Time-resolved Fluorescence Immunoassay Research Articles

Time-resolved fluorescence immunoassay (TRFIA) for the simultaneous detection of hs-CRP and lipoprotein(a) in serum.

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 0.052μg/ml for hs-CRP and 0.64μg/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.

Establishment of Galectin-3 Time-resolved Fluoroimmunoassay and its Application in Idiopathic Membranous Nephropathy.

The aim of this study was to establish a time-resolved fluorescent immunoassay (TRFIA) for the detection of serum Galectin-3 (Gal-3) and apply this method to evaluate the clinical significance of serum Gal-3 in predicting Idiopathic Membranous Nephropathy (IMN) progression.The Gal-3-TRFIA was established using the double antibody sandwich method, with the capture antibodies coated on a 96-well microplate and the detection antibodies chelated with Europium (III) (Eu3+). Serum Gal-3 was detected in 81 patients with IMN and 123 healthy controls to further evaluate the value of the Gal-3 in staging of IMN.The sensitivity of the Gal-3-TRFIA assay was 0.85ng/mL, and the detection range was 0.85-1000ng/mL. The Gal-3 intra-batch and inter-batch coefficients of variation were 3.45% and 5.12%, respectively. The correlation coefficient (R) between the Gal-3-TRFIA assay and commercially available enzyme-linked immunosorbent assay kits was 0.83. The serum Gal-3 concentration was higher in patients with IMN (65.57 ± 55.90ng/mL) compared to healthy controls (16.29 ± 9.91ng/mL, P < 0.0001).In this study, a wide detection range Gal-3-TRFIA assay was developed using lanthanide (Eu3+) chelates for the detection of Gal-3 concentrations in serum. Gal-3 concentration is elevated in patients with IMN.

A novel time-resolved fluorescent lateral flow immunoassay for quantitative detection of the trauma brain injury biomarker-glial fibrillary acidic protein

A highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) was developed to quantify glial fibrillary acidic protein (GFAP), a trauma brain injury (TBI) biomarker in blood, for the purpose of providing a diagnosis of mild brain injury.

Detection of anti-Neospora caninum antibodies in sheep’s full-cream milk by a time-resolved fluorescence immunoassay

Ovine neosporosis, caused by the Apicomplexan parasite Neospora caninum, leads to reproductive failure worldwide. Nowadays, there is a trend to develop diagnostic techniques using non-invasive samples, such as milk, in order to reduce animal stress, sample collection effort, and costs. The objective of this study was to develop and validate a highly sensitive and specific serological technique, based on a time resolved-fluorescence immunoassay using a N. caninum GRA7 antigen (GRA7-TRFIA), for the detection of anti-N. caninum immunoglobulins G on sheep’ full-cream milk samples. An analytical validation was performed, including intra- and inter-assay precision, analytical sensitivity and accuracy. The diagnostic performance of the assay was evaluated by studying the positive-negative discrimination by Mann Whitney U tests. In additon optimal cut-offs, diagnostic sensitivity and specificity, and areas under the curve were calculated by three Receiver Operating Curve (ROC) analyses, using GRA7-TRFIA and a N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET-ELISA) in blood sera, and the coinciding results of both techniques, as reference techniques. Moreover, Spearman’s correlation of GRA7-TRFIA in milk with the techniques in sera and agreement (kappa values) were also estimated. GRA7-TRFIA for milk samples showed an adequate precision, with high analytical sensitivity and accuracy. Regarding ROC analyses, at the optimal cut-offs, the diagnostic sensitivity and specificity were more than 90 % in all cases. In addition, GRA7-TRFIA values in milk were more positively correlated to GRA7-TRFIA values in blood sera than in the case of values obtained with NcSALUVET-ELISA. GRA7-TRFIA in milk showed an almost perfect agreement with GRA7-TRFIA in blood sera (kappa = 0.98) and with the coinciding results of GRA7-TRFIA and NcSALUVET in blood sera (kappa = 1.00), while it has a substantial agreement with NcSALUVET-ELISA (kappa = 0.69). In the light of these results, GRA7-TRFIA in full-cream milk samples is a highly sensitive technique that could be used for screening anti-N. caninum antibodies in sheep flocks.

