Time-resolved Fluoroimmunoassay Research Articles

Autoluminescence-Free Prostate-Specific Antigen Detection by Persistent Luminous Nanorods and Au@Ag@SiO2 Nanoparticles.

The sensitive detection of biomarker with high selectivity and accuracy is of remarkable significance for the early screening and clinical diagnosis of malignant cancer. Persistent phosphors play a role in illumination-free biosensing owing to their unparalleled optical properties, which produces long-lasting luminescence without the illumination to eliminate the interference of autofluorescence and scattering light from biological matrix. In this study, ZnGeO:Mo persistent luminescence nanorods (PLNRs) were prepared and showed tunable luminescence intensity and decay patterns. An excitation-free luminescent aptasensor was proposed for the detection of prostate specific antigen (PSA) by using ZnGeO:Mo PLNRs as probes and Au@Ag@SiO2 nanoparticles as quenchers. Under optimal conditions, the limit of detection for PSA was 9.2 pg mL-1. This work creates a possibility for the preparation of multiplex colored persistent luminescence nanomaterials with desired decay patterns for multiple bioassay and time-resolved fluoroimmunoassay.

Validation and evaluation of a newly developed time-resolved fluoroimmunoassay for cyclosporine quantitative analysis in human whole blood

Various immunoassay methods have been developed and used for the therapeutic drug monitoring (TDM) of cyclosporine (CsA). However, there is no report on the application of a time-resolved fluoroimmunoassay (TRFIA) in routine CsA TDM. The aim of this study was to evaluate the feasibility and validate the performance of a newly developed TRFIA method for CsA analysis in human whole blood. The TRFIA method was then compared with the method of chemiluminescent microparticle immunoassay (CMIA). The calibration range of the CsA-TRFIA method was 0–1000 ng/mL. The linear range and correlation coefficients were 30–1000 ng/mL and more than 0.990, respectively. The accuracy, precision, and inter-batch range were 90.0%–110.0%, less than 10%, and no more than 15%, respectively. The lowest limit of detection was less than 10 ng/mL. The linear regression equation was YCMIA = 0.961XTRFIA + 3.357, which showed that the measurements of CMIA and TRFIA were strongly correlated (r = 0.980). The results demonstrate that TRFIA is a precise and reproducible method for detecting the CsA concentration and can be used for routinely CsA TDM.

A pilot study on newborn screening for congenital adrenal hyperplasia in Beijing

Background A provisionary screening programme for 21-hydroxylase deficiency (21-OHD) was initiated in Beijing in 2014. The aim of this study was to investigate the incidence and the associated clinical characteristics of neonatal congenital adrenal hyperplasia (CAH) in Beijing and to provide evidence-based guidance for its application in CAH screening. Methods Live birth newborns (n=44,360) were screened for CAH in Beijing from July 2014 to April 2018. The levels of 17-hydroxyprogesterone (17-OHP) in the blood were estimated using the time-resolved fluoroimmunoassay. Neonates with a positive result and a level >30 nmol/L of 17-OHP were called for a retest. CAH was diagnosed based on further laboratory findings combined with clinical signs, such as weight loss, feeding difficulties, skin pigmentation, and atypical genitalia. Through a review of medical records, the clinical findings including molecular data were reported. Results Of the 44,360 neonates screened, 280 cases were deemed positive. Of these, 203 neonates were recalled for further tests and six patients (three boys and three girls) were diagnosed with CAH. Five cases of classic salt-wasting and one case of simple virilising 21-OHD were identified. The incidence of CAH in Beijing was 1:7393. The most frequent 21-OHD mutation was c.293-13C/A>G. Conclusions The incidence of CAH in Beijing was higher than the national average. The results support the need for neonatal CAH screening in Beijing. This pilot study demonstrates the clinical characteristics of 21-OHD through newborn screening. Early detection and treatment through neonatal screening may reduce mortality rates and optimise developmental outcomes.

Serum AKR1B10 predicts the risk of hepatocellular carcinoma – A retrospective single-center study

ObjectivesAKR1B10, first cloned from liver cancer tissues, has recently been reported to be up-regulated significantly in hepatocellular carcinoma (HCC) tissues, but the relationship between serum level of AKR1B10 and the risk of HCC is not understood. Methods170 HCC patients and 120 health donors from October 2014 to March 2017 were recruited in the affiliated hospital of Guilin Medical University. Serum AKR1B10 in all cases were detected and in 30 HCC patients were analyzed preoperatively and postoperatively by Time-resolved fluoroimmunoassay. ResultsThe level of serum AKR1B10 was significantly higher in HCC patients (1800.24±2793.79) than in health donors (129.34±194.129), and downregulation of serum AKR1B10 in HCC patients was observed after hepatectomy. When samples were grouped according to the serum level of AKR1B10 (≥232.7pg/ml), serum AKR1B10 positively correlated to serum AFP (χ2=6.295, P=0.012), ALT (χ2=18.803, P=0.000), AST (χ2=33.421, P=0.000), tumor nodule number (χ2=6.777, P=0.009), cirrhosis (χ2=43.458, P=0.000), and tumor size (χ2=6.042, P=0.014) in the Chi-square test. ConclusionsDiagnosis of HCC could be improved using the both predictors of serum AKR1B10 and AFP. AKR1B10 was thus considered to be a new serological biomarker for HCC.

