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@#Objective To develop and verify a double antibody sandwich ELISA method for quantitative detection of TrypLE.Methods The optimal concentration of capture antibody and detection antibody were determined by orthogonal experiments to develop TrypLE double antibody sandwich ELISA quantitative detection method,which was verified for linear range,specificity,limit of detection(LOD),limit of quantitation(LOQ),accuracy and reproducibility.A variety of biological products were detected by the developed method to verify the applicability.Results The TrypLE double antibody sandwich ELISA quantitative detection method was established by using 3 μg/mL capture antibody and 15 000 times dilution of detection antibody,with a linear range of 0.41 ～ 40.00 ng/mL,a LOD of 0.258 ng/mL,a LOQ of 0.5 ng/mL.The measurement deviation was less than 5% and the CV of reproducibility verification was less than 5% when detecting standards and samples.The recovery rates of different types of samples were within 80% ～ 120%.Conclusion The established TrypLE double antibody sandwich ELISA quantitative detection method accurately,effectively and quickly detected residual amount of TrypLE in various types of biological products with good specificity,accuracy and reproducibility.
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Background & objectives: The pandemic caused by the SARS-CoV-2 has been a threat to humankind due to the rapid spread of infection and appearance of multiple new variants. In the present study, we report the dynamics and persistence of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies in asymptomatic and symptomatic COVID-19 patients by chemiluminescent assay. Methods: A total of 463 serum samples from 218 SARS-CoV-2 PCR-positive patients were collected over a period of 124 days post-onset of disease (POD). Antibody levels were measured by chemiluminescence bioanalyzer. Neutralizing antibody titres were assessed by plaque reduction neutralization test (PRNT) for SARS-CoV-2. Results: Both IgM and IgG started appearing from day five post-infection in symptomatic and asymptomatic patients. IgM antibody response peaked around day 35 POD and rapidly diminished thereafter, with the last IgM-positive sample observed at 90 days POD. IgG antibody response peaked around 45 days POD and persisted till 124 days. The chemiluminescence immunoassay (CLIA) results showed a moderate correlation (R=0.5846, P<0.001) compared with PRNT. Additional analysis indicated a neutralizing titre of 250 corresponded to 12.948 AU/ml of YHLO iFlash SARS-CoV-2 IgG units. Interpretation & conclusions: Both symptomatic and asymptomatic COVID-19 patients seem to initiate production of antibody responses from day five of onset of disease. Although the CLIA gives high sensitivity and specificity and also its binding IgG antibody titres may correlate moderately with protective immunity, our results indicate that the values of binding antibody alone may not be a perfect guide to represent virus neutralization titre during donor selection for plasma therapy. However, IgM and IgG antibody detection may help in monitoring the status of disease progression and burden in the community.
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Chronic HBV infection can generally be divided into four stages according to the natural course of disease. Clinically, the determination of different natural stages of chronic HBV infection is crucial for patients to start antiviral therapy and to avoid missing the antiviral opportunity and progressing to cirrhosis. In particular, it is a challenge for clinicians to distinguish the immune control stage from the reactive stage. As a novel marker of HBV, the quantitative detection of HBV core-associated antigen (HBcrAg) is of value for the identification of the HBV infection stages. This article reviews the research progress of HBcrAg in the identification of different stages of chronic HBV infection.
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As specific serological markers for autoimmune diseases, autoantibodies are significantly related to diseases or their clinical phenotypes with exclusiveness. Therefore, autoantibodies can not only favor clinical comprehension of different pathophysiological processes of autoimmune diseases, but also contribute to the early screening or diagnosis, severity evaluation, prognosis prediction and the decision-making process for treatment. An increasing number of international clinical practice guidelines have included autoantibodies for disease classification indicators, which further promoted the laboratory utility of them. However, there still exists many problems in the quality management of the determination and clinical application of autoantibodies in China. Thus, attentions should be paid to the standardized detection of autoantibodies and the promotion of new clinical applications.
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Liquid chromatography tandem mass spectrometry (LC-MS/MS) is rapidly applied and developed in the field of laboratory medicine. High resolution mass spectrometry promotes efficient screening of biomarkers. Triple quadrupole mass spectrometry helps verify the diagnosis of disease and monitoring efficacy of biomarkers. Its application runs through different fields and links from omics research to experimental diagnosis, and has gradually become an indispensable technical means of disease research and diagnosis. At present, most LC-MS platforms are in the stage of medical research, and there are still some challenges in clinical promotion and application.
