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The serum biomarker copeptin, an innovative and stable substitute biomarker of vasopressin, is associated with stroke. Therefore, establishing a highly sensitive time-resolved fluorescence immunoassay for copeptin (copeptin-TRFIA) is helpful to measure stroke and evaluate its value in clinical applications. Double antibody sandwich was used to establish copeptin-TRFIA. The established method was then assessed. Two coated and Eu3+-labeled copeptin monoclonal specific antibodies targeting different antigen epitopes were employed. The serum fluorescence counts of patients with stroke and healthy volunteers were detected by using the well-established copeptin-TRFIA. Serum copeptin levels were measured and analyzed statistically. The actual measurement linearity range of the proposed method was 0.13–44.66 ng/mL. Copeptin-TRFIA had the inter-assay coefficient of variation (CV) of 6.49%–9.08% and the intra-assay CV of 4.75%–7.77%. Patients with cerebral infarction (CI) and intracerebral hemorrhage (ICH) had significantly higher serum copeptin levels than healthy subjects. Copeptin concentrations in the serum of patients with stroke were significantly correlated with the scores of the National Institute for Healthy Stroke Scale (NIHSS) and modified Rankin Scale (mRS). A highly sensitive copeptin-TRFIA was successfully established. Serum copeptin has a certain value in the clinical diagnosis and prognosis of stroke. [ABSTRACT FROM AUTHOR]

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Objective Canine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases. Methods A sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated. Results A double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than − 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%. Conclusion In this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future.

Objective: Canine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases. Methods: A sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated. Results: A double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than − 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%. Conclusion: In this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future. [ABSTRACT FROM AUTHOR]

ABSTRACTTobacco mosaic virus (TMV) causes a significant yield loss in solanaceous crops. Accurate and rapid diagnosis of TMV is an essential pre-requisite for proactive and tailored disease management strategies, filling the gap between more rigorous laws regarding pesticide use and maintaining crop production. Herein, we developed a time-resolved fluorescence immunoassay (TRFIA) to quantitatively detect TMV in tobacco. TMV virions were used as immunogens to develop monoclonal antibodies, which were then conjugated to Eu3+ nanospheres. The detection limit of the TRFIA platform was 0.24 ng/mL, with recovery rates in the range of 90.71–108.11% in spiked tobacco. The specificity was confirmed using ten species of tobacco-infecting phytopathogens. In-field applicability was implemented by screening for TMV in 10,000 tobacco leaf samples collected from five fields in Kunming, China. Overall, the proposed TRFIA platform is rapid, accurate, and end-user friendly, demonstrating its application in TMV onsite testing in tobacco.

Copyright of Chinese Medical Equipment Journal is the property of Chinese Medical Equipment Journal Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Jianfeng Hong,1 Xindong Chen,2 Lingli Chen,2 Yigang Wang,2 Biao Huang,2 Hongming Fang1 1Affiliated Xiaoshan Hospital, Hangzhou Normal University, Hangzhou, People’s Republic of China; 2Immunological Analysis Laboratory of Academy of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, People’s Republic of ChinaCorrespondence: Hongming Fang, Affiliated Xiaoshan Hospital, Hangzhou Normal University, Hangzhou, 310016, People’s Republic of China, Tel + 86 0571-83865858, Email 3295988078@qq.com Biao Huang, Immunological Analysis Laboratory of Academy of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, 310016, People’s Republic of China, Tel +86 0571-86843187, Email jswxhb@163.comPurpose: The present study aimed to evaluate the clinical value of Combined Detection of serum soluble T-cell immunoglobulin 3 (sTim-3) with carcinoembryonic antigen (CEA) or glycotype antigen 19– 9 (CA19-9) for Postoperative Recurrence of Colorectal Cancer (CRC) Diagnosis.Patients and Methods: The serum sTim-3 was measured by highly sensitivity TRFIA, and serum CEA and CA19-9 were obtained through the collection of clinical data. Quantitative detection of serum sTim-3, CEA, CA19-9 in 90 patients after the CRC surgery (52 postoperative recurrence and 38 no-postoperative recurrence), 21 patients with colorectal benign tumors, and 67 healthy controls. To analyze the clinical diagnostic value of combined detection of sTim-3 with CEA or CA19-9 to test whether patients have recurrence after CRC surgery.Results: The sTim-3 (15.94± 11.24ng/mL) in patients after CRC surgery was significantly higher than in healthy controls (8.95± 3.34ng/mL) and colorectal benign tumors (8.39± 2.28ng/mL) (P < 0.05), and sTim-3 (20.33± 13.04ng/mL) in CRC postoperative recurrent group was significantly higher than in the group without recurrence after CRC surgery (9.94± 2.36ng/mL) (P < 0.05). In terms of detecting postoperative recurrence after CRC surgery, combined detection of sTim-3 and CEA (AUC: 0.819, sensitivity: 80.77%, specificity: 65.79%), sTim-3 and CA19-9 test (AUC: 0.813, sensitivity: 69.23%, specificity: 97.30%) was significantly better than the CEA single test (AUC: 0.547, sensitivity: 63.16%, specificity: 48.08%) and CA19-9 single test (AUC: 0.675 sensitivity: 65.38%, specificity: 67.57%), Delong test P < 0.05.Conclusion: The efficacy of CEA and CA19-9 single test was not optimal, and the combination of sTim-3 in serum could significantly improve the sensitivity and specificity of detecting patient recurrence after CRC surgery.Keywords: serum tumor markers, Tim-3, time-resolved fluorescence immunoassay, colorectal cancer recurrence

Abstract Background Lymphocytic leukemia (LL) is a primary malignant tumor of hematopoietic tissue, which seriously affects the health of children and the elderly. The study aims to establish a new detection method for screening acute/chronic LL using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1) in serum. Methods Here a sandwich TRFIA was optimized and established: Anti-S100A8/LRG1 caputre antibodies immobilized on 96-well plates captured S100A8/LRG1, and then banded together with the anti-S100A8/LRG1 detection antibodies labeled with Europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally time resolved fluorometry measured the fluorescence intensity. Results The sensitivity of S100A8 was 1.15 ng/mL(LogY = 3.4027 + 0.4091 × LogX, R 2 = 0.9828, P