A highly sensitive and quantitative time resolved fluorescent microspheres lateral flow immunoassay for streptomycin and dihydrostreptomycin in milk, honey, muscle, liver, and kidney

Streptomycin (STR) and dihydrostreptomycin (DHSTR) are clinically widely used in the prevention and treatment of animal diseases. Unreasonable use or abuse can easily cause adverse effects on human health. Thus, it is very necessary to establish a rapid detection method for STR and DHSTR in animal-derived foods. In this study, a time-resolved fluorescent microspheres lateral flow immunoassay (TRFM-LFIA) integrated with a portable fluorescence reader was developed for STR and DHSTR detection. The cut-off values of the TRFM-LFIA for STR and DHSTR in milk/honey/muscle/liver/kidney were both 30/15/80/25/60 μg kg−1, respectively, the limits of detection were 1.10/0.28/2.82/1.52/3.02, 0.75/0.23/1.76/1.21/2.35 μg kg−1, respectively, and the limits of quantitation were 3.62/0.93/9.31/5.02/9.96, 2.48/0.76/5.81/3.99/7.76 μg kg−1, respectively. The recoveries ranged from 80.0% to 120.1%, with coefficient of variation from 2.8% to 14.3%, respectively. The parallel experiment of 25 samples showed excellent correlation (R2 > 0.99) between ELISA kit and TRFM-LFIA. The sample pretreatment is very simple, and the results can be achieved in 8 min. This sensitive, simple, rapid and portable analysis strategy can provide universal technical support for the residue detection of veterinary drugs and even small molecular hazard factors.

A chemiluminescence immunoassay for precise automatic quality control of glycoprotein in human rabies vaccine

Currently, quality control of glycoprotein in the human rabies vaccine is based on enzyme-linked immunosorbent assay (ELISA). However, ELISA does not match the needs of a modernised quality control system. For a long time, human rabies virus vaccine manufacturers have been devoted to seeking a detection platform that is sensitive, accurate, automatic, and feasible for practical applications. Therefore, our team invested major efforts into establishing a fully automated micromagnetic particle (MMP)-based chemiluminescence immunoassay (CLIA) platform. For vaccine quality control, MMP-coupled rabies virus glycoprotein monoclonal antibodies (S037) were used to capture the rabies virus. Another rabies virus glycoprotein antibody (S053) labelled with acridinium ester was added as a signal tracer. After pretreating the vaccine sample, the entire analysis was performed using a fully automated machine, which had a limited detection time (only 30 min) and eliminated manual error. Multiple experiments have identified the optimal conditions allowing valid and reliable assessment of vaccine potency. The CLIA platform has exhibited merits in terms of speed, robustness, high sensitivity (with a minimum detection value of 0.45 mIU/mL), considerable accuracy, and a wide linear range of detection (9.4–1200 mIU/mL). Furthermore, the results showed that the CLIA platform is consistent with the National Institutes of Health test and time-resolved fluorescent immunoassay (TRFIA) in quantitative analysis, and had a better analytic performance than TRFIA. Therefore, the CLIA platform presented here may be important for application in modern vaccine quality control.

Development of a time-resolved fluorescence immunoassay based on immunomagnetic beads for gastrin-17

ObjectiveIn this study, a novel, simple, and rapid immunoassay for the determination of gastrin-17 (G-17) in human serum was established by combining immunomagnetic beads with time-resolved fluorescence immunoassay (TRFIA). MethodsImmunomagnetic beads were coated with anti-G-17 M01 antibody, anti-G-17 M02 antibody was labeled with Eu3+ chelates. The concentration of G-17 in the serum was detected with the double-antibody sandwich method. ResultsThe limit of background(LOB), limit of detection (LOD), and limit of quantification (LOQ) were 0.09, 0.104, and 0.39 pmol/L, respectively. The detection range of G-17-TRFIA was 0.39–100 pmol/L. The average intra- and inter-assay coefficients of variation (CV) were 5.95%–9.07% and 6.09%–8.14%, respectively. The recoveries for the serum samples ranged from 94.70% to 100.95%. The specificity of our G-17-TRFIA was acceptable. The correlation coefficient between G-17-TRFIA and commercial G-17-ELISA methods was R2 = 0.9092. ConclusionsA novel G-17-TRFIA detection method was successfully established to provide a reference for the early diagnosis of patients with atrophic gastritis in clinical research.

