteristics were determined in patients with AAV (n = 248), with special attention for those patients with C-ANCA (n = 132), as well as disease controls (n = 585) and healthy controls (n = 429). Additionally, for prediction of relapses serial samples of 46 patients with PR3-AAV were analysed.Results:At a predefined specificity of 99% both ELISAs containing hr-PR3 revealed a substantial increase in sensitivity. For the prediction of relapses by rises in PR3-ANCA titres the capture ELISA was most optimal (odds ratio 12.5). With an odds ratio of 8.9 the novel anti-PR3-hn-hr ELISA was second best.Conclusions:Owing to the very high sensitivity of the novel anti-PR3-hn-hr ELISA for the detection of PR3-ANCA in C-ANCA-positive samples of patients with AAV this assay has an excellent diagnostic performance. This feature is combined with a good predictability of clinical relapses in patients with PR3-AAV. These characteristics challenge the dogma that, for detection of PR3-ANCA, capture ELISAs are superior for diagnosis and follow-up.

scholarly journals Monitoring Proteinase 3 Antineutrophil Cytoplasmic Antibodies for Detection of Relapses in Small Vessel Vasculitis

2003 ◽  
Vol 10 (5) ◽  
pp. 769-774 ◽  
Author(s):  
Mårten Segelmark ◽  
Brian D. Phillips ◽  
Susan L. Hogan ◽  
Ronald J. Falk ◽  
J. Charles Jennette
Keyword(s):  
Small Vessel ◽  
Antineutrophil Cytoplasmic Antibodies ◽  
Small Vessel Vasculitis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Published Data ◽  
Proteinase 3 ◽  
Medline Search ◽  
Capture Elisa ◽  
Negative Results ◽  
Sensitive Tool

ABSTRACT The clinical usefulness of antineutrophil cytoplasmic antibodies (ANCAs) in the monitoring of patients treated for small vessel vasculitis is debated. A capture enzyme-linked immunosorbent assay (ELISA) based on anti-proteinase 3 (anti-PR3) monoclonal antibody 4A3 has previously been proven to be superior to indirect immunofluorescence (IIF) and standard ELISA for the diagnosis of vasculitis. The present study compared the effectiveness of the capture ELISA for the detection of disease relapse. Samples from patients with relapses and remissions (relapse and remission samples, respectively) were identified through the database of the Glomerular Disease Collaborate Network. Twenty-one relapse samples and 49 remission samples were analyzed by the capture PR3-ANCA ELISA from Wieslab AB, the standard PR3-ANCA ELISA from Inova, and IIF. A Medline search was performed to identify published data on ANCA status at relapse. The capture ELISA was positive for 21 instances of relapses in 14 patients, while the standard ELISA and IIF each failed to detect 2 relapses (P was not significant). By using a higher cutoff value, the capture ELISA correctly categorized 84% of the remission samples and 81% of the relapse samples. Similar degrees of discrimination could be achieved by IIF but not by the standard ELISA. In previously published series, the median proportions of patients positive at relapse were 100% by IIF (range, 75 to 100%) and 86% by standard ELISA (range, 38 to 100%). The corresponding values for a rise that accompanied or preceded a relapse were 75% (range, 20 to 100%) for IIF and 50% (range, 25 to 81%) for ELISA. The capture PR3-ANCA ELISA is a sensitive tool for the detection of relapses. Larger studies are needed to detect differences between methods. Negative results by tests for ANCAs are rare during relapses.

scholarly journals Rapid, Quantitative, and High-Sensitivity Detection of Anti-Phospholipase A2 Receptor Antibodies Using Novel Cdse/Zns-Based Fluorescence Immunosorbent Assay

2020 ◽  
Author(s):  
Chenxi Li ◽  
Manyun Qian ◽  
Qiaozhen Hong ◽  
Xiaohong Xin ◽  
Zichun Sun ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Detection Sensitivity ◽  
Response To Treatment ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Serum Samples ◽  
Phospholipase A2 Receptor