Dual-label time-resolved fluoroimmunoassay as an advantageous approach for investigation of diethyl phthalate & dibutyl phthalate in surface water

Due to the extensive presence of phthalate esters (PAEs) in the environment, it is very important to develop highly efficient methods for determining their environmental concentrations and for risk assessment. In this study, europium (Eu3+) and samarium (Sm3+) were employed as fluorescent labels to develop a dual-labeled time-resolved fluoroimmunoassay (TRFIA) for the sensitive detection of diethyl phthalate (DEP) and dibutyl phthalate (DBP) in aquatic environments. Under optimum conditions, the half-maximal inhibition concentration (IC50) and limit of detection (LOD, IC10) of the TRFIA were 28.1 and 4.9ng/mL for DEP, and 33.4 and 3.9ng/mL for DBP, respectively. The cross-reactivities of the TRFIA between these two phthalates and with their analogues were negligible. The proposed method indicated satisfactory accuracy with recoveries of 78.30–120.13% for DEP and 78.58–113.07% for DBP, which were in good agreement with the gas chromatography tandem mass spectrometry analysis results of the same samples. Meanwhile, the results of the immunoassay were used to evaluate the presence and environmental risk of those pollutants in the inner rivers of Zhenjiang city. The concentrations of DEP and DBP ranged from non-detectable to 61.31ng/mL and from non-detectable to 94.57ng/mL, respectively. The results of the potential ecological risk assessment by the risk quotient method showed that there were some sampling points exceeding risk limits, and the total ecological risk of DBP was higher than that of DEP.

Development of a highly sensitive time-resolved fluoroimmunoassay for the determination of trace salbutamol in environmental samples

Veterinary drug residues have become a major source of environmental pollutants. To monitor trace salbutamol (SAL) in the environment, a highly sensitive and reliable time-resolved fluoroimmunoassay (TRFIA) was developed. Under the optimum parameters, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC10) were determined to be 0.08 μg/L and 0.66 ng/L with a linear range (IC20 - IC80) of 0.0028–2.25 μg/L for SAL. The IC50 and LOD of the TRFIA improved approximately 5-fold and 31-fold, respectively, when compared with our previously reported ELISA data. When compared to most other conventional methods, the TRFIA also showed an excellent sensitivity and accuracy for the detection of SAL. Recoveries from 83.4 to 111.3% and standard deviations (RSDs) from 3.9 to 14.0% were observed in various environmental SAL-spiked samples, including river water, paddy water, livestock wastewater, vegetable field soil and rice paddy soil. In addition, the developed TRFIA showed largely consistent with analytic results from UPLC-MS/MS (R2 = 0.9825, n = 15). These results suggest that the proposed TRFIA can be applied as a sensitive and reliable monitoring method to detect trace SAL in environmental samples.

Development of a novel immunoassay for the simple and fast quantitation of neutrophil gelatinase-associated lipocalin using europium(III) chelate microparticles and magnetic beads

Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for diagnosing acute kidney injury (AKI). Currently, there are few assays for determining NGAL and they are complex, time-consuming or expensive. We aimed to establish an efficient immunoassay to measure NGAL in human urine simply and rapidly. A novel immunoassay for NGAL determination was established by combining a dissociation-enhanced-free time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a “sandwich”-type immunoassay format, analytes in samples were captured by a pair of monoclonal antibodies (mAb) in which one mAb was coated in magnetic beads and the other mAb was labeled with europium(III) chelate microparticles (CM-EUs) as “fluorescent reporters”. NGAL concentrations were determined in a linear range (10–1500 ng mL−1) with a limit of detection of 0.32 ng mL−1. The reproducibility, recovery, and specificity of our TRFIA were acceptable. Our method was compared with that of a chemiluminescence immunoassay (CMIA) using 115 urine samples, and the results showed good correlation (R2 = 0.8677). We expect our novel method to be useful for the early diagnosis of AKI.

Does sex hormone-binding globulin cause insulin resistance during pubertal growth?