Clinical Value of Combined Detection of Serum sTim-3 and Pepsinogen for Gastric Cancer Diagnosis.

PurposeThe 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 MethodsThe 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.ResultsSerum 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).ConclusionsTim-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.

Time-resolved Fluorescence Immunoassay (TRFIA) for the Simultaneous Detection of MMP-9 and Lp-PLA2 in Serum.

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.

Establishment of heparin-binding protein time-resolved immunoassay and some potential clinical applications

AimTo establish a highly sensitive time-resolved fluorescence immunoassay of heparin-binding protein (HBP-TRFIA) and evaluate its application value for bacterial or fungal infections in tumor patients. MethodsTwo types of HBP monoclonal specific antibodies against different epitopes of the antigen molecule were used as coating antibodies and Eu3+-labeled antibodies, respectively. The double-antibody sandwich method was used in establishing HBP-TRFIA, and the methodology was evaluated. The established HBP-TRFIA was used in detecting HBP concentration in the plasma samples of healthy individuals, patients with bacterial or fungal infections, and infected or uninfected patients with various types of tumors. ResultsThe linear range of HBP-TRFIA was (0.11–530 ng/mL). Plasma HBP concentrations detected through HBP-TRFIA were consistent with the results of fluorescence quantitative immunochromatography (ρ = 0.964). The plasma HBP concentrations of infected tumor patients were significantly higher than those of uninfected tumor patients (P < 0.01). ConclusionThis study successfully established a highly sensitive HBP-TRFIA, which was highly comparable to commercially available fluorescent quantitative immunochromatographic kits and was able to facilitate the timely diagnosis of bacterial or fungal infections in patients with tumor.

Determination of human COVID-19 total antibodies in serum using a time-resolved fluorescence immunoassay.

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.

Time-resolved fluorescence immunoassay for urine retinol-binding protein is more sensitive than polyclonal and monoclonal assays.

Retinol-binding protein4 (RBP) assays using polyclonal antibodies (pRBP) have major problems of non-linearity of dilution and a very small useable dynamic range. Our objective was to develop a specific assay with a wider dynamic range to detect tubular proteinuria. mRBP (monoclonal capture and second antibody with colorimetric detection) and fluoroimmunoassays for RBP (fRBP) (polyclonal capture and monoclonal second antibody with fluorescence detection) were developed and compared with pRBP. Four hundred and eighty-eight patient samples were collected; 290 samples were analysed by mRBP and 198 samples with fRBP and compared with pRBP. mRBP assay has the advantages of better linearity on dilution and wider analytical range over pRBP. It is limited by poor signal in the patients with albuminuria and glomerular proteinuria and inferior discrimination between patient groups. fRBP had an intra-assay and inter-assay CV of <6% and <8%, respectively, and analytical range was 2.3-599 µg/L. fRBP was linear on dilution within the analytical range. Correlation (r) was 0.8722 (95% CI 0.7621 to 0.9333, P< 0.0001); Mann-Whitney test revealed no significant difference (U = 18,877, n = 198, P = 0.5244) asserting that the medians of the two samples were identical. Bland-Altman test between pRBP and fRBP showed a mean negative bias of 16.43 (CI -994 to 1027) µg/mmol. The combination assay with fluorescence detection (fRBP) proved more discriminatory than a purely monoclonal system especially in patients with significant proteinuria and has advantages of better linearity on dilution and wider analytical range than the existing pRBP assay and compared extremely well with pRBP.

Research Progress on Application of New Labeling Materials Based Immunoassay on the Detection of Mycotoxin

In recent years, mycotoxin contamination in food has become a hot and difficult issue in the field of food safety. Immunoassay has the advantages of short detection time, simple operation steps, low cost and environmental friendliness, which is suitable for food safety detection. The research of new labeling materials-based immunoassay techniques in rapid detection of mycotoxin in cereal products is reviewed in this paper, including enzyme linked immunosorbent assay, immunochromatography, electrochemical immunosensor, immunochips, cytometric bead array based on immunoassay and time resolved fluorescence immunoassay. The advantages and disadvantages of the above immunoassay are systematically analyzed, which provides references for the application and development of immunoassay techniques in the detection of mycotoxins, and also provides new ideas for ensuring safety of grain products.

