scholarly journals Development and Evaluation of an Electrochemical Biosensor for Detection of Dengue-Specific IgM Antibody in Serum Samples

Diagnostics ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 33
Author(s):  
Om Parkash ◽  
Muhammad Amiruddin Abdullah ◽  
Chan Yean Yean ◽  
Shamala Devi Sekaran ◽  
Rafidah Hanim Shueb
Keyword(s):  
Sensitivity And Specificity ◽  
Predictive Value ◽  
Clinical Symptoms ◽  
Limit Of Detection ◽  
Protein A ◽  
Immunoglobulin M ◽  
Ease Of Use ◽  
Analytical Sensitivity ◽  
Serum Samples ◽  
Igm Antibody

Dengue is an arbovirus disease transmitted mainly by Aedes mosquitoes. As dengue shares similar clinical symptoms with other infectious diseases, prompt and accurate diagnosis is pivotal to clinicians’ decisions on appropriate management. Conventional diagnostic tests to detect the dengue-specific IgM antibody are limited in their performance and ease of use. To address these issues, we developed and evaluated a biosensor based on screen-printed carbon electrodes (SPCEs) for the detection of dengue-specific immunoglobulin M (IgM) antibodies. Various optimisations were performed in order to increase the sensitivity and specificity of the biosensor. For optimal and proper orientation of the paratope sites of goat anti-human IgM capture antibodies (GAHICA), various antibody techniques, including passive, covalent, protein A, protein G and streptavidin/biotin systems, were tested on the SPCEs. The assay reagents for the biosensor were also optimised prior to its evaluation. Analytical sensitivity evaluation was carried out using pooled sera, while analytical specificity evaluation was conducted on a panel of six non-dengue serum samples. Subsequently, diagnostic sensitivity and specificity evaluation were performed using 144 reference samples. Electrochemical current signals generated from H2O2 catalysed by HRP-labelled anti-dengue detection antibodies were measured using the chronoamperometric technique. With a limit of detection (LOD) of 106 serum dilution, the analytical sensitivity of the developed biosensor was 10 times higher than commercial ELISA. The analytical specificity of this dengue IgM biosensor was 100%. Similarly, the biosensor’s diagnostic performance was 100% for sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). These findings suggest that the developed biosensor has a great potential to be used to diagnose dengue after seroconversion.

scholarly journals Quantification of Proteolytically Active Pregnancy-Associated Plasma Protein-A with an Assay Based on Quenched Fluorescence

2007 ◽  
Vol 53 (5) ◽  
pp. 947-954 ◽  
Author(s):  
Claus Gyrup ◽  
Michael Christiansen ◽  
Claus Oxvig
Keyword(s):  
Proteolytic Activity ◽  
Plasma Protein ◽  
Basic Protein ◽  
Biological Function ◽  
Limit Of Detection ◽  
Protein A ◽  
Maternal Serum ◽  
Serum Samples ◽  
A Subunit ◽  
Eosinophil Major Basic Protein

Abstract Background: Maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) are used to predict the occurrence of Down syndrome. In pregnancy, PAPP-A primarily circulates as a covalent 2:2 complex with the proform of eosinophil major basic protein (proMBP), which inhibits the proteolytic activity of PAPP-A. At term, however, ∼1% of PAPP-A exists as an active, uncomplexed dimer with proteolytic activity directed specifically toward insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5. No assays have been developed that allow quantification of PAPP-A proteolytic activity. Methods: We developed a sensitive and specific immunocapture assay for PAPP-A activity based on intramolecular quenched fluorescence. We used a 26-residue synthetic peptide derived from IGFBP-4 in which specific positions on each side of the PAPP-A cleavage site were substituted with 3-nitrotyrosine and o-aminobenzoic acid. Results: The assay detected the activity of recombinant PAPP-A as well as PAPP-A in serum samples from pregnant women. The limit of detection (mean signal of blank plus 3 SD) of the active PAPP-A subunit was 13 pmol/L, and the intra- and interassay CVs were <10% and <15%, respectively. Interestingly, the fraction of active PAPP-A decreased gradually from week 7 to week 19 of pregnancy. Conclusions: This method allows the measurement of PAPP-A enzymatic activity and because of its specificity it is relevant to the study of the biological function of PAPP-A. The method may also be useful in the diagnosis of pregnancy disorders.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for Immunoglobulin M Antibodies against Measles Virus