Abstract Autoantibodies against M-type phospholipase A2 receptor (PLA2R) are specific biomarkers for idiopathic membranous nephropathy (IMN) and their quantification has been helpful to monitor disease activity. In this study, we describe a highly sensitive and rapid quantum dots-based immunochromatography assay (QD-ICA) for quantifying PLA2R autoantibodies. Serum samples from 135 biopsy-confirmed patients with nephrotic syndrome were analyzed for PLA2R autoantibodies using the novel QD-ICA as well as enzyme-linked immunosorbent assay (ELISA). The detection sensitivity and specificity of QD-ICA (80.9 and 100%, respectively) exceeded those of ELISA (72.1 and 98.5%, respectively). The optimum cut-off value of QD-ICA was 18.18 RU/mL and limit of detection was 2.86 relative units/mL. The novel QD-ICA outperforms ELISA in detecting PLA2R autoantibodies, with shorter detection time, fewer steps, smaller equipment size, and broader testing application, suggesting its capability to improve IMN diagnosis and monitor patient response to treatment.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Antibody to Epizootic Hemorrhagic Disease of Deer Virus

2000 ◽  
Vol 12 (2) ◽  
pp. 142-145 ◽  
Author(s):  
James O. Mecham ◽  
Michael M. Jochim
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hemorrhagic Disease ◽  
Structural Protein ◽  
Serum Samples ◽  
Epizootic Hemorrhagic Disease ◽  
Diagnostic Potential ◽  
The Us ◽  
Serotype 1 ◽  
Infected Cells

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1-and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.

scholarly journals Interpretations of Antibody Responses toSalmonella enterica Serotype Enteritidis gm Flagellin in Poultry Flocks Are Enhanced by a Kinetics-Based Enzyme-Linked Immunosorbent Assay

1998 ◽  
Vol 5 (4) ◽  
pp. 550-555 ◽  
Author(s):  
Patrick L. McDonough ◽  
Richard H. Jacobson ◽  
John F. Timoney ◽  
Ahmed Mutalib ◽  
David C. Kradel ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
High Sensitivity ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Analytical Sensitivity ◽  
Department Of Agriculture ◽  
Trace Back ◽  
Elisa Format ◽  
Commercial Poultry ◽  
Computer Controlled

ABSTRACT Many regulatory and diagnostic programs for the detection ofSalmonella enterica serotype Enteritidis infection in commercial poultry flocks have relied on rapid Pullorum agglutination tests to screen birds because of the shared antigens of S. enterica Enteritidis and S. enterica Pullorum and Gallinarum; however, the use of the enzyme-linked immunosorbent assay (ELISA) format affords better analytical sensitivity than crude agglutination tests. In this study, we adapted our earlier conventional indirect ELISA, using gm flagellin as the antigen, to a kinetics-based, computer-controlled ELISA (KELA). The KELA was used to screen for flagellin antibody from three commercial flocks: (i) a large flock involved in a U.S. Department of Agriculture trace back from a humanS. enterica Enteritidis foodborne outbreak (n = 3,209), (ii) a flock infected with the endemicS. enterica Enteritidis serotype but which also had multiple other salmonella serotypes (n = 65), and (iii) an S. enterica Pullorum-infected flock (n = 12). The first flock (S. entericaEnteritidis prevalence of 2.45% based on culture) provided a field test of the KELA and allowed the calculation of diagnostic sensitivity (D-Sn) and diagnostic specificity (D-Sp). With a cutoff of 10 (used for screening flocks [i.e., high sensitivity]), the KELA has a D-Sn of 95.2% and a D-Sp of 18.5%; with a cutoff of 140 (used in confirmatory flock testing [i.e., high specificity]), the KELA has a D-Sn of 28.0% and a D-Sp of 99.1%. We found that with a cutoff of 60 (D-Sn = 63.1%; D-Sp = 91.6%), we could eliminate reactions in the KELA caused by other non-S. enterica Enteritidis salmonellae. The KELA was also compared to two commercial rapid Pullorum tests, the Solvay (D-Sn = 94.9%; D-Sp = 55.5%) and the Vineland (D-Sn = 62.0%; D-Sp = 75.3%).

scholarly journals Clinical evaluation of a capture ELISA for detection of proteinase-3 antineutrophil cytoplasmic antibody: Technical Note

1998 ◽  
Vol 53 (5) ◽  
pp. 1230-1236 ◽  
Author(s):  
Kerstin W.A. Westman ◽  
Daina Selga ◽  
Per Bygren ◽  
Mårten Segelmark ◽  
Bo Baslund ◽  
...  
Keyword(s):  
Clinical Evaluation ◽  
Antineutrophil Cytoplasmic Antibody ◽  
Technical Note ◽  
Proteinase 3 ◽  
Capture Elisa

scholarly journals Cystatin Capture Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Human Clonorchiasis and Profile of Captured Antigenic Protein of Clonorchis sinensis