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Objective To evaluate the diagnostic value of quantitative detection of cytomegalovirus (CMV) DNA from different sources [plasma, sputum and bronchoalveolar lavage fluid(BALF)] for CMV pneumonia after allogeneic hematopoietic stem cell transplantation. Methods Clinical data of 405 recipients undergoing allogeneic hematopoietic stem cell transplantation were retrospectively analyzed. Among them, 19 recipients diagnosed with CMV pneumonia were assigned into the CMV pneumonia group, and 229 recipients with CMV viremia alone, 11 recipients without CMV pneumonia who received fiberoptic bronchoscopy and 16 recipients diagnosed with bacterial or fungal pneumonia based on pathogenic evidence receiving sputum culture were assigned into the control A, B and C groups, respectively. The incidence of CMV pneumonia was summarized. The CMV DNA load of specimens from different sources (plasma, sputum and BALF) of recipients with CMV pneumonia was analyzed. The clinical prognosis of recipients with CMV pneumonia was evaluated. Results Among 405 recipients undergoing allogeneic hematopoietic stem cell transplantation, 19 cases developed CMV pneumonia, and the overall incidence of CMV pneumonia was 4.7%(19/405). The CMV DNA load in the plasma, sputum and BALF of recipients with CMV pneumonia was higher than those in the control A, B and C groups (all P < 0.05). In the 19 recipients, 12 cases were cured after antiviral treatment and 7 died from treatment failure(3 cases abandoned treatment). The fatality was 37%(7/19). Conclusions Quantitative detection of CMV DNA in the plasma, sputum and BALF may increase the diagnostic rate of CMV pneumonia, thereby improving clinical prognosis of recipients undergoing allogeneic hematopoietic stem cell transplantation.
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Nonalcoholic fatty liver disease (NAFLD) is a multi-system disease that also affects extrahepatic organs and overall metabolic regulation pathways, increasing the risk of type 2 diabetes, cardiovascular disease, and chronic kidney disease. The progressive form of NAFLD is called nonalcoholic steatohepatitis (NASH), which can be further developed into liver cirrhosis and liver cancer. Early screening, grading the disease and assessment of drug treatment response are the focus of current research. The detection of liver fat content is the key to early diagnosis of NAFLD, and it is also a reliable indicator for evaluating the drug efficacy in clinical trials. This article reviewed the noninvasive diagnostic techniques for the quantitative detection of liver fat content.
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Objective@#To explore the clinical value of magnetic resonance T2-mapping imaging in the diagnosis of early intervertebral disc degeneration in obese people.@*Methods@#From January 2018 to June 2019, 30 obese volunteers and 30 healthy volunteers with normal weight underwent T2-mapping scan, while their routine MRI examination showed no abnormalities were slected.The T2 value of nucleus pulposus in both two groups were measured and compared.@*Results@#The T2 values of nucleus pulposus of intervertebral disc of lumbar 1-2, lumbar 2-3, lumbar 3-4, lumbar 4-5 and lumbar 5-sacral 1 in the control group were (108.17±10.87)ms, (113.93±11.54)ms, (126.65±10.22)ms, (118.62±8.86)ms and (111.61±10.65)ms, respectively, which of nucleus pulposus in different segments was statistically significant(F=14.28, P<0.001). The T2 values of nucleus pulposus of intervertebral disc of lumbar 1-2, lumbar 2-3, lumbar 3-4, lumbar 4-5, lumbar 5-sacral 1 in the simple obesity group were (104.90±7.67)ms, (101.10±6.61)ms, (112.65±5.75)ms, (98.27±6.18)ms, (89.82±6.34)ms, respectively, , which of nucleus pulposus in different segments was statistically significant(F=49.52, P<0.001). There were statistically significant differences in the T2 values of nucleus pulposus of intervertebral disc of lumbar 2-3, lumbar 3-4, lumbar 4-5, lumbar 5-sacral 1 between the two groups(t=5.283, 6.535, 10.327, 9.626, all P<0.001).@*Conclusion@#Magnetic resonance T2-mapping can detect early changes in tissue composition of intervertebral disc degeneration.Therefore, magnetic resonance T2-mapping imaging technology can provide an important reference for the early diagnosis of intervertebral disc degeneration in obese people.