Deoxynivalenol (DON) contamination of food crops and feeds is almost impossible to avoid completely; however, through best management practices, this risk can be effectively managed and maximumly mitigated. Accurate and rapid detection of DON contamination as early in the entire value chain as possible is critical. To achieve this goal, we developed a DON test strip based on time-resolved fluorescence immunoassay (TRFIA) and a specific DON monoclonal antibody for the rapid quantification of DON in food crops and feeds. The strip displayed a good linearity (R2 = 0.9926), with a limit of quantification of 28.16 μg/kg, a wide linear range of 50 ~ 10,000 μg/kg. The intra-batch coefficient of variation (CV) and the inter-batch CV was

This study aimed to establish time-resolved fluorescence immunoassays to quantitatively detect the autoantibodies targeting different epitopes of M-type phospholipase A2 receptor (PLA2R) and evaluate its clinical application in primary membranous nephropathy (PMN). PLA2R and its reactive epitope-specific IgG/IgG4 time-resolved fluorescence immunoassays (TRFIAs) were established using europium-labeled anti-human IgG/IgG4 antibodies, recombinant proteins, and patient serum. The levels of IgG/IgG4 targeting PLA2R and its epitopes in PMN patient serum were detected, and the relationship between epitope spreading of PLA2R and the severity of patients with PMN was evaluated. The TRFIAs established in this study could quantitatively detect PLA2R and its epitope-specific IgG and IgG4. Sera from 59 patients with PMN were subjected to detection using anti-PLA2R IgG and anti-PLA2R IgG4. Among them, 46 and 54 patients were found positive for PLA2R antibodies, respectively. Moreover, the levels of PLA2R antibodies were strongly correlated with the severity of patients with PMN. Patients who were detected to have two or more epitopes had more serious renal injury. PLA2R domain-specific IgG/IgG4 TRFIAs were established in this study, and detection with anti-PLA2R IgG4 could more sensitively screen the reactivity of patients to the PLA2R domain. Moreover, detection epitope spreading of PLA2R was confirmed which is related to the severity of patients with PMN. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Modern Laboratory Medicine is the property of Journal of Modern Laboratory Medicine Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Elevated serum high‐sensitivity C‐reactive protein (hs‐CRP) and lipoprotein(a) (Lp(a)) levels are associated with the development of native coronary atherosclerosis. We aimed to establish a new method for the simultaneous detection of hs‐CRP and Lp(a) to predict the development of atherosclerosis. A one‐step time‐resolved fluorescence immunoassay (TRFIA) with europium(III) (Eu3+) or samarium(III) (Sm3+) labels was established, and the performance of this TRFIA (in terms of sensitivity, specificity, accuracy, and cutoff values) was evaluated using clinical serum samples and compared with those of registered kits. The sensitivity was 0.052 μg/ml for hs‐CRP and 0.64 μg/ml for Lp(a). The intra‐assay and inter‐assay cross‐reactivities (CVs) were very low, ranging from 2.05% to 4.67% for hs‐CRP and from 2.42% to 6.43% for Lp(a). The CVs were very low (<0.34% and <2.65%, respectively) with five interferents. Additionally, there was a high Pearson coefficient between the present TRFIA method and the registered kits (R2 = 0.9967 and 0.9906, respectively). These data indicate that this study developed a TRFIA method that can be used for the quantitative detection of hs‐CRP and Lp(a) in serum with high sensitivity, specificity, and accuracy. This TRFIA provides a new method for predicting the development of atherosclerosis. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is a global disease that is harder to treat at a later stage. Therefore, early diagnosis and monitoring of CKD are crucial. T cell immunoglobulin and mucin domain molecule 3 (Tim-3) is a negative regulator of the T cell responses and it is involved in the immunomodulation of kidney disease. To date, only a small number of reports regarding serum soluble Tim-3 (sTim-3) in CKD are available. In the present study, the serum levels of sTim-3 in patients with CKD at different stages and the levels of sTim-3 in the early diagnosis and monitoring of CKD were analyzed. A highly sensitive time-resolved fluorescence immunoassay was performed to quantify sTim-3 levels in 318 patients with CKD and 114 healthy individuals. The serum levels of sTim-3 in patients with CKD (33.47±20.77 ng/ml) were significantly higher than those in the healthy individuals group (8.32±3.23 ng/ml; P<0.0001). As CKD progressed from stage G1 to G5, the serum sTim-3 level gradually increased (P<0.0001). A cut-off value of 13.63 ng/ml for the sTim-3 concentration was effective in diagnosing patients with CKD (area under the receiver operating characteristic curve, 0.9176; sensitivity, 79.87%; specificity, 96.49%). At this critical value, the positive detection rate of CKD in the early stages (G1 + G2), G3, G4 and G5 was 55.70, 77.78, 84.44 and 92.86%, respectively. In conclusion, the serum sTim-3 levels in patients with CKD were significantly higher than those in the healthy individuals group. As CKD progressed from G1 to G5, the serum sTim-3 concentration gradually increased, facilitating the monitoring of the progression of CKD. In addition, serum sTim-3 had an auxiliary effect that was useful in the early diagnosis of CKD. The positive detection rate of CKD in the early stages was 55.70%, which can assist other clinically common kidney disease indicators. [ABSTRACT FROM AUTHOR]