2001 ◽  
Vol 8 (6) ◽  
pp. 1076-1080 ◽  
Author(s):  
Tae Yun Kim ◽  
Shin-Yong Kang ◽  
Sun Hyo Park ◽  
Kom Sukontason ◽  
Kabkaew Sukontason ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Proteinase Inhibitors ◽  
Binding Capacity ◽  
Clonorchis Sinensis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Moderate Degree ◽  
Cysteine Proteinases ◽  
Capture Elisa ◽  
Crude Extracts ◽  
High Degree

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) with crude extracts of adult Clonorchis sinensis has been reported to have a high degree of sensitivity with a moderate degree of specificity for the serodiagnosis of clonorchiasis. The cystatin capture ELISA was investigated for its usefulness for the serodiagnosis of human clonorchiasis. Cystatin bound specifically to cysteine proteinases in crude extracts of adult C. sinensis worms, and its binding capacity was not hindered competitively by the other proteinase inhibitors tested. The cystatin capture ELISA for clonorchiasis showed a higher degree of specificity than the conventional ELISA, which produced some cross-reactivities to sera from patients with cysticercosis, sparganosis, and opisthorchiasis. Immunoglobulin G antibodies to C. sinensis cysteine proteinases were produced in experimental rabbits at week 3, and their levels increased rapidly and remained at a plateau after 8 weeks of infection. Of the proteins from the C. sinensis crude extract captured with cystatin, seven proteins were reactive with the serum from patients with clonorchiasis. The cystatin capture ELISA is indicated to be a sensitive and highly specific immunodiagnostic assay for serodiagnosis of human clonorchiasis.

Evaluation of immunoglobulin G4 subclass antibody in a peptide-based enzyme-linked immunosorbent assay for the serodiagnosis of human fascioliasis

Parasitology ◽  
2007 ◽  
Vol 134 (14) ◽  
pp. 2021-2026 ◽  
Author(s):  
C. TANTRAWATPAN ◽  
W. MALEEWONG ◽  
C. WONGKHAM ◽  
S. WONGKHAM ◽  
P. M. INTAPAN ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Large Scale ◽  
Laboratory Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Parasitic Infections ◽  
Immunoglobulin G4 ◽  
Predictive Values ◽  
Diagnostic Potential ◽  
Human Fascioliasis ◽  
Specific Igg4

SUMMARYTo improve the diagnosis of human fascioliasis caused byFasciola gigantica, we developed a peptide-based enzyme-linked immunosorbent assay (peptide-based ELISA) based on the detection of specific IgG4 subclass antibody. Two identified B-cell epitopes ofF. giganticacathepsin L1 were synthesized as single synthetic peptides, acetyl-DKIDWRESGYVTELKDQGNC-carboxamide (peptide L) and acetyl-DKIDWRESGYVTEVKDQGNC-carboxamide (peptide V), and their diagnostic potential was evaluated. The sera of 25 patients infected withF. gigantica, 212 patients with other parasitic infections, 32 cholangiocarcinoma patients and 57 healthy controls were analysed. The sensitivity, specificity, accuracy, and positive and negative predictive values of this assay were the same with both peptides at 100%, 99·7%, 99·7%, 96·2% and 100%, respectively. These highly sensitive and specific peptide-based ELISAs for the detection of specific IgG4 antibody could be useful for laboratory diagnosis of human fascioliasis in future large-scale surveys throughout Southeast Asia where this disease is prevalent.

scholarly journals Evaluation of an Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Orientia tsutsugamushi Infection

2003 ◽  
Vol 10 (3) ◽  
pp. 394-398 ◽  
Author(s):  
Won-Jong Jang ◽  
Myung-Suk Huh ◽  
Kyung-Hee Park ◽  
Myung-Sik Choi ◽  
Ik-Sang Kim
Keyword(s):  
Immunosorbent Assay ◽  
Scrub Typhus ◽  
Microtiter Plate ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Orientia Tsutsugamushi ◽  
Serum Samples ◽  
Capture Elisa ◽  
Igm Antibodies ◽  
Immunodominant Antigen

ABSTRACT To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli. In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.

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