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Objective To explore the clinical value of magnetic resonance T 2 -mapping imaging in the diagnosis of early intervertebral disc degeneration in obese people.Methods From January 2018 to June 2019, 30 obese volunteers and 30 healthy volunteers with normal weight underwent T 2 -mapping scan,while their routine MRI examination showed no abnormalities were slected.The T2 value of nucleus pulposus in both two groups were measured and compared.Results The T2 values of nucleus pulposus of intervertebral disc of lumbar 1-2,lumbar 2-3,lumbar 3-4,lumbar 4-5 and lumbar 5-sacral 1 in the control group were (108.17 ±10.87)ms,(113.93 ± 11.54)ms,(126.65 ±10.22) ms,(118.62 ±8.86) ms and (111.61 ±10.65) ms,respectively,which of nucleus pulposus in different segments was statistically significant (F＝14.28,P＜0.001).The T2 values of nucleus pulposus of intervertebral disc of lumbar 1-2,lumbar 2-3,lumbar 3 -4,lumbar 4-5,lumbar 5 -sacral 1 in the simple obesity group were (104.90 ±7.67)ms,(101.10 ±6.61) ms,(112.65 ±5.75) ms,(98.27 ±6.18)ms,(89.82 ± 6.34)ms,respectively,,which of nucleus pulposus in different segments was statistically significant (F＝49.52,P＜0.001).There were statistically significant differences in the T 2 values of nucleus pulposus of intervertebral disc of lumbar 2-3,lumbar 3-4,lumbar 4 -5,lumbar 5 -sacral 1 between the two groups (t＝5.283,6.535,10.327, 9.626,all P＜0.001).Conclusion Magnetic resonance T2 -mapping can detect early changes in tissue composition of intervertebral disc degeneration.Therefore,magnetic resonance T2 -mapping imaging technology can provide an important reference for the early diagnosis of intervertebral disc degeneration in obese people .
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Elimination of HBV cccDNA from hepatocytes infected with chronic HBV virus is considered to be the key to eradicating HBV. Monitoring HBV cccDNA before, during, and after viral treatment is essential for routine treatment of patients with chronic hepatitis B. With the introduction of new anti-HBV treatment technologies and new drugs targeting HBV cccDNA, Accurate and sensitive HBV cccDNA assays are urgently needed to evaluate efficacy. In recent years, HBV cccDNA detection methods have achieved gratifying results in both traditional PCR methods and digital PCR methods popular in recent years. In this paper, the advances in HBV cccDNA quantitative detection by qPCR, Magnetic bead capture hybridization, rolling circle amplification combined with in situ PCR, digital PCR and digital PCR assay in single cells were reviewed.
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OBJECTIVE: To establish a high sensitivity method for rapid quantitative detection of morphine in the biological samples including serum, saliva and urine. METHODS: With the immunochromatographic lateral flow strip as reaction method, the luminescent lanthanide europium nanoparticles covalently conjugated with morphine monoclonal antibody were adopted as reporters. The morphine antigen and goat anti-mouse antibody were coated at the nitrocellulose membrane separately as the test line and control line. The strip based on competitive inhibition immunoassay principle was detected by the fluorescent reader for rapid quantitative detection of morphine in the biological samples. RESULTS: After experiment optimization, the improved strip could provide the line range 3-3 000 ng·mL-1; precise quality morphine control materials verified CV<10%. There were no cross reactions with amphetamine, methylamphetamine, ketamine and so on; keep the strips at 37 ℃ for 7 d, the performance of the strips did not decline markedly. CONCLUSION: The preliminary established method shows high linearity, precision, specificity and stability. The method could quantitatively detect the morphine in the biological samples in short time. It is supposed to be applied into the grassroots unit.