A New Method for Detection African Swine Fever Virus: Time-resolved Fluorescence Immunoassay.

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.

Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles.

Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear. Urine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization. Urine particle and creatinine concentrations were highly correlated in the water-loading study (R2 0.96) and in random spot urines from healthy subjects (R2 0.47-0.95) and patients (R2 0.41-0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers. Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment-specific EVs.

Development and validation of a time-resolved fluorescence immunoassay for the detection of anti-Toxoplasma gondii antibodies in goats

Toxoplasma gondii is a worldwide distributed parasite causing abortions and fetal malformations in small ruminants. The aim of this study was to design and validate a new immunoassay based on the use of TgSAG1-GRA8 chimeric antigen for the detection of anti-T. gondii antibodies in serum of goats. First, a time-resolved fluorescence immunoassay (TgSAG1-GRA8-TRFIA) was developed. In addition, the diagnostic performance of TgSAG1-GRA8-TRFIA was compared with an optimized enzyme-linked immunosorbent assay (TgSALUVET-ELISA) and a Western Blot (WB), both based on whole T. gondii tachyzoite antigenic extract. The TgSAG1-GRA8-TRFIA has shown a high intra- and inter-assay precision, analytical sensitivity and accuracy. The ROC analysis of this assay showed an optimal cut-off of 217.4 Units of Fluorometry for T. gondii (UFT), with 92 % of sensitivity and 90.48 % of specificity. A positive and statistically significant Spearman’s correlation with TgSALUVET-ELISA was detected, and kappa value was 0.83, presenting high agreement with both methods. However, TgSAG1-GRA8 protein showed cross-reactivity with specific anti-Neospora caninum antibodies. Thus, TgSAG-1-GRA8 chimeric antigen seems not to be an ideal option for the serodiagnosis of T. gondii infection in goats unless combined with the serodiagnosis of N. caninum infection in parallel. In the light of the results obtained, a comprehensive study on the existence of cross-reactivities between T. gondii antigens used in serological tests employed in animal health and specific antibodies directed against Toxoplasmatinae parasites should be performed.

Establishment of interleukin-18 time-resolved fluorescence immunoassay and its preliminary application in liver disease.

ABSTRACTBackgroundTo establish a time‐resolved fluorescence immunoassay of interleukin (IL)‐18 (IL‐18‐TRFIA) and detect its concentration in different liver disease serum samples.MethodsThe IL‐18 coating antibody and the Eu3+‐labeled detection antibody were used for the IL‐18‐TRFIA to detect serum IL‐18 concentration in patients with liver cancer, hepatitis B, hepatitis C, autoimmune hepatitis, fatty liver disease, and healthy controls. The double‐antibody sandwich method was used and methodological evaluation was performed.ResultsThe average intra‐ and inter‐assay coefficient of variation for IL‐18‐TRFIA was 4.80% and 5.90%, respectively. The average recovery rate was 106.19 ± 3.44%. The sensitivity (10.96 pg/mL) was higher than that obtained using the ELISA method (62.5 pg/mL). The detection range was 10.96–1000 pg/mL. IL‐6 and galectin‐3 did not cross‐react with IL‐18‐TRFIA. The serum concentration of IL‐18 was (776.99; 653.48–952.39 pg/mL) in hepatitis C, (911; 775.55–1130.03 pg/mL) in fatty liver, (1048.88; 730.04–1185.10 pg/mL) in liver cancer, and (949.12; 723.70–1160.28 pg/mL) in hepatitis B. Moreover, IL‐18 serum levels were significantly higher in patients than the healthy controls (483.09; 402.52–599.70/mL) (p < 0.0001). Autoimmune hepatitis with a serum IL‐18 concentration of 571.62; 502.47–730.31 pg/mL was not significantly different from the healthy controls (p > 0.05).ConclusionWe established a highly sensitive IL‐18‐TRFIA method that successfully detected serum IL‐18 concentrations in different liver diseases. Furthermore, IL‐18 serum concentration was higher in patients with liver cancer, hepatitis C, hepatitis B, and fatty liver disease compared to healthy controls.

