scholarly journals INDIRECT ELISA FOR DETECTION OF ANTIBODIES TO FMDV NON-STRUCTURAL PROTEINS IN PORCINE SERA

2018 ◽  
pp. 13-20
Author(s):  
A. S. Yakovleva ◽  
A. V. Kanshina ◽  
A. V. Scherbakov
Keyword(s):  
Indirect Elisa ◽  
High Sensitivity ◽  
High Specificity ◽  
Structural Proteins ◽  
Post Inoculation ◽  
Nonstructural Proteins ◽  
Diagnostic Specificity ◽  
Fmd Virus ◽  
Specificity And Sensitivity ◽  
Sensitivity Specificity

An indirect variant of ELISA used for detection of antibodies to nonstructural proteins of the FMD virus in porcine blood sera was developed. The results of the validation showed that the developed method is characterized by high sensitivity, specificity and reproducibility. When testing the blood serum panel obtained from experimentally infected animals, the method allowed to detect antibodies to FMD virus in 7 of 18 sera collected on day 6 post inoculation, in 13 of 19 sera – on day 7 post inoculation, in 16 of 19 sera – on day 8 post inoculation and in all 76 sera obtained on days 9–12 post inoculation. The diagnostic specificity of 3AB-ELISA was 100% when testing 100 knowingly negative blood sera from pigs imported to Russia from Norway. High specificity and sensitivity of the method, established during the development of the method, are confirmed in the course of routine diagnostic tests.

scholarly journals Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1207
Author(s):  
Hong Jae Cheon ◽  
Quynh Huong Nguyen ◽  
Moon Il Kim
Keyword(s):  
Magnetic Nanoparticles ◽  
High Sensitivity ◽  
High Specificity ◽  
Choline Oxidase ◽  
Fluorescent Detection ◽  
One Pot ◽  
Site Structure ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Peroxidase Mimics

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

scholarly journals Crude antigen from Taenia crassiceps cysticercus used as heterologous antigen in ELISA and in EITB for neurocysticercosis diagnosis of patients from Paraná-Brazil

2008 ◽  
Vol 51 (6) ◽  
pp. 1127-1137 ◽  
Author(s):  
João Carlos Minozzo ◽  
Juliana de Moura ◽  
Sérgio Monteiro Almeida ◽  
Vanete Thomaz-Soccol
Keyword(s):  
Public Health ◽  
Neurological Disorders ◽  
Indirect Elisa ◽  
Taenia Solium ◽  
High Specificity ◽  
Heterologous Antigen ◽  
Public Health Systems ◽  
Crude Antigen ◽  
Specificity And Sensitivity ◽  
Cysticercus Cellulosae

Neurocysticercosis (NCC), the cerebral presence of Taenia solium metacestode (Cysticercus cellulosae), is responsible for neurological disorders worldwide. In order to validate an immunodiagnosis for public-health patients in the State of Parana-Brazil, crude antigen of Taenia crassicepsmetacestode (Cysticercus longicollis) was used as an alternative heterologous antigen to be used in ELISA and in electroimmunotransfer blotting (EITB) for active and inactive NCC diagnosis. Indirect ELISA was able to discriminate between active and inactive samples and presented high specificity and sensitivity. Any immunodominant band was able to distinguish the NCC stages, although the EITB showed 100% specificity. The immunological results proved to be an important auxiliary toll for NCC diagnosis, mainly for public-health systems in developing countries, where either the neuroimage techniques are not accessible or the resources are scarce.

scholarly journals A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

2022 ◽  
Vol 12 ◽  
Author(s):  
Katharina Radakovics ◽  
Claire Battin ◽  
Judith Leitner ◽  
Sabine Geiselhart ◽  
Wolfgang Paster ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Ectopic Expression ◽  
High Sensitivity ◽  
High Specificity ◽  
Reporter System ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Definition Of ◽  
The Individual ◽  
Allergen Extracts

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

scholarly journals Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Zhang Tie ◽  
Wang Chunguang ◽  
Wei Xiaoyuan ◽  
Zhao Xinghua ◽  
Zhong Xiuhui
Keyword(s):  
Staphylococcus Aureus ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Reaction Tube ◽  
Specificity And Sensitivity ◽  
Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

A Preliminary Evaluation of a Prototype Western Blot Confirmatory Test Kit for Syphilis