Background: Lymphocytic leukemia (LL) is a primary malignant tumor of hematopoietic tissue, which seriously affects the health of children and the elderly. The study aims to establish a new detection method for screening acute/chronic LL using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1) in serum. Methods: Here a sandwich TRFIA was optimized and established: Anti-S100A8/LRG1 caputre antibodies immobilized on 96-well plates captured S100A8/LRG1, and then banded together with the anti-S100A8/LRG1 detection antibodies labeled with Europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally time resolved fluorometry measured the fluorescence intensity. Results: The sensitivity of S100A8 was 1.15 ng/mL(LogY = 3.4027 + 0.4091 × LogX, R2 = 0.9828, P < 0.001, dynamic range: 2.1–10,000 ng/mL), and 3.2 ng/mL for LRG1 (LogY = 3.3009 + 0.4082 × LogX, R2 = 0.9748, P < 0.001, dynamic range: 4.0–10,000 ng/mL). The intra-assay and inter-assay CVs were low, ranging from 5.75% to 8.23% for S100A8 and 5.30% to 9.45% for LRG1 with high specificity and affinity in serum samples. Bland–Altman plots indicated TRFIA and ELISA kits have good agreement in clinical serum samples. Additionally, the cutoff values for S100A8 and LRG1 were 1849.18 ng/mL and 588.08 ng/mL, respectively. Conclusion: The present TRFIA method could be used for the quantitative detection of S100A8 and LRG1 in serum, and it has high sensitivity, accuracy and specificity. Clinically, this TRFIA method could be suitable for screening of LL via the quantitative detection of S100A8 and LRG1. [ABSTRACT FROM AUTHOR]

Introduction: The objective of this study was to develop a highly sensitive time-resolved fluorescence immunoassay (TRFIA) method to detect phospholipase A2 receptor (PLA2R)-IgG1 antibodies and evaluate its clinical relevance in predicting the prognosis of individuals with idiopathic membranous nephropathy (IMN)., Materials and Methods: A three-step indirect TRFIA method was established using a PLA2R antigen-coated microtiter plate to capture PLA2R-IgG antibodies, followed by detection using mouse anti-human IgG1 and Eu 3+ -labeled goat anti-mouse IgG antibodies. This method was applied to the initial serum of 56 patients with PLA2R-IMN to investigate the clinical value of PLA2R-IgG1 antibody levels in predicting IMN prognosis., Results: The detection range of PLA2R-IgG1-TRFIA was 0.85-300RU/mL, with intra-assay precision of 3.54-5.93 % and inter-assay precision of 4.39-9.36 %. Recoveries were 101.77-108.04 %. A PLA2R-IgG1 level above 2.21RU/mL indicated PLA2R-IMN. At initial diagnosis, the median PLA2R-IgG level was 51.24Ru/mL in the remission group and 93.27Ru/mL in the non-remission group. The median PLA2R-IgG1 level was 603.32RU/mL in the non-remission group, which was 4.29 times higher than that in the remission group (140.67RU/mL). PLA2R-IgG1 levels (P = 0.001) more effectively distinguished between remission and non-remission groups compared with PLA2R-IgG levels (P = 0.094)., Conclusions: The first quantitative TRFIA for PLA2R-IgG1 was established, showing greater clinical value in predicting IMN prognosis, compared to that for PLA2R-IgG levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lingli Chen,1,&ast; Jianfeng Hong,2,&ast; Renjing Hu,3,&ast; Xiaomei Yu,1 Xindong Chen,1 Shaoxiong Zheng,1 Yuan Qin,1 Xiumei Zhou,1 Yigang Wang,1 Liping Zheng,2 Hongming Fang,2 Pengfei Liu,4 Biao Huang1 1Department of Immunoassay Laboratory, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, People’s Republic of China; 2Department of Laboratory, Affiliated Xiaoshan Hospital, Hangzhou Normal University, Hangzhou, People’s Republic of China; 3Department of Laboratory, The Affiliated Wuxi No.2 People’s Hospital of Nanjing Medical University, Wuxi, People’s Republic of China; 4Department of Gastroenterology, The Jiangyin Clinical College of Xuzhou Medical University, Wuxi, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Biao HuangImmunoassay Laboratory, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, 310016, People’s Republic of ChinaTel +86 571-86843187Email jswxhb@163.comHongming FangDepartment of Laboratory, Affiliated Xiaoshan Hospital, Hangzhou Normal University, Hangzhou, 310016, People’s Republic of ChinaTel +86 571 83865858Email 3295988078@qq.comPurpose: The present study aimed to evaluate the clinical value of the combined detection of soluble T cell immunoglobulinand mucin domain molecule 3 (sTim-3) and pepsinogen (PG) in sera for gastric cancer (GC) diagnosis.Patients and Methods: The double antibody sandwich method was used to establish a highly sensitive time-resolved fluorescence immunoassay for the detection of sTim-3. Serum sTim-3, PGI, and PGII levels in 149 GC patients (123 first-diagnosis GC patients and 26 post-GC patients), 81 patients with benign gastric disease (BGD), and 73 healthy controls were quantitatively detected. The clinical diagnostic value of the combined detection of sTim-3 and PG in GC was analyzed.Results: Serum sTim-3 levels in GC (20.41 ± 9.55 ng/mL) and BGD (16.50 ± 9.76 ng/mL) patients were significantly higher (P < 0.001) than those in healthy controls (9.22 ± 3.40 ng/mL). Combined detection of sTim-3 and PGI/PGII (AUC: 0.9330, sensitivity: 86.44%, and specificity: 91.78%) showed a high diagnostic value for GC. When the level of PGI/PGII was less than 12.11 and that of sTim-3 was greater than 14.30 ng/mL, the positive rate of the control group was reduced to 0%, and the positive detection rate of GC was 54.47%. In addition, in post-operative patients, serum sTim-3 levels in the recurrence group (33.56 ± 4.91 ng/mL) were significantly higher than those in the no recurrence group (11.95 ± 5.16 ng/mL).Conclusion: sTim-3 levels in BGD and GC sera were significantly higher than those in the control group sera. Additionally, sTim-3 serum levels can predict recurrence in post-operative patients. Compared with PG alone, the combined detection of serum PG and sTim-3 can significantly improve the detection sensitivity and specificity of BGD and GC.Keywords: T cell immunoglobulin and mucin domain molecule 3, time-resolved fluorescence immunoassay, biomarker, gastric cancer

Coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is spreading rapidly around the world. Antibody detection plays an important role in the diagnosis of COVID‐19. Here, we established a new time‐resolved fluorescence immunoassay (TRFIA) to determine COVID‐19 total antibodies. A double‐antigen sandwich TRFIA was optimized and established: recombinant nucleocapsid phosphoprotein (N protein) and spike protein (S protein) of COVID‐19 immobilized on 96‐well plates captured human COVID‐19 antibodies and then banded together with the N/S proteins labeled with europium(III) (Eu3+) chelates, and finally, time‐resolved fluorometry was used to measure the fluorescence values. We successfully established a TRFIA method for the detection of human COVID‐19 total antibodies, and the cutoff value was 2.02. There was no cross‐reactivity with the negative reference of the National Reference Panel for IgM and IgG antibodies to COVID‐19. The CV of the precision assay was 3.19%, and the assay could be stored stably for 15 days at 37°C. Compared with that of the colloidal gold method and chemiluminescence method, the sensitivity of the TRFIA method was higher, and the false positive/negative rate was lower. This established TRFIA has high sensitivity, accuracy, and specificity, which indicates that this method provides a new detection method for the high‐throughput routine diagnosis of COVID‐19. [ABSTRACT FROM AUTHOR]

Objective To investigate the value of double-labeled time-resolved fluorescence immunoassay (TRFIA) for quantitatively detecting serum levels of tumor necrosis factor-α (TNF-α) and high-sensitivity C-reactive protein (hs- CRP). Methods Anti-TNF-α and hs-CRP monoclonal antibodies were coated on the 96-well plate, meanwhile prepared the europium (Eu3+) and samarium (Sm3+)-detection antibody conjugates, and then established the double-antibody sandwich TRFIA method and assembly into a kit, and finally evaluated the detection performance of this kit, such as sensitivity, linear range, spike recovery rate. Results A new method for detecting serum TNF- α and hs-CRP levels by TRFIA was successfully established and assembled into a kit. The linear range of the prepared TRFIA kit for TNF-α was 0-100 ng/L, sensitivity was 0.05 ng/L, the linear range for hs-CRP was 0-100 mg/L, sensitivity was 0.02 mg/L. The spiked recovery rate of TNF-α was between 92.00% and 107.00%, and that of hs-CRP was between 95.00% and 106.82%. There was no obvious cross-reaction with the symptom detection indexes. The TNF-α CV of intra-assay was between 4.57% and 9.24%. The interassay was between 5.13% and 9.27%. The hs-CRP CV of intra-assay was between 5.12% and 7.69%, and the inter-assay was 6.07%-10.00%. Additionally, the kit can be stored stably at 4 ℃ for half a year and at 37 ℃ for 7 days. The detection threshold of TNF-α was 0.44 ng/L, and the detection threshold of hs-CRP was 1.41 mg/L. The test results of the kit were consistent with the clinical situation, and the coincidence rate reached 100%. Conclusion The double-labeled TRFIA method can quantitatively detect TNF-α and hs-CRP levels, which has the advantages of high sensitivity, high specificity, convenience and fast. This TRFIA kit provides a new detection method for the early screening, efficacy evaluation and prognostic assessment of clinical samples of sepsis. [ABSTRACT FROM AUTHOR]

To establish a rapid and highly sensitive assay for tumor-associated trypsinogen-2 (TAT-2) based on the time-resolved fluorescence immunoassay (TRFIA) and evaluate its potential clinical value in patients with lung cancer. The double-antibody sandwich method was used in detecting TAT-2 antigen concentrations, and two types of TAT-2 antibodies (coating antibodies and Eu3+ labeled antibodies) were used. A TAT-2–TRFIA method was then established, evaluated, and used in detecting the serum TAT-2 levels of healthy subjects and patients with lung cancer. The linear range of the TAT-2–TRFIA method was 1.53–300 ng/mL, the intra-assay coefficient of variation (CV) were between 1.67% and 8.42%, and the inter-assay CV were between 4.29% and 11.44%. The recovery rates of TAT-2–TRFIA were between 99.17% and 107.06%. The cross-reactivities of trypsin and T-cell immunoglobulin mucin 3 were 0.02% and 0.82%, respectively. The serum TAT-2 levels of patients with lung cancer were higher than those of healthy subjects (P < 0.001). Combined with TAT-2, the sensitivity and specificity of CEA and CA-125 for lung cancer improved significantly. Conclusion: We successfully established a highly sensitive TAT-2–TRFIA method, which was able to facilitate the timely diagnosis of lung cancer. [ABSTRACT FROM AUTHOR]

The primary pollutants in cereal products are aflatoxins B1 (AFB1), zearalenone (ZEN), and deoxynivalenol (DON). In this study, anti-AFB1 MAb (4C9), anti-ZEN MAb (2A3), and anti-DON MAb (1F10) were developed and used in time-resolved fluorescence immunoassay microfluidics to determine AFB1, ZEN, and DON in agricultural products. The linear range for AFB1, ZEN, and DON were 0.05~2.2 μg/kg, 1.45~375.75 μg/kg, and 11.1~124.2 μg/kg, respectively. In maize, the recoveries of AFB1/ZEN/DON were 92~101%, 102~105%, and 103~108%, respectively. High-performance liquid chromatography and the proposed approach had a good correlation. Time-resolved fluorescence immunoassay microfluidics is a highly efficient and sensitive field detection method for fungal toxins in agricultural products. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

BackgroundViral hepatitis is a widespread and serious infectious disease, and most patients with liver cirrhosis and hepatocellular carcinoma are prone to viral infections. T cell immunoglobulin-and mucin-domain-containing molecule-3 (Tim-3) is an immune checkpoint molecule that negatively regulates T cell responses, playing an extremely important role in controlling infectious diseases. However, reports about the role of serum soluble Tim-3 (sTim-3) in hepatitis virus infection are limited. Therefore, this study explored changes in sTim-3 levels in patients infected with hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis E virus (HEV).MethodsThis study applied high-sensitivity time-resolved fluorescence immunoassay for the detection of sTim-3 levels. A total of 205 cases of viral hepatitis infection (68 cases of HBV infection, 60 cases of HCV infection, and 77 cases of HEV virus infection) and 88 healthy controls were quantitatively determined. The changes in serum sTim-3 level and its clinical value in hepatitis virus infection were analyzed.ResultsPatients with HBV infection (14.00, 10.78–20.45 ng/mL), HCV infection (15.99, 11.83–27.00 ng/mL), or HEV infection (19.09, 10.85–33.93 ng/mL) had significantly higher sTim-3 levels than that in the healthy control group (7.69, 6.14–10.22 ng/mL, P < 0.0001). Patients with hepatitis and fibrosis infected with HBV (22.76, 12.82–37.53 ng/mL), HCV (33.06, 16.36–39.30 ng/mL), and HEV (28.90, 17.95–35.94 ng/mL) had significantly higher sTim-3 levels than patients with hepatitis without fibrosis (13.29, 7.75–17.28; 13.86, 11.48–18.64; 14.77, 9.79–29.79 ng/mL; P < 0.05).ConclusionsTim-3 level was elevated in patients infected with HBV, HCV, or HEV and gradually increased in patients with either hepatitis or hepatitis with hepatic fibrosis. It has a certain role in the evaluation of the course of a disease after hepatitis virus infection.