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<p><b>OBJECTIVE</b>Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China.</p><p><b>METHODS</b>Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR).</p><p><b>RESULTS</b>A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias.</p><p><b>CONCLUSION</b>This study provides a systematic analysis of norovirus contamination 'from the farm to the fork' in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.</p>
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To establish a time-resolved fluorescence immunochromatographic assay for quantitative determination of carbohydrate antigen 19-9 (CA19-9) in serum, we prepared CA19-9 test strips by integrating double-antibody sandwich method and fluorescence immunochromatography technique. Carboxy fluorescent microspheres and nitrocellulose membrane were used as carriers for labeling and coating CA19-9 pairing antibodies. We optimized the process by adjusting the amount of labeling and coating antibody. According to the linear range, lowest detection limit and precision, We evaluated the time-resolved fluorescence immunochromatographic assay of CA19-9. When the amount of labeled antibody was 80 μg for 20 μL fluorescent microspheres, and the concentration of coated antibody on the test line was 1.5 mg/mL, the optimal reaction time was 15 minutes. Assay linear range was 12.5 to 800 U/mL and the minimum detection limit was 6.32 U/mL. The Within-run and between-run coefficient of variation were less than 15%. Average recovery rate was 101%. By detecting 50 clinical samples in parallel with Roche electrochemical luminescence detection kit, correlation coefficient was 0.980 6. The experiment, initially established a fluorescence immunochromatographic detection method to quantitative detection of serum CA19-9, which has a good clinical application prospect.
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Near infrared spectroscopy (NIR) was used to detect trans fatty acids (TFA) in edible vegetable oils quantitatively. And prediction model of TFA was optimized through band selection, pretreatment method, variable selection and modeling method. NIR spectra of 98 edible vegetable oil samples were collected in spectral range of 4000-10000 cm-1 using an Antaris Ⅱ Fourier transform near infrared spectrometer, and the true content of TFA was measured by gas chromatography. First, optimization of waveband and pretreatment method was conducted on original spectra. On this basis, competitive adaptive reweighted sampling (CARS) was used to select important variables that related to TFA. Finally, the prediction models of TFA content in edible vegetable oils were established using principal component regression ( PCR), partial least square (PLS) and least square support vector machine (LS-SVM). The results indicated that NIR spectroscopy was feasible for detecting TFA content in edible vegetable oils, R2 of the best prediction model after optimized in calibration and prediction sets were 0. 992 and 0. 989, and root mean square error of calibration (RMSEC) and root mean square error of prediction ( RMSEP) were 0. 071% and 0. 075% , respectively. Only 26 variables were used in the best prediction model, accounting for 0. 854% of the whole waveband variables. In addition, compared with the full waveband PLS prediction model, the R2 in prediction set increased from 0. 904 to 0. 989, and RMSEP decreased from 0. 230% to 0. 075% . It shows that model optimization is very necessary, CARS method can select important variables related to TFA effectively and immensely reduce the number of modeling variables, so it can simplify the prediction model, and greatly improve the accuracy and stability of prediction model.
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Objective To achieve the goal of the qualitatively and relatively quantitatively analyze of the target pro-tein in a time-saving,labor-saving and reagents-saving way by the microfluidic paper-based immunoassay. Methods We chose MGMT as the target protein and compared the qualitative and semi-quantitative results of the MGMT ex-pression in the MCF7 cells which was treated with MGMT inhibitor, lomeguatrib, in both the traditional Western blot and the paper chip immunoassay. Results Microfluidic paper-based immunoassay can make the qualitative and relative quantitative detection on protein expression. Compared to the sensitivity of 1-5 ng of the traditional Western blot,the microfluidic paper-based immunoassay could detect as low as 10-25 pg of the protein. The sensitivity could be improved by 3 orders of magnitude. The entire operational duration took only 1 hour with less costed rea-gents being consumed. It maked the high-throughput protein detection as sensitive as the reverse phase protein as-says (RPPA) does. Conclusions The paper chip immunoassay could be performed qualitatively and semi-quanti-tatively to detect protein expression,and is more effective than that of traditional Western blot.
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A double quenching molecular beacon ( MB) with simple structure was designed based on organic quencher and G bases, and a simple detection method for thrombin was developed using this MB. In this MB, FAM and BHQ-1 were selected as fluorophore and organic quencher, three continuous nucleotides with G base were connected with BHQ-1, and the loop of MB was designed as a nucleic acid aptamer of thrombin. In the absence of thrombin, the MB was in the stem-loop structure, the fluorophore FAM was close to BHQ-1 and G bases, the fluorescence of FAM was dual quenched by BHQ-1 and G bases, and the fluorescence signal of FAM was very weak. In the presence of thrombin, MB specifically bound thrombin and formed a G-quadruplex structure. The stem-loop structure of the MB was destroyed, and FAM was separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. Under the optimal conditions, the fluorescence intensity of FAM exhibited a good linear relationship with concentration of thrombin in the range of 0. 4-40. 0 nmol/L, and regression equation was △I=24. 63C (nmol/L)+ 13. 06 (R2 =0. 9972) with the detection limit of 0. 18 nmol/L (3σ, n=9). The average recoveries of this method in serum samples were 96. 3%-98. 7%, which indicated that the method had high accuracy.