1994 ◽  
Vol 5 (6) ◽  
pp. 409-414 ◽  
Author(s):  
H Young ◽  
P J Walker ◽  
D Merry ◽  
A Mifsud
Keyword(s):  
Western Blot ◽  
False Positive ◽  
High Sensitivity ◽  
High Specificity ◽  
Confirmatory Test ◽  
Preliminary Evaluation ◽  
Serological Tests ◽  
Western Blot Test ◽  
Specificity And Sensitivity ◽  
Test Kit

A prototype Western blot kit was evaluated as a confirmatory test for syphilis using 131 sera characterized by other serological tests for syphilis. There were 114 treponemal sera (including 94 cases of early syphilis, 83 of which were untreated) and 17 non-treponemal problem sera (11 gave false positive reactions on screening with the TmpA recombinant antigen enzyme immunoassay (EIA), 3 gave false positive fluorescent treponemal antibody absorbed (FTA-abs) tests, and 3 false positive Captia Syphilis G EIA results). Based on the manufacturer's criteria of reactivity in multiple bands for designating a positive result the Western blot test gave a sensitivity of 99.1% (113/114) and a specificity of 88.2% (15/17) when indeterminate reactions were scored positive and 98.2% (112/114) and 100% (17/17) when indeterminate reactions were scored negative. Sensitivity was high in both treated and untreated infection. Corresponding sensitivities for the TPHA and FTA-abs when equivocal reactions were scored negative were 97.5% (111/114) and 99.1% (113/114). The high sensitivity of the FTA-abs in this study is probably due to the large number of untreated primary infections. Our results with the Western blot, confirm earlier studies using ‘in-house’ test systems and, support a role for a commercial Western blot test in the confirmatory diagnosis of syphilis. Further studies are required to confirm the high specificity and sensitivity of the kit in a larger series including a wider variety of non-treponemal cases as well as patients with untreated and treated infection.

scholarly journals Bolus Residue Scale: An Easy-to-Use and Reliable Videofluoroscopic Analysis Tool to Score Bolus Residue in Patients with Dysphagia

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Nathalie Rommel ◽  
Charlotte Borgers ◽  
Dirk Van Beckevoort ◽  
Ann Goeleven ◽  
Eddy Dejaeger ◽  
...  
Keyword(s):  
Rating Scale ◽  
Intraclass Correlation ◽  
Correlation Coefficients ◽  
Posterior Pharyngeal Wall ◽  
High Specificity ◽  
Analysis Tool ◽  
Pharyngeal Wall ◽  
Specificity And Sensitivity ◽  
Low Sensitivity ◽  
Sensitivity Specificity

Background. We aimed to validate an easy-to-use videofluoroscopic analysis tool, the bolus residue scale (BRS), for detection and classification of pharyngeal retention in the valleculae, piriform sinuses, and/or the posterior pharyngeal wall.Methods. 50 randomly selected videofluoroscopic images of 10 mL swallows (recorded in 18 dysphagia patients and 8 controls) were analyzed by 4 experts and 6 nonexpert observers. A score from 1 to 6 was assigned according to the number of structures affected by residue. Inter- and intrarater reliabilities were assessed by calculation of intraclass correlation coefficients (ICCs) for expert and nonexpert observers. Sensitivity, specificity, and interrater agreement were analyzed for different BRS levels.Results. Intrarater reproducibility was almost perfect for experts (mean ICC 0.972) and ranged from substantial to almost perfect for nonexperts (mean ICC 0.835). Interjudge agreement of the experts ranged from substantial to almost perfect (mean ICC 0.780), but interrater reliability of nonexperts ranged from substantial to good (mean 0.719). BRS shows for experts a high specificity and sensitivity and for nonexperts a low sensitivity and high specificity.Conclusions. The BRS is a simple, easy-to-carry-out, and accessible rating scale to locate pharyngeal retention on videofluoroscopic images with a good specificity and reproducibility for observers of different expertise levels.

