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The detection of serum antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a new tool for the coronavirus disease-2019 (COVID-19) diagnosis. Since many coronaviruses are sensitive to heat, heating inactivation of samples at 56 {degrees}C prior to testing is considered a possible method to reduce the risk of transmission, but the effect of heating on the measurement of SARS-CoV-2 antibodies is still unclear. By comparing the levels of SARS-CoV-2 antibodies before and after heat inactivation of serum at 56 {degrees}C for 30 minutes using a quantitative fluorescence immunochromatographic assay, we shown that heat inactivation significantly interferes with the levels of antibodies to SARS-CoV-2. The IgM levels of all the 34 serum samples (100%) from COVID-19 patients decreased by an average level of 53.56%. The IgG levels were decreased in 22 of 34 samples (64.71%) by an average level of 49.54%. Similar changes can also be observed in the non-COVID-19 diseases group (n=9). Of note, 44.12% of the detected IgM levels were dropped below the cut-off value after heating, suggesting heat inactivation can lead to false-negative results of these samples. Our results indicate that heat inactivation of serum at 56 {degrees}C for 30 minutes interferes with the immunoanalysis of antibodies to SARS-CoV-2. Heat inactivation prior to immunoanalysis is not recommended and the possibility of false-negative results should be considered if the sample was pre-inactivated by heating.
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Extracellular vesicles (EVs), one of the basic ways of intercellular interactions, carry a large number of biological substances and show great potential in disease diagnosis and treatment. Compared with tissue biopsy, its detection has the advantages of non-invasive, convenient sampling and real-time monitoring. Therefore, the research and clinical application of EVs biomarkers have become an international research hotspot. In order to encourage the development of screening and detection technologies for EVs biomarkers, effectively improve the accuracy of EVs biomarker diagnosis, and promote the transformation of EVs biomarker research results into the clinical application of disease, this review summarizes the significance of EVs in disease diagnosis and its biomarker screening strategy and clinical validation.
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Objective@#To establish a colloidal gold technique assay for the rapid detection of immunoglobulin(Ig) M and IgG antibodies against 2019 novel coronavirus (2019-nCoV) and to evaluate its clinical performance.@*Methods@#A total of 278 patients who were treated at Wuhan Hankou Hospital and the People's Hospital of Honghu from February 12, 2020 to February 20, 2020 were collected. According to the diagnostic criteria, 89 patients were confirmed with 2019-nCoV nucleic acid positive diagnosis, and 189 were 2019-nCoV nucleic acid-negative suspected patients. A total of 273 medical examiners from Nanfang Hospital, Southern Medical University from 2015 to 2018 were selected as controls. The serum samples of patients were collected. 2019-nCoV nucleic proteins were obtained from prokaryotic expression vectors. Indirect IgM and IgG colloidal gold techniques were established by using recombinant N protein. 2019-nCoV nucleic acid detection by reverse transcription-polymerase chain reaction (RT-PCR) was used as control. Serum specimens were tested for 2019-nCoV IgM and IgG. The specificity and sensitivity of colloidal gold assay were analyzed.@*Results@#The sensitivity and specificity of IgM detection reagents were 78.7% and 98.2%, respectively, those of IgG detection reagents were 73.0% and 99.3%, respectively, and those of IgM combined with IgG detection were 87.6% and 98.2%, respectively. For suspected patients with negative 2019-nCoV nucleic acid, the positive rates of IgM and IgG were 59.8% (113/189) and 52.9% (100/189), respectively, and the positive rate of IgM combined with IgG detection was 66.1% (125/189).@*Conclusion@#This reagent of 2019-nCoV antibodies detection (colloidal gold technique) fulfills the requirement for clinical application with high specificity and sensitivity, which can be served as a supplementary detection method for 2019-nCoV nucleic acid detection by RT-PCR.