ABSTRACTCOVID-19 is caused by SARS-CoV-2 infection and was initially discovered in Wuhan. This outbreak quickly spread all over China and then to more than 20 other countries. SARS-CoV-2 fluorescent microsphere immunochromatographic test strips were prepared by the combination of time-resolved fluorescence immunoassay with a lateral flow assay. The analytical performance and clinical evaluation of this testing method was done and the clinical significance of the testing method was verified. The LLOD of SARS-CoV-2 antibody IgG and IgM was 0.121U/L and 0.366U/L. The specificity of IgM and IgG strips in healthy people and in patients with non-COVID-19 disease was 94%, 96.72% and 95.50%, 99.49%, respectively; and sensitivity of IgM and IgG strips for patients during treatment and follow-up was 63.02%, 37.61% and 87.28%, 90.17%, respectively. The SARS-CoV-2 antibody test strip can provide rapid, flexible and accurate testing, and is able to meet the clinical requirement for rapid on-site testing of virus. The ability to detect IgM and IgG provided a significant benefit for the detection and prediction of clinical course with COVID-19 patients.

Aflatoxin M1 (AFM1) in milk is a problem of great concern. Current methods of detection require large instruments and need specific test sites. Therefore, it is necessary to establish a fast, convenient, and accurate detection method for AFM1. We established a system based on fluorescent microspheres containing a Eu3+ chelate named AFM1-POCT. These components comprised the AFM1-POCT kits. After refrigeration at 4°C for 12 months, the intra and inter assay coefficients of variability (CVs) for the kits were 4% and 5%, respectively. AFM1-POCT compared well with ultra-high-performance liquid chromatography (UHPLC) determination in the range 0.0121–2 μg/kg (paired samples test, P > 0.05). Aflatoxin B1 and G1 do not react with aflatoxin M1. By using the AFM1-POCT method, the detection time is shortened to 5 min, the accuracy is comparable to that of UHPLC, and convenience and range of application are improved.

Currently, atherosclerosis accounts for the majority of cardiovascular morbidity and mortality worldwide, and predicting the stability of atherosclerotic plaque is the main method to prevent atherosclerotic death. This study aims to establish a dual-label time-resolved fluorescence immunoassay (TRFIA) of matrix metalloprotein-9 (MMP-9) and lipoprotein-associated phospholipaseA2 (Lp-PLA2) to predict atherosclerotic plaque stability. A dual-label TRFIA was introduced for the simultaneous quantification of MMP-9 and Lp-PLA2 using fluorescent lanthanide (Eu3+ and Sm3+) chelates. The performance (sensitivity, specificity, accuracy, precision and reference intervals in different subjects) of this TRFIA was evaluated and compared with commercial kit. The sensitivity of the TRFIA for MMP-9 was 0.85 ng/mL and for Lp-PLA2 was 0.68 ng/mL with high affinity and specificity. The average recoveries were 94.58% to 109.82%, and 104.32% to 109.26%, respectively. All intra- and inter-assay CVs ranged from 3.10% to 5.46%. For the normal subjects, the cutoff value was 160.70 ng/mL for MMP-9 and 183.73 ng/mL for LP-PLA2; for the subjects with stable plaque, the cutoff value was 181.98~309.22 ng/mL for MMP-9 and 194.73~337.89 ng/mL for LP-PLA2; for the subjects with unstable plaque, the cutoff value was 330.43 ng/mL for MMP-9 and 343.23 ng/mL for LP-PLA2. This TRFIA detection results agreed well with the results of commercial kit (R2=0.9567 and R2=0.9771, respectively) in clinical serum samples. The TRFIA developed has a wide detection range and good sensitivity for the high-throughput simultaneous detection of MMP-9 and Lp-PLA2 in serum, which provides a new method for predicting the stability of atherosclerotic plaque. [ABSTRACT FROM AUTHOR]