Targeted next generation sequencing to expand HER2 status detection: Implication for newer HER2-directed agents.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 1047-1047
Author(s):  
Ning Liao ◽  
RongFeng Song ◽  
FangQin Xue ◽  
Shuirong Zhang ◽  
Samuel J Klempner ◽  
...  
Keyword(s):  
Clinical Laboratory ◽  
False Negative ◽  
High Sensitivity ◽  
Expression Level ◽  
Her2 Status ◽  
Genomic Alteration ◽  
Her2 Expression ◽  
Specificity And Sensitivity ◽  
Sensitivity Specificity ◽  
Erbb2 Amplification

1047 Background: Establishment of ERBB2 ( HER2) amplification status in breast carcinoma (BC) and gastric carcinoma (GC) is essential for treatment selection, but no anti- HER2 therapies have been approved for tumors with low level of HER2 expression. The clinical trial ( NCT02564900 ) was initiated to evaluate the safety and efficacy of trastuzumab deruxtecan (DS-8201a) in BC patients with lower HER2 expression by current methods. This study aims to validate NGS-based detection for HER2 low expression. Methods: 275 BC and 425 GC were collected and subjected to NGS for genomic alteration detection. The testing was carried out by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory, Shanghai, China. Protein expression was analyzed by using IHC. FISH was carried out on 108 samples, including 63 BC and 45 GC. To set up NGS cutoff, FISH was performed on additional 34 samples. Sensitivity, specificity and accuracy were evaluated based on FISH as a gold-standard reference. Results: In BC, the expression level of HER2 protein detected by IHC was overall IHC 0 in 28.7%, 1+ in 18.9%, 2+ in 27.3% and 3+ in 25.1%, respectively, while in GC, the expression level was 60.7%, 18.6%, 14.8% and 5.9%, respectively. Log2ratio was used to assess ERBB2 amplification status detected by NGS. According to the FISH results of 34 other samples with high sensitivity (98%) and specificity (100%), the threshold was determined as 0.5. 8 and 10 samples of IHC 1+/2+ met the cutoff in BC and GC, respectively. In 63 BC, there were 17 positive and 46 negative by FISH. According to the threshold, the sensitivity and specificity of NGS detection was 94.1% and 97.8%, respectively. The proportion of samples with IHC 2+ that couldn't determine NGS ERBB2 status was 49.2%. However, except for false positive and false negative, the NGS results were concordant with FISH. In 45 GC, there were 5 positive and 40 negative by FISH. The specificity and sensitivity was 97.5% and 40%, respectively. In 4 samples with IHC 2+, 2 of them were discordant with the results of NGS and FISH. All IHC 1+ didn't meet the cutoff of NGS and was FISH negative in 108 samples. The accuracy of NGS in ERBB2 detection was 96.8% and 91.1% for BC and GC, respectively. Conclusions: Our data indicated that the NGS-based detection of ERBB2 amplification had high sensitivity, specificity and accuracy. In samples with IHC 1+/2+, the results of NGS detection were high concordant with FISH detection.

scholarly journals DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

2015 ◽  
Vol 8 ◽  
pp. MBI.S29736 ◽  
Author(s):  
Kenjiro Nagamine ◽  
Guo-Chiuan Hung ◽  
Bingjie Li ◽  
Shyh-Ching Lo
Keyword(s):  
Safety Concern ◽  
Rapid Development ◽  
High Sensitivity ◽  
High Specificity ◽  
Clostridium Tetani ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Pcr Assays ◽  
Primer Sets ◽  
Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

scholarly journals An alternative PCR assay with high sensitivity and specificity for the detection of swine toxoplasmosis based on the GRA14 gene

2021 ◽  
Author(s):  
Xi He ◽  
Derong Zhou ◽  
Yanwu Sun ◽  
Yuan Zhang ◽  
Xiaogang Zhang ◽  
...  
Keyword(s):  
Detection Method ◽  
Southern China ◽  
Acute Infection ◽  
High Sensitivity ◽  
High Specificity ◽  
Pcr Detection ◽  
Pcr Assay ◽  
Specific Primers ◽  
Specificity And Sensitivity ◽  
Pcr Method