2003 ◽  
Vol 10 (3) ◽  
pp. 439-442 ◽  
Author(s):  
F. Roodbari ◽  
M. H. Roustai ◽  
A. Mostafaie ◽  
H. Soleimanjdahi ◽  
R. Sarrami Foroshani ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Neutralizing Antibodies ◽  
Measles Virus ◽  
Clinical Symptoms ◽  
Maculopapular Rash ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Vaccination Programs ◽  
Elisa System

ABSTRACT Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926).

scholarly journals Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Tian Du ◽  
Ji-hong Lin ◽  
Jun-hua Zhao ◽  
Hai-bo Wang ◽  
Qiu-hua Mo
Keyword(s):  
Predictive Value ◽  
Large Scale ◽  
Limit Of Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Preliminary Screening ◽  
Oral Transmission ◽  
Resource Limited ◽  
Clinical Accuracy ◽  
Insulated Isothermal Pcr

Background. Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. Methods. In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. Results. The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. Conclusion. Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.

scholarly journals Quantifying the bias associated with use of discrepant analysis

1998 ◽  
Vol 44 (1) ◽  
pp. 108-115 ◽  
Author(s):  
Harvey B Lipman ◽  
J Rex Astles
Keyword(s):  
Positive Predictive Value ◽  
Laboratory Test ◽  
Sensitivity And Specificity ◽  
Predictive Value ◽  
Comparison Method ◽  
Confirmatory Test ◽  
Analytical Sensitivity ◽  
Performance Parameters ◽  
Alternative Approaches ◽  
Biased Estimates

Abstract Discrepant analysis is a widely used technique for estimating the performance parameters of a laboratory test. In discrepant analysis, each specimen is initially tested with the candidate test and a comparison method, and when the results of the two tests disagree, a confirmatory test is used to resolve the discrepancy. Discrepant analysis usually produces biased estimates. This report quantifies this bias and shows that it is usually positive, leading to overestimation of the performance parameters of a laboratory test. The direction and magnitude of the bias are predictably influenced by the analytical sensitivity and specificity of the candidate test, comparison method, and confirmatory test. The proportion of abnormal specimens tested also affects the magnitude of the bias, particularly the estimates of analytical sensitivity and positive predictive value when this proportion is low. Alternative approaches are suggested.

scholarly journals Detection of Bovine Herpesvirus-1-Specific IgM Using a Capture Enzyme Immune Assay with Isotype-Specific Monoclonal Antibodies

1989 ◽  
Vol 1 (2) ◽  
pp. 139-145 ◽  
Author(s):  
Fernando Osorio ◽  
Subramaniam Srikumaran ◽  
Marvin Rhodes ◽  
David Christensen ◽  
Pushpa Srikumaran
Keyword(s):  
Monoclonal Antibodies ◽  
Solid Phase ◽  
Bovine Herpesvirus ◽  
Immunoglobulin M ◽  
Viral Reactivation ◽  
Clinical Samples ◽  
Serum Samples ◽  
Bovine Herpesvirus 1 ◽  
Igm Antibody ◽  
Herpesvirus 1

The detection of virus-specific immunoglobulin M (IgM) antibodies in acute-phase serum samples offers the possibility of making an accurate and rapid serologic diagnosis. We have developed a solid-phase capture assay that uses murine monoclonal antibodies specific for bovine IgM to separate the whole IgM fraction of a bovine serum sample. The IgM specific for bovine herpesvirus-1 (BHV-1) is then detected by the addition of viral antigen, which in turn is detected by BHV-1-specific monoclonal antibodies conjugated to horseradish peroxidase. A BHV-1 IgM antibody response was detected during the early postinfection period (7–40 days PI). Bovine herpesvirus-1 IgM antibody was not detected in sera taken from 3 animals following dexamethasone-induced viral reactivation. This method compares favorably with viral isolation, antigen detection in the clinical samples, and paired serology in the diagnosis of BHV-1 infection at a herd level.

scholarly journals Development of a Genus-Specific Brucella Real-Time PCR Assay Targeting the 16S-23S rDNA Internal Transcribed Spacer from Different Specimen Types

2020 ◽  
Vol 7 (4) ◽  
pp. 175
Author(s):  
Rejoice Nyarku ◽  
Ayesha Hassim ◽  
Annelize Jonker ◽  
Melvyn Quan
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Transcribed Spacer ◽  
Bacterial Culture ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Surface Protein ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Analytical Sensitivity ◽  
Tissue Samples