• We established a dual-labeled PLA2R-IgG4/IgG-TRFIA for simultaneous detection. • Accurate diagnosis of IMN could be achieved using the anti-PLA2R-IgG4/IgG ratio. • Anti-PLA2R-IgG/IgG4 TRFIA contributes to the accurate diagnosis of IMN. • Combined detection approach increases the positive rate of IMN from 86.5 % to 91.7 %. • The false-positive rates of other diseases decreased from 25 to 50 % to 0 %. The renal biopsy is an accurate and reliable gold standard for membranous nephropathy (MN) diagnosis. However, it is an invasive procedure involving the risk of hemorrhage or infection. Thus, an alternative approach that can facilitate the effective diagnosis and treatment monitoring of idiopathic membranous nephropathy (IMN) is urgently needed. We established a dual-labeled time-resolved fluoroimmunoassay (TRFIA) to simultaneously detect phospholipase A2 receptor (PLA2R)-IgG4 and PLA2R-IgG antibodies. Utilizing this assay, we determined the ratio of autoantibodies in the serum of patients with different kidney diseases and normal controls. The sensitivity of TRFIA for detecting anti-PLA2R-IgG and anti-PLA2R-IgG4 was 0.12 µg/mL and 0.001 µg/mL, respectively. Human IgA did not interfere with the assay. Compared to anti-PLA2R-IgG alone, the positive rate of IMN could be increased from 86.5 % to 91.7 % through the combined use of anti-PLA2R-IgG4 and the PLA2R-IgG4/IgG ratio. In contrast, the false-positive rates for the detection of IgA nephropathy, lupus nephropathy, diabetic nephropathy, and minimal change nephropathy decreased from 25 to 50 % to 0 %. The dual-labeled PLA2R-IgG4/IgG-TRFIA for simultaneous detection of anti-PLA2R-IgG4 and anti-PLA2R-IgG will contribute to improved accuracy of IMN diagnosis. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) has severely influenced the swine industry of the whole world. Fast and accurate African swine fever virus (ASFV) antigen detection is very important for ASF prevention. This study aims to establish a new detection method for detection ASFV antigen using time-resolved fluorescence immunoassay (TRFIA) in the nose and mouth discharge. A double antibody sandwich TRFIA method was optimized and established. Recombinant P30 recombinant antigen was captured by its antibodies immobilized on 96-well plate, and then banded together with another detection antibodies labeled with Europium(III) (Eu3+) chelates, finally time-resolved analyzer measured the fluorescence intensity. The performance of this TRFIA (sensitivity, specificity and accuracy) was evaluated using the clinical samples and compared with the nucleic acid testing method. The sensitivity of this TRFIA was 0.015 ng/mL (dynamic range 0.24–500 ng/mL) with high specificity. The recovery ranged from 92.00 to 103.62 %, the inter-assay CVs ranged from 5.50 to 11.96 %, and the intra-assay CVs was between 5.20 and 10.53 %. Additionally, the cutoff value was 0.016. TRFIA took only 45 min to generate results, and its detection capability comparable to the nucleic acid detection. This study developed a TRFIA method that could be used for qualitative/quantitative detection of ASFV antigen in pigs nasal discharge, which has high sensitivity, specificity and accuracy. This TRFIA provides a new method for rapidly screening ASFV infection in pigs industry. [ABSTRACT FROM AUTHOR]

As a highly contagious and potentially fatal disease of dogs, canine parvovirus type 2 (CPV‐2) usually causes severe myocarditis and gastroenteritis, while vaccine injection has greatly reduced the incidence of CPV‐2 diseases. However, there is currently a lack of simple and effective method for quantitative detection of CPV‐2 in vaccine. Therefore, this study aims to prepare an accurate method to determine the CPV‐2 antigen (CPV‐2‐Ag) in vaccine. Here, a sandwich time‐resolved fluorescence immunoassay (TRFIA) was established and optimized. Anti‐CPV‐2 antibodies were immobilized on 96‐well plates to capture CPV‐2‐Ag, and then bound together with the detection antibodies labeled with Europium(III) (Eu3+) chelates; finally, time‐resolved fluorometry was employed to measure the fluorescence intensity. Vaccination was performed to evaluate the relationship between CPV‐2‐Ag concentration and antibody titer. The sensitivity is 1.15 mEU/mL (LogY = 1.524 + 0.8667 × LogX, R2 = 0.9933), and the average recovery is among 91.00% to 106.39% without cross‐reactions with the other canine viral antigen. The correlation between ELISA assay and this method is up to 0.9861. And, there is high correlation between the CPV‐2‐Ag concentration and antibody titers (R2 = 0.9234). This immunoassay established has high sensitivity, accuracy, and specificity, which indicate that this method could be suitable for quantitative detection of CPV‐2‐Ag in vaccine evaluation. [ABSTRACT FROM AUTHOR]

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation. [Display omitted] • Rapid and Sensitive Detection. • High-Affinity Monoclonal Antibodies. • Time-Resolved Fluorescence Immunoassay (TRFIA). • Cloud System and Practical Implications. [ABSTRACT FROM AUTHOR]

The direct and indirect competition time-resolved fluorescence immunoassays (dc-TRFIA, ic-TRFIA) were established by combining the autofluorescence properties of lanthanide europium (Eu) with the monoclonal antibody of oxyfluorfen. The purified Eu antibody was optimized and the conditions such as the working concentration of the Eu antibody, monoclonal antibody, and working buffer were optimized. In the optimal condition, the IC50 of dc-TRFIA was 10.27 ng/mL, the lowest detection limit IC10 was 0.071 ng/mL, the detection range (IC10-IC90) was 0.071–1074.3 ng/mL, and the detection range (IC10-IC90) and IC50 of ic-TRFIA were 0.024–504.6 and 2.76 ng/mL, respectively. The comparison showed that the sensitivity and detection limit of ic-TRFIA were superior to dc-TRFIA. The cross reaction (CR) tests showed that the CR with other oxyfluorfen structure analogs was

The primary pollutants in cereal products are aflatoxins B1 (AFB1), zearalenone (ZEN), and deoxynivalenol (DON). In this study, anti-AFB1 MAb (4C9), anti-ZEN MAb (2A3), and anti-DON MAb (1F10) were developed and used in time-resolved fluorescence immunoassay microfluidics to determine AFB1, ZEN, and DON in agricultural products. The linear range for AFB1, ZEN, and DON were 0.05~2.2 μg/kg, 1.45~375.75 μg/kg, and 11.1~124.2 μg/kg, respectively. In maize, the recoveries of AFB1/ZEN/DON were 92~101%, 102~105%, and 103~108%, respectively. High-performance liquid chromatography and the proposed approach had a good correlation. Time-resolved fluorescence immunoassay microfluidics is a highly efficient and sensitive field detection method for fungal toxins in agricultural products.

Neonatal infectious diseases are a serious threat to the health of newborns. The aim was to establish a new detection method for the simultaneous measurement of (1,3)‐β‐d‐glucan and procalcitonin in serum for the early screening and efficacy testing of neonatal infectious diseases. We established a sandwich dual‐label time‐resolved fluorescence immunoassay (TRFIA): anti‐(1,3)‐β‐d‐glucan/procalcitonin antibodies immobilized on 96‐well plates captured (1,3)‐β‐d‐glucan/procalcitonin antigens and then banded together with the detection antibodies labeled with europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally, time‐resolved fluorometry was used to measure the fluorescence intensity. The linear correlation coefficient (R2) of the (1,3)‐β‐d‐glucan standard curve was 0.9913, and the R2 of the procalcitonin standard curve was 0.9911. The detection sensitivity for (1,3)‐β‐d‐glucan was 0.4 pg/mL (dynamic range: 0.6–90 pg/mL), and the average recovery was 101.55%. The detection sensitivity for procalcitonin was 0.02 ng/mL (dynamic range: 0.05–95 ng/mL), and the average recovery was 104.61%. There was a high R2 between the present TRFIA method and a commercially available assay (R2 = 0.9829 for (1,3)‐β‐d‐glucan and R2 = 0.9704 for procalcitonin). Additionally, the cutoff values for (1,3)‐β‐d‐glucan and procalcitonin were 23.95 pg/mL and 0.055 ng/mL, respectively. The present TRFIA method has high sensitivity, accuracy, and specificity and is an effective method for early screening and efficient testing of neonatal invasive fungal infection. [ABSTRACT FROM AUTHOR]