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Objective:To establish a colloidal gold technique assay for the rapid detection of immunoglobulin(Ig)M and IgG antibodies against 2019 novel coronavirus (2019-nCoV) and to evaluate its clinical performance.Methods:A total of 278 patients who were respectively treated at Wuhan Hankou Hospital and the People′s Hospital of Honghu from February 12, 2020 to February 20, 2020 were collected. According to the diagnostic criteria, 89 patients were confirmed with positive 2019-nCoV nucleic acid, and 189 were 2019-nCoV nucleic acid-negative suspected patients. A total of 273 medical examiners from Nanfang Hospital, Southern Medical University from 2015 to 2018 were selected as controls. The serum samples of patients were collected. 2019-nCoV nucleic proteins were obtained from prokaryotic expression vectors. Indirect IgM and IgG colloidal gold techniques were established by using recombinant nuclear protein. 2019-nCoV nucleic acid detection by reverse transcription-polymerase chain reaction (RT-PCR) was used as control. Serum specimens were tested for 2019-nCoV IgM and IgG. The specificity and sensitivity of colloidal gold assay were analyzed.Results:The positive rates of IgM and IgG with the colloidal gold detection in confirmed patients with positive 2019-nCoV nucleic acid were 78.7%(70/89) and 73.0%(65/89), respectively. The positive rates of IgM and IgG in medical examiners were 1.8%(5/273) and 0.7%(2/273), respectively. The sensitivity and specificity of IgM detection reagents were 78.7% and 98.2%, respectively, those of IgG detection reagents were 73.0% and 99.3%, respectively, and those of IgM combined with IgG detection were 87.6% and 98.2%, respectively. For suspected patients with negative 2019-nCoV nucleic acid, the positive rates of IgM and IgG were 59.8%(113/189) and 52.9%(100/189), respectively, and the positive rate of IgM combined with IgG detection was 66.1%(125/189).Conclusion:This reagent of 2019-nCoV antibodies detection (colloidal gold technique) fulfills the requirement for clinical application with high specificity and sensitivity, which can be served as a supplementary detection method for 2019-nCoV nucleic acid detection by RT-PCR.
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Extracellular vesicles (EVs), one of the basic ways of intercellular interactions, carry a large number of biological substances and show great potential in disease diagnosis and treatment. Compared with tissue biopsy, its detection has the advantages of non-invasive, convenient sampling and real-time monitoring. Therefore, the research and clinical application of EVs biomarkers have become an international research hotspot. In order to encourage the development of screening and detection technologies for EVs biomarkers, effectively improve the accuracy of EVs biomarker diagnosis, and promote the transformation of EVs biomarker research results into the clinical application of disease, this review summarizes the significance of EVs in disease diagnosis and its biomarker screening strategy and clinical validation.
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Objective To observe the expression level and diagnosis performance of serum exosomal miR-21 in lung cancer. Method Sera of 94 lung cancer patients,29 pulmonary tuberculosis patients,and 26 healthy controls from Nan Fang Hospital were collected. Exosomes in serum were purified and characterized ,and miRNA was abstracted from exosomes. Levels of exosomal miR-21 in three groups were compared with relative quantitative RT-PCR. Diagnosis performance of exosomal miR-21 was evaluated by ROC curve. Results Purified serum exosome sample possesses typical exosome features. There were significant difference among exosomal miR-21 levels from lung cancer ,tuberculosis ,and health control group. Exosomal miR-21 obviously increased in lung cancer patients,and decreased in tuberculosis patients. Exosomal miR-21 were increased in early and late stage lung cancer patients. The diagnostic performance of exosomal miR-21 was evaluated by ROC curve. Its area under the curve was 0.702 and specificity was 0.923. Conclusions Serum exosomal miR-21 was increased in many kinds of lung can-cer patients. It possesses excellent diagnosis performance ,which may be developed as new biomarkers to assist lung diagnosis.
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Objective The effect of serum exosome isolation method on obtained exosomal RNA is not yet clear .The aim of this study was to provide evidences to selected exosome isolation methods .Methods This was a method comparison study to assess three methods .Vein blood samples were collected from 3 healthy donors from Nanfang hospital , 4 ℃ 12 h was taken to make blood natural coagulated and the supernatant was taken as the serum . Exosomes extracted by Ultracentrifugation , ExoQuick and Total Exosome Isolation Reagent ( TEI ) kits from serum were assessed by transmission electron microscopy , nanoparticle tracking analysis and protein analysis to identify morphology , protein expression , concentration and size distribution of particles .ExoRNA extracted by Trizol-LS, SeraMir, Total Exosome RNA Isolation ( TER) and exoRNeasy were assessed by Bioanalyzer 2100 and qPCR to ascertain RNA distribution and miRNA expression.Results For exosomes isolation , two commercial kits ( ExoQuick and TEI ) showed higher exosomes recovery and protein concentration than ultracentrifugation , but exosome isolated by ultracentrifugation expressed abundant protein makers mostly .For exo-RNA isolation, the distribution of RNA and expression of miRNAinfluenced by three methods , but there was no effect on the relative expression trend of miRNA.ExoRNeasy resulted in the highest yield and most narrow size distribution pattern of small RNA with higher level of miRNA expression .Results of TEI with TER kits showed no obvious bands of small RNA and moderate miRNA expression among methods .Ultra with Trizol-LS or ExoQuick with SeraMir showed low concentration measurable bands around 100 nt, with changeable miRNA profiling irregularly . Conclusions Extraction of exosomes using traditional ultracentrifugation method is applicable for proteomics research work .Extraction using ExoQuick and TEI kits is suitable for preparing exosomes from scarce samples such as serum.