The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for the early diagnosis and rapid screening of brucellosis. Blood, milk, and tissue samples were spiked with B. abortus biovar 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The 95% limit of detection of the ITS qPCR assay was highest in tissue, followed by blood, then milk, i.e., 0.48, 4.43, and 15.18 bacteria/PCR reaction, respectively. The diagnostic performance of the assay was compared to the Brucella cell surface protein (BCSP) 31 qPCR assay and bacterial culture. Out of 56 aborted foetal tissue samples from bovine, ovine, and caprine, 33% (19/56) were positive for Brucella spp. The sensitivity and specificity of the ITS qPCR assay was 87% and 95% respectively, compared to 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The assay was efficient, sensitive, and specific, making it a valuable tool in the early detection of the Brucella pathogen.

scholarly journals Detection of antibodies to decorin-binding protein A (DbpA) and DbpB after infection of dogs with Borrelia burgdorferi by tick challenge

2020 ◽  
Vol 32 (3) ◽  
pp. 481-485
Author(s):  
Darby G. Oldenburg ◽  
Dean A. Jobe ◽  
Steven D. Lovrich ◽  
Rhonda L. LaFleur ◽  
Douglas W. White ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Ixodes Scapularis ◽  
Binding Protein ◽  
Protein A ◽  
Immune Serum ◽  
Immunoglobulin M ◽  
Antibody Responses ◽  
Igg Antibodies ◽  
Serum Samples

We characterized the antibody response to decorin-binding protein A (DbpA) or DbpB from immune serum samples collected from 27 dogs infected with Borrelia burgdorferi by Ixodes scapularis ticks. Immunoglobulin M (IgM) antibodies to DbpA or DbpB were rarely detected, but high levels of IgG antibodies to DbpA were detected in 16 of 27 of the immune sera collected 1 mo after infection, 20 of 25 of the sera collected after 2 mo, and each of the 23, 17, or 11 serum samples evaluated after 3, 4, or 5 mo, respectively. In addition, IgG antibodies to DbpB were detected in 22 of 27 ( p = 0.005) tested dogs after 1 mo, and the frequency of detecting the antibodies thereafter closely mimicked the antibody responses to DbpA. Moreover, antibodies to DbpA or DbpB were not produced by dogs vaccinated with a whole-cell B. burgdorferi bacterin; removing the antibodies to DbpA by adsorption to recombinant DbpA (rDbpA) did not affect the reactivity detected by a rDbpB ELISA. Therefore, the findings from our preliminary study showed that antigenically distinct antibodies to DbpA or DbpB are produced reliably during canine infection with B. burgdorferi, and the response is not confounded by vaccination with a Lyme disease bacterin. Larger studies are warranted to more critically evaluate whether detecting the antibody responses can improve serodiagnostic confirmation of canine Lyme disease.

scholarly journals Modified Staphylococcal Absorption Method Used for Detecting Rubella-Specific Immunoglobulin M Antibodies During a Rubella Epidemic

1977 ◽  
Vol 5 (6) ◽  
pp. 588-592
Author(s):  
R. Handsher ◽  
A. Fogel
Keyword(s):  
Density Gradient ◽  
Clinical Symptoms ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Original Method ◽  
Immunoglobulin M ◽  
Absorption Method ◽  
Igm Antibody ◽  
Specific Immunoglobulin ◽  
History Of

A recently described method for detecting rubella-specific immunoglobulin M (IgM) antibody based on absorption of IgG by Staphylococcus aureus strain Cowan I has been applied to 198 sera collected during a recent rubella epidemic in Israel. Modification of the original method introduced for the present study includes treatment with 2-mercaptoethanol of antibody remaining after absorption by staphylococci. This treatment confirms that the residual antibody is IgM (sensitive to 2-mercaptoethanol) rather than IgG (2-mercaptoethanol resistant). None of the 67 control patients (seropositive for rubella but without history of recent illness or contact) had specific IgM when tested by this method, though 15 showed some residual antibody after staphylococcal absorption. A total of 125 of 131 rubella convalescents (95%) were positive 4 to 49 days after onset of the clinical symptoms. Six patients had no IgM antibodies when tested by the method described, and all were convalescents tested late in relation to onset of clinical symptoms (beyond 3 weeks). When density gradient centrifugation was applied to clarify some results, 2 of 3 convalescents classified as IgM negative by the staphylococcal absorption method did in fact possess IgM antibody. None of 10 controls tested by density gradient centrifugation was IgM positive. This combination of staphylococcal absorption and 2-mercaptoethanol treatment is recommended as a screening test for selection of IgM positives, in addition to the use of a more sensitive method (such as density gradient centrifugation) on at least some samples classified as IgM negative.

scholarly journals Performance evaluation of the point-of-care SAMBA II SARS-CoV-2 Test for detection of SARS-CoV-2