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Objective To achieve the goal of the qualitatively and relatively quantitatively analyze of the target pro-tein in a time-saving,labor-saving and reagents-saving way by the microfluidic paper-based immunoassay. Methods We chose MGMT as the target protein and compared the qualitative and semi-quantitative results of the MGMT ex-pression in the MCF7 cells which was treated with MGMT inhibitor, lomeguatrib, in both the traditional Western blot and the paper chip immunoassay. Results Microfluidic paper-based immunoassay can make the qualitative and relative quantitative detection on protein expression. Compared to the sensitivity of 1-5 ng of the traditional Western blot,the microfluidic paper-based immunoassay could detect as low as 10-25 pg of the protein. The sensitivity could be improved by 3 orders of magnitude. The entire operational duration took only 1 hour with less costed rea-gents being consumed. It maked the high-throughput protein detection as sensitive as the reverse phase protein as-says (RPPA) does. Conclusions The paper chip immunoassay could be performed qualitatively and semi-quanti-tatively to detect protein expression,and is more effective than that of traditional Western blot.
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A double quenching molecular beacon ( MB) with simple structure was designed based on organic quencher and G bases, and a simple detection method for thrombin was developed using this MB. In this MB, FAM and BHQ-1 were selected as fluorophore and organic quencher, three continuous nucleotides with G base were connected with BHQ-1, and the loop of MB was designed as a nucleic acid aptamer of thrombin. In the absence of thrombin, the MB was in the stem-loop structure, the fluorophore FAM was close to BHQ-1 and G bases, the fluorescence of FAM was dual quenched by BHQ-1 and G bases, and the fluorescence signal of FAM was very weak. In the presence of thrombin, MB specifically bound thrombin and formed a G-quadruplex structure. The stem-loop structure of the MB was destroyed, and FAM was separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. Under the optimal conditions, the fluorescence intensity of FAM exhibited a good linear relationship with concentration of thrombin in the range of 0. 4-40. 0 nmol/L, and regression equation was △I=24. 63C (nmol/L)+ 13. 06 (R2 =0. 9972) with the detection limit of 0. 18 nmol/L (3σ, n=9). The average recoveries of this method in serum samples were 96. 3%-98. 7%, which indicated that the method had high accuracy.
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A method for quantitative detection of florfenicol by colloidal gold lateral flow immunoassay was developed.The experimental conditions including pH value, concentrations of antibody in the process of conjugation between the colloidal gold and antibody, amount of gold-labeled antibody, concentration of the antigen sprayed on test lines (T line), and detection time were optimized.With a colloidal gold strip reader, the signal intensity of T lines and control lines (C line) on lateral flow strips was recorded.The T/C ratio of negative control and positive samples was defined as B0 and Bx, and the standard curve was established by plotting the Bx/B0 ratio against the concentration of florfenicol.This assay showed a good linear range from 0.1 to 1.5 ng/mL with the limit of detection of 0.08 ng/mL, while the result could be obtained within 15 min.The result showed that this quantitative method was convenient and rapid, and could be used in screening a large amount of samples on site.
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Objective To establish a rapid, simple, sensitive, and specific multiplex real-time PCR method for quantitative detection of Campylobacter jejuni, Salmonella and Shigella in tree shrews.Methods Specific primers and probes were designed, according to the HipO gene of Campylobacter jejuni, inV gene of Salmonella and ipaH gene of Shigella.The primers were confirmed by single pathogen quantitative PCR, and the sensitivity and specificity of the multiplex PCR were analyzed.Finall, the samples of experimental tree shrews were detected by this multiplex PCR method.Results The PCR element of TaqMan-MGB real-time PCR assay was able to quantitatively amplify the Campylobacter jejuni, Salmonella or Shigella.Appropriate standard amplification curves of Campylobacter jejuni, Salmonella and Shigella in the multiplex quantitative PCR were obtained.The sensitivity of this method was 1×103 ng/μL.There was no false positive detection from other bacterial strains.Conclusions This multiplex quantitative real-time PCR method has good application and development prospects in the detection of microorganisms in tree shrews.