Abstract Background Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect all warm-blooded animals. Infected swine are considered one of the most important sources of T. gondii infection in humans. Rapidly and effectively diagnosing T. gondii infection in swine is essential. PCR-based diagnostic tests have been fully developed, and very sensitive and specific PCR is crucial for the diagnosis of swine toxoplasmosis. Methods To established a high specificity and sensitivity PCR detection method for swine toxoplasmosis, we used T. gondii GRA14 gene as target to design specific primers and established a PCR detection method for swine toxoplasmosis. A total of 5462 blood specimens collected from pigs in 5 provinces and autonomous regions in southern China during 2016–2017 were assessed by the newly established GRA14 gene PCR method. Result Altogether, we used T. gondii GRA14 gene as target to design specific primers and established a high specificity and sensitivity PCR detection method for swine toxoplasmosis; in particular, this PCR method could detect T. gondii tachyzoite DNA in the acute infection phase. The GRA14 gene PCR assay detected a minimum of 2.35 tachyzoites of T. gondii, and it could be used for T. gondii detection in blood, tissue, semen, urine and waste feed specimens. The overall T. gondii infection rate was 18.9% (1033/5462) by the newly established GRA14 gene PCR method. According to statistical analysis among different regions, the positive rates of swine toxoplasmosis in the Shaanxi, Fujian and Guangdong areas in China from 2016 to 2017 were the highest, at 31.7% (44/139), 21.9% (86/391) and 18.8% (874/4645), respectively (χ2 = 84.2, P < 0.0001). Specimens collected in 2017 had a higher positive rate (19.1% or 886/4639) than those collected in 2016 (16.1% or 155/963) (χ2 = 4.5, P < 0.05). Specimens collected in autumn (39.4% or 187/474), spring (22.8% or 670/2940) and winter (18.2% or 129/709) also had higher positive rates than those collected in summer (3.8% or 57/1479) (χ2 = 427.7, P < 0.0001). Conclusions These results indicate that the new PCR method based on the T. gondii GRA14 gene would be useful for the diagnosis of swine toxoplasmosis and that it would facilitate the diagnosis of toxoplasmosis in clinical laboratories.

Expression of circulating miR375 and miR371 to differentiate teratoma and viable germ cell tumor in patients with post-chemotherapy residual disease.

2020 ◽  
Vol 38 (6_suppl) ◽  
pp. 414-414
Author(s):  
Lucia Nappi ◽  
Marisa Thi ◽  
Nabil Adra ◽  
Ricardo Romao Nazario Leao ◽  
Bernhard J. Eigl ◽  
...  
Keyword(s):  
Germ Cell ◽  
Residual Disease ◽  
Germ Cell Tumors ◽  
High Specificity ◽  
Internal Controls ◽  
Youden Index ◽  
Specificity And Sensitivity ◽  
Simultaneous Evaluation ◽  
Mirnas Expression ◽  
Sensitivity Specificity

414 Background: Viable germ cell tumors (vGCT) express high levels of certain circulating microRNAs, including miR-371a-3p (miR371) that has shown high specificity and sensitivity. However, neither tissue nor serum/plasma from patients with only teratoma are miR371 positive. miR375 is overexpressed in teratoma tissue, but detectability in blood is unknown. Methods: miR371 and miR375 expression was analyzed in 100 patients with various stages and histology of GCT. miR375 expression in teratoma was validated in patients with post-chemotherapy pathologically confirmed teratoma (PCPCT). The miRNAs expression was assessed by RT-PCR and quantified by ΔΔCT method. The optimal cut-off for miR375 expression was estimated by Youden index ( > 20). Spike-in cel-miR-39-3p, miR-451 and miR-30b-5p were used as internal controls. Sensitivity, specificity, AUC of the ROC of miR375 in detecting teratoma was analyzed. Results: In the discovery cohort miR371 and miR375 were measured in 62 pts: 27 CSI NED, 15 chemo-naïve metastatic seminoma and 20 with PCPCT. miR375 was over-expressed in pts with teratoma compared to CSI and seminoma pts (p = 0.002), while miR371 was expressed in the seminoma pts and undetectable in the PCPCT and CSI pts (p < 0.001). In the post-chemotherapy setting, 38 pts were analyzed: 21 PCPCT, 6 vGCT and 11 complete remission (CR). Also in this cohort, miR375 was over-expressed in pts with teratoma compared to the pts presenting vGCT and post-chemotherapy CR (p = 0.01), while miR371 was detectable only in the pts with vGCT (p < 0.001). Overall, sensitivity and specificity of miR375 in identifying teratoma were 78% and 80%, respectively; the AUC was 0.7 (95% CI: 0.5490-0.8186; p < 0.01). Conclusions: Pts with residual post-chemotherapy teratoma present higher plasma levels of miR375 compared to pts with vGCT in whom miR375 is low but miR371 is expressed at high levels. The simultaneous evaluation of miR371 and miR375 may be clinically useful to predict the histology of the GCT components in pts with post-chemotherapy residual disease to inform the best therapeutic options (surgery or chemotherapy). Further validation within larger studies is warranted.

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