2020 ◽  
Author(s):  
Sonny M Assennato ◽  
Allyson V Ritchie ◽  
Cesar Nadala ◽  
Neha Goel ◽  
Hongyi Zhang ◽  
...  
Keyword(s):  
Public Health ◽  
Infection Control ◽  
Sensitivity And Specificity ◽  
Patient Management ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Point Of Care Testing ◽  
Clinical Sensitivity

AbstractNucleic acid amplification for the detection of SARS-CoV-2 RNA in respiratory samples is the standard method for diagnosis. These tests are centralised and therefore turnaround times can be 2-5 days. Point-of-care testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly.Inclusivity and specificity of the SAMBA II SARS-CoV-2 assay was determined by in silico analyses of the primers and probes. Analytical and clinical sensitivity and specificity of the SAMBA II SARS-CoV-2 Test was evaluated for analytical sensitivity and specificity. Clinical performance was evaluated in residual clinical samples compared to the Public Health England reference tests.The limit of detection of the SAMBA II SARS-CoV-2 Test is 250 cp/mL and is specific for detection of 2 regions of the SARS-CoV-2 genome. The clinical sensitivity was evaluated in 172 clinical samples provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge (CMPHL), which showed a sensitivity of 98.9% (95% CI 94.03-99.97%), specificity of 100% (95% CI 95.55-100%), PPV of 100% and NPV of 98.78% (92.02-99.82%) compared to testing by CMPHLSAMBA detected 3 positive samples that were initially negative by PHE Test. The data shows that the SAMBA II SARS-CoV-2 Test performs equivalently to the centralised testing methods with a much quicker turnaround time. Point of care testing, such as SAMBA, should enable rapid patient management and effective implementation of infection control measures.

scholarly journals Value of Examination Under Fluoroscopy for the Assessment of Sacroiliac Joint Dysfunction

2015 ◽  
Vol 5;18 (5;9) ◽  
pp. E781-E786
Author(s):  
Burton D Beakley
Keyword(s):  
Sensitivity And Specificity ◽  
Roc Curve ◽  
Predictive Value ◽  
Joint Pain ◽  
Clinical Symptoms ◽  
Small Sample ◽  
Joint Disease ◽  
Pain Clinic ◽  
Predictive Values ◽  
Si Joint

Background: Pain emanating from the sacroiliac (SI) joint can have variable radiation patterns. Single physical examination tests for SI joint pain are inconsistent with multiple tests increasing both sensitivity and specificity. Objective: To evaluate the use of fluoroscopy in the diagnosis of SI joint pain. Study Design: Prospective double blind comparison study Setting: Pain clinic and radiology setting in urban Veterans Administration (VA) in New Orleans, Louisiana. Methods: Twenty-two adult men, patients at a southeastern United States VA interventional pain clinic, presented with unilateral low back pain of more than 2 months’ duration. Patients with previous back surgery were excluded from the study. Each patient was given a Gapping test, Patrick (FABERE) test, and Gaenslen test. A second blinded physician placed each patient prone under fluoroscopic guidance, asking each patient to point to the most painful area. Pain was provoked by applying pressure with the heel of the palm in that area to determine the point of maximum tenderness. The area was marked with a radio-opaque object and was placed on the mark with a fluoroscopic imgage. A site within 1 cm of the SI joint was considered as a positive test. This was followed by a diagnostic injection under fluoroscopy with 1 mL 2% lidocaine. A positive result was considered as more than 2 hours of greater than 75% reduction in pain. Then, in 2-3 days this was followed by a therapeutic injection under fluoroscopy with 1 mL 0.5% bupivacaine and 40 mg methylprednisolone. Results: Each patient was reassessed after 6 weeks. The sensitivity and specificity in addition to the positive and negative predictive values were determined for both the conventional examinations, as well as the examination under fluoroscopy. Finally, a receiver operating characteristic (ROC) curve was constructed to evaluate test performance. The sensitivity and specificity of the fluoroscopic examination were 0.82 and 0.80 respectively; Positive predictive value and negative predictive value were 0.93 and 0.57 respectively. The area under ROC curve was 0.812 which is considered a “good” test; however the area under ROC for the conventional examination were between 0.52 -0.58 which is considered “poor to fail”. Limitations: Variation in anatomy of the SI joint, small sample size. Conclusions: Multiple structures of the SI joint complex can result in clinical symptoms of pain. These include intra-articular structures (degenerative arthritis, and inflammatory conditions) as well as extraarticular structures (ligaments, muscles, etc.). Key words: Sacroilliac joint disease, radicular pain, thigh thrust test, compression test, distraction test, Gaenslen test, Patrick test (FABER test)

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