Aflatoxin M1 (AFM1) in milk is a problem of great concern. Current methods of detection require large instruments and need specific test sites. Therefore, it is necessary to establish a fast, convenient, and accurate detection method for AFM1. We established a system based on fluorescent microspheres containing a Eu3+ chelate named AFM1-POCT. These components comprised the AFM1-POCT kits. After refrigeration at 4°C for 12 months, the intra and inter assay coefficients of variability (CVs) for the kits were 4% and 5%, respectively. AFM1-POCT compared well with ultra-high-performance liquid chromatography (UHPLC) determination in the range 0.0121–2 μg/kg (paired samples test, P > 0.05). Aflatoxin B1 and G1 do not react with aflatoxin M1. By using the AFM1-POCT method, the detection time is shortened to 5 min, the accuracy is comparable to that of UHPLC, and convenience and range of application are improved. [ABSTRACT FROM AUTHOR]

11-Dehydro-thromboxane B2 (11-dehydro-TXB2) is the final stable metabolite of thromboxane A2 (TXA2) and is involved in thrombus formation. Patients with membranous nephropathy (MN) are prone to thromboembolism events. Time-resolved fluorescence immunoassay (TRFIA) for 11-dehydro-TXB2 was established by indirect competitive method. The coated 11-dehydro-TXB2-BSA conjugate was used to bind the 11-dehydro-TXB2 antibody competitively to the 11-dehydro-TXB2 antigen in the samples, followed by Eu3+-labeled goat anti-mouse IgG antibody, to detect 11-dehydro-TXB2. This study measured 11-dehydro-TXB2 concentrations in serum samples from healthy individuals and patients with MN. The linear range of TRFIA was 16.38–2000 pg/mL, the sensitivity was 4.70 pg/mL, the average coefficients of variation from intra-assay and inter-assay were 3.50% and 4.95%, respectively, and the recovery was 99.38%. The serum level of 11-dehydro-TXB2 in patients with MN was significantly higher than that in healthy subjects (P < 0.05). The serum 11-dehydro-TXB2 concentration detected by TRFIA was highly consistent with that by ELISA (ρ = 0.900). This study successfully established a new highly sensitive method for the detection of 11-dehydro-TXB2 in serum. 11-Dehydro-TXB2 has great potential in evaluating the risk of thromboembolic events in patients with MN and is expected to be applied to other thromboembolic-related diseases. [Display omitted] • Establishment of 11-dehydro-thromboxane B2 time-resolved immunoassay. • We found the levels of serum 11-Dehydro-thromboxane B2 were higher in patients with membranous nephropathy than healthy subjects. [ABSTRACT FROM AUTHOR]

This study aimed to establish a highly sensitive time-resolved fluorescence immunoassay of growth stimulating express gene 2 protein (ST2-TRFIA) and evaluate its application value for sepsis. Two types of ST2 monoclonal specific antibodies against different epitopes of antigen molecule were used as coating and Eu3+-labeled antibodies. The double-antibody sandwich method was used in establishing ST2-TRFIA, and the methodology was evaluated. The established ST2-TRFIA was used in detecting ST2 concentration in the plasma samples of healthy controls and sepsis. The linear range of ST2-TRFIA was 1.446–500 ng/mL. Plasma ST2 concentrations detected through ST2-TRFIA were consistent with the results of fluorescence quantitative immunochromatography (ρ = 0.946). The plasma ST2 concentrations of patients with sepsis were significantly higher than those of healthy controls (P < 0.01). This study successfully established a highly sensitive ST2-TRFIA, which was highly comparable to commercially available fluorescent quantitative immunochromatographic kits and can facilitate the timely diagnosis of sepsis. [Display omitted] • A novel TRFIA method was developed to detect ST2 in human plasma • ST2-TRFIA achieved more sensitivity and detection linearity (1.446–500 ng/mL). • Helps in timely and accurate diagnosis of sepsis. • Differentiated diagnosis of sepsis and septic shock in patients detected by combined ST2 and CRP. • Excellent performance, can be used clinically [ABSTRACT FROM AUTHOR]

Detection of specific viral antibody or nucleic acid produced by infection or immunization, using oral fluid samples, offers increased potential for wider population uptake compared to blood sampling. This methodology is well established for the control of HIV and measles infections, but can also be applied to the control of other vaccine preventable infections, and this review describes the application of oral fluid assays in support of mumps, rubella and varicella national immunization programs. In England and Wales individuals with suspected mumps or rubella, based on clinical presentation, can have an oral fluid swab sample taken for case confirmation. Universal varicella immunization of children has led to a drastic reduction of chickenpox in those countries where it is used; however, in England and Wales such a policy has not been instigated. Consequently, in England and Wales most children have had chickenpox by age 10 years; however, small, but significant, numbers of adults remain susceptible. Targeted varicella zoster virus (VZV) immunization of susceptible adolescents offers the potential to reduce the pool of susceptible adults and oral fluid determination of VZV immunity in adolescents is a potential means of identifying susceptible individuals in need of VZV vaccination. The main application of oral fluid testing is in those circumstances where blood sampling is deemed not necessary, or is undesirable, and when the documented sensitivity and specificity of the oral fluid assay methodology to be used is considered sufficient for the purpose intended.