Rapid Quantification of Chlorpromazine Residues in Pork Using Nanosphere-Based Time-Resolved Fluorescence Immunoassay Analyzer.

Immunochromatographic assays are good analytical tools for the detection of drug residues. We report a nanosphere-based time-resolved fluorescence immunoassay (nano-TRFIA) based on a monoclonal antibody and a portable TRFIA analyzer for the rapid quantification of chlorpromazine (CPZ) residues in pork. Under optimal conditions, the nano-TRFIA detected CPZ residues within 6 min of sample pretreatment. The results showed good linearity (R2 = 0.991), with a limit of detection (LOD) of 0.32 μg/kg, a wide dynamic range of 0.46–10.0 μg/kg, and coefficients of variation (CVs) of the overall intrabatch and interbatch assays of 7.34% and 7.65%, respectively. The nano-TRFIA was also used to detect CPZ at different spiked concentrations in pork, and the results were confirmed via ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The nano-TRFIA was evaluated for the analysis of six commercial pork samples, and the results agreed well with those obtained via UPLC-MS/MS, without significant differences (P > 0.05). Therefore, the proposed nano-TRFIA is a powerful alternative for the rapid and accurate quantification of CPZ residues in pork to meet the required Chinese maximum residue limits for veterinary drugs in foods.

Establishment and Application of a Dual-Labeling Time-Resolved Fluorescence Immunoassay Method for Simultaneous Detection of the Troponin I-C Complex and Full-Size-Troponin I.

Background: The measurement of cardiac troponin I (cTnI) is widely used in the diagnosis of acute myocardial infarction (AMI). Although existing cTnI detection methods measure total cTnI, the significance of undegraded full-size-cTnI levels is still not well-understood. In this study, we have established a novel dual-labeling time-resolved fluorescence immunoassay (TRFIA) technique that simultaneously detects the cTnI-C complex and full-size-cTnI, allowing us to explore the clinical value of full-size-cTnI determination.Methods: An antibody against the 23–43 amino acid region of cTnI protected by endogenous cTnC is coupled to magnetic beads to provide a solid-phase antibody for capturing all cTnI. An antibody against cTnC in the cTnI-C complex labeled with Eu3+ was used to detect the cTnI-C complex, and an antibody labeled with Sm3+ near the C-terminal 190–203 amino acids of cTnI was used to detect full-size-cTnI. Through dual-labeling TRFIA, cTnI-C complex, full-size-cTnI, and the full-size-cTnI/cTnI-C ratio can be detected simultaneously. The dual-labeling TRFIA technique was used to analyze serum samples collected at different times during treatment and compare their full-size-cTnI/cTnI-C ratios.Results: The sensitivity for the cTnI-C-TRFIA complex was 0.02 ng/mL, the measurement range was 0.02–40 ng/mL, the average intra-batch coefficient of variation (CV) was 4.35%, and the inter-average CV was 6.23%. The correlation coefficient between cTnI-C-TRFIA and commercial cTnI-CLIA methods was R2 = 0.8887. The sensitivity for full-size-cTnI-TRFIA was 0.04 ng/mL, the measurement range was 0.04–40 ng/mL, the average intra-batch CV was 4.95%, and the average inter-batch CV was 7.79%. The correlation coefficient between full-size-cTnI-TRFIA and commercial cTnI-CLIA methods was R2 = 0.7247.Conclusions: Dual-labeling full-size-cTnI/cTnI-C-TRFIA analysis is helpful for determining the length of time of chest pain before admission and the degree of continuous release of cTnI in the myocardium. Thus, it is more for early prognosis than just detecting cTnI.

Sensitive Time-Resolved Fluorescence Immunoassay for Quantitative Determination of Oxyfluorfen in Food and Environmental Samples.

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 <0.02%, except that there was a certain CR with the benzofluorfen (CR = 11.58) and the bifenox (CR = 8.23%). The average recoveries of ic-TRFIA were 74.6–108.3%, and the RSDs were between 2.1 and 10.9%, in the addition recovery test with five substrates. The results of the correlation test with the real samples of GC-ECD showed that they were highly correlated (y = 0.975x – 0.4446, R2 = 0.9901), which proved that the TRFIA method established in this study had high reliability and accuracy and could be used in environment and agricultural products for rapid detection of oxyfluorfen residues.