Abstract: Introduction: Paclitaxel (Tax) is a diterpene alkaloid isolated from Taxus species and has proved clinically effective in treating a number of malignancies. Current quantitative analytical methods for Tax such as high‐performance liquid chromatography (HPLC) often involve complicated sample preparation procedures with low recovery rates. Objective: To establish a rapid and sensitive time‐resolved fluoroimmunoassay (TRFIA) for measuring Tax in Taxus materials with convenient sample preparation and a high recovery rate. Methods: Rabbit anti‐mouse IgG was coated onto a 96‐well microplate, which was then incubated with standard solutions of Tax and anti‐Tax monoclonal antibody 3A3. A Eu3+‐labelled conjugate of Tax and human serum albumin was used as the tracer. The luminescent system was enhanced with a solution containing 2‐naphthoyltrifluoroacetone. Results: The established TRFIA showed a linear response within the Tax concentration range of 3.2 to 80 ng/mL, with a limit of detection of 1.4 ng/mL. The intra‐ and inter‐assay coefficient of variation of the assay was 9.6% and 9.7%, respectively, with an average recovery rate from spiked samples of 108.5%. Tax contents in Taxus samples were determined using both the established TRFIA system and a previously established enzyme‐linked immunosorbent (ELISA), and the results of two assays were well correlated. Conclusion: This TRFIA system shows a high sensitivity, precision and accuracy for detection of Tax. This assay, which is convenient and less time‐consuming, allows rapid analysis of Tax and provides another option for Tax measurement for quality control of Taxus materials and products. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu3+) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections. [ABSTRACT FROM AUTHOR]

The effectiveness and necessity of human papillomavirus (HPV) vaccination to prevent HPV infection and cervical cancer are increasingly recognized by people. The 15-valent HPV vaccine, which protects against almost high-risk types of HPV viruses identified by WHO, has attracted much attention. However, as the valence of vaccines increases, quality control in the HPV vaccine production process is facing more challenges. The precise quality control of the HPV type 68 virus-like particles (VLPs), one of the unique components of the 15-valent HPV vaccine that distinguishes it from existing vaccines, is the new requirement for vaccine manufacturers. Here we developed a novel time-resolved fluorescence immunoassay (TRFIA) for rapid and precise automatic quality control of HPV68 VLPs in HPV vaccine. Two murine monoclonal antibodies specifically targeting the HPV68 L1 protein were used to establish a classical sandwich assay. Except for pretreating the vaccine sample, the whole analysis process was performed by a fully automated machine, which saves detection time and gets rid of manual error. Multiple experiments established that the current novel TRFIA can efficiently and reliably analyses HPV68 VLPs. Present novel TRFIA has exhibited merits with speed, robustness, high sensitivity with a minimum detection value of 0.08 ng/mL, considerable accuracy, a wide detection range (up to 1000 ng/mL) and excellent specificity. It is also expected to provide a new detection method for quality control for each HPV type VLPs. To summarize, the novel TRFIA is of great interest for application in HPV vaccine quality control. [ABSTRACT FROM AUTHOR]

Deoxynivalenol (DON) contamination of food crops and feeds is almost impossible to avoid completely; however, through best management practices, this risk can be effectively managed and maximumly mitigated. Accurate and rapid detection of DON contamination as early in the entire value chain as possible is critical. To achieve this goal, we developed a DON test strip based on time-resolved fluorescence immunoassay (TRFIA) and a specific DON monoclonal antibody for the rapid quantification of DON in food crops and feeds. The strip displayed a good linearity ( R 2 = 0.9926), with a limit of quantification of 28.16 μg/kg, a wide linear range of 50 ~ 10,000 μg/kg. The intra-batch coefficient of variation (CV) and the inter-batch CV was <5.00 and 6.60%, respectively. This TRFIA-DON test strip was applied to detect DON in real samples, and the accuracy and reliability were confirmed by liquid chromatography-mass spectrometry (LC-MS/MS). Results showed that the relative standard deviation between the DON strips and LC-MS/MS was <9%. The recovery rates in corn samples ranged from 92 to 104%. The established TRFIA-DON test strip had the characteristics of high sensitivity, high accuracy, and a wide linear range which was suitable for rapid and quantitative determination of DON in food crops and feeds at both on-site and laboratory., Competing Interests: JW, XM, LQ, and YZ were employed by Shanghai Xiongtu Biotechnology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Wang, Zhang, Mei, Qiu, Liu and Zhou.)

We demonstrate an ultrasensitive time-resolved fluorescence immunoassay for bisphenol A (BPA) where gold nanoparticles (Au-NPs) were modified with anti-BPA antibody and thiolated dsDNA-biotin acting as a signal amplifier. In a competitive reaction, the analyte (BPA) competes with immobilized BPA-ovalbumin conjugate on the surface of microtiter plates to bind to the anti-BPA antibodies on the surface of the Au-NPs. In the next step, a Eu(III)-labeled streptavidin is added to link to the SH-dsDNA-biotin as a tracer. Fluorescence is amplified via both the Au-NPs and the biotin-streptavidin systems, and its intensity is measured in a time-resolved fluorescence immunoassay. The linear range is from 1.0 fg∙mL to 1.0 ng∙mL, and the detection limit is 0.3 fg mL, respectively. The method was applied to the determination of BPA in spiked water sample, with recoveries ranging from 90.2 to 106.4 %. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Background High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test the clinical relevance of using these as cut-points in colorectal cancer (CRC) prognosis. Methods uPAR forms were measured in citrated and EDTA plasma samples using time-resolved fluorescence immunoassays. Diurnal, intra- and inter-individual variations were assessed in plasma samples from cohorts of healthy individuals. Reference intervals were determined in plasma from healthy individuals randomly selected from a Danish multi-center cross-sectional study. A cohort of CRC patients was selected from the same cross-sectional study. Results The reference intervals showed a slight increase with age and women had ~ 20% higher levels. The intra- and inter-individual variations were ~ 10% and ~ 20–30%, respectively and the measured levels of the uPAR forms were within the determined 95% reference intervals. No diurnal variation was found. Applying the normal upper limit of the reference intervals as cut-point for dichotomizing CRC patients revealed significantly decreased overall survival of patients with levels above this cut-point of any uPAR form. Conclusions The reference intervals for the different uPAR forms are valid and the upper normal limits are clinically relevant cut-points for CRC prognosis. [ABSTRACT FROM AUTHOR]