BackgroundThe directional influences between serum sex hormone-binding globulin (SHBG), adiposity and insulin resistance during pubertal growth remain unclear. The aim of this study was to investigate bidirectional associations between SHBG and insulin resistance (HOMA-IR) and adiposity from childhood to early adulthood.MethodsParticipants were 396 healthy girls measured at baseline (age 11.2 years) and at 1, 2, 4 and 7.5 years. Serum concentrations of estradiol, testosterone and SHBG were determined by ELISA, glucose and insulin by enzymatic photometry, insulin-like growth factor 1 (IGF-1) by time-resolved fluoroimmunoassays, whole-body fat mass by dual-energy X-ray absorptiometry and HOMA-IR were determined by homeostatic model assessment. The associations were examined using cross-lagged path models.ResultsIn a cross-lagged path model, SHBG predicted HOMA-IR before menarche β = −0.320 (95% CI: −0.552 to −0.089), P = 0.007, independent of adiposity and IGF-1. After menarche, no directional effect was found between SHBG and insulin resistance or adiposity.ConclusionsOur results suggest that in early puberty, decline in SHBG predicts development of insulin resistance, independent of adiposity. However, after menarche, no directional influences between SHBG, adiposity and insulin resistance were found, suggesting that observational associations between SHBG, adiposity and insulin resistance in pubertal children may be subject to confounding. Further research is needed to understand the underlying mechanisms of the associations between SHBG and cardiometabolic risk markers in peripubertal children.

A pilot study on newborn screening for congenital adrenal hyperplasia in Beijing.

Background A provisionary screening programme for 21-hydroxylase deficiency (21-OHD) was initiated in Beijing in 2014. The aim of this study was to investigate the incidence and the associated clinical characteristics of neonatal congenital adrenal hyperplasia (CAH) in Beijing and to provide evidence-based guidance for its application in CAH screening. Methods Live birth newborns (n=44,360) were screened for CAH in Beijing from July 2014 to April 2018. The levels of 17-hydroxyprogesterone (17-OHP) in the blood were estimated using the time-resolved fluoroimmunoassay. Neonates with a positive result and a level >30 nmol/L of 17-OHP were called for a retest. CAH was diagnosed based on further laboratory findings combined with clinical signs, such as weight loss, feeding difficulties, skin pigmentation, and atypical genitalia. Through a review of medical records, the clinical findings including molecular data were reported. Results Of the 44,360 neonates screened, 280 cases were deemed positive. Of these, 203 neonates were recalled for further tests and six patients (three boys and three girls) were diagnosed with CAH. Five cases of classic salt-wasting and one case of simple virilising 21-OHD were identified. The incidence of CAH in Beijing was 1:7393. The most frequent 21-OHD mutation was c.293-13C/A>G. Conclusions The incidence of CAH in Beijing was higher than the national average. The results support the need for neonatal CAH screening in Beijing. This pilot study demonstrates the clinical characteristics of 21-OHD through newborn screening. Early detection and treatment through neonatal screening may reduce mortality rates and optimise developmental outcomes.

HBV T1719G mutation reduced HBV replication through mutant Enh II and HBx protein in vitro.

It was repeatedly reported that the hepatitis B virus (HBV) T1719G mutation was very common and related to progression and malignancy of liver disease. However, its effect on viral replication efficiency remains unclear. In this study, we aimed to evaluate the function and mechanisms of the T1719G mutation on viral replication capacity. Wild-type and T1719G mutation-bearing HBV1.2× plasmids were transfected into Huh7 and HepG2 cells, respectively, and HBV total RNA, 3.5kb RNA and supernatant HBV DNA were assessed using real-time PCR, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured by time-resolved fluoroimmunoassay. In order to assess Enh II activity and the binding capacity of HNF3β to Enh II sequence, dual-luciferase assay and Chromatin immunoprecipitation (ChIP)-PCR were employed, respectively. Simultaneously, the HBx or HBx-mut (T1719G) plasmid was co-transfected to evaluate the effect of HBx on viral replication. Our results showed that the T1719G mutation impaired viral replication efficacy compared with the wild type both by reducing Enh II activity and binding capacity of HNF3β with Enh II. And such reduction caused by T1719G mutation could be rescued by HBx protein. Our results show that the T1719G mutation decreases HBV viral replication capacity possibly by mutant HBx protein and altered Enh II activity.

Establishment of an ultrasensitive indirect competitive time-resolved fluoroimmunoassay for vancomycin determination

ABSTRACT Vancomycin (Van) is a common antibiotic that pollutes food, feed and ecological environment. In the present study, an anti-Van polyclonal antibodies (pAbs) and Eu3+-labeled goat anti-rabbit IgG monoclonal antibody based indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for ultrasensitive determination of Van, and its half-maximum inhibition concentrations (IC50) was 13.74 ng/L and the limit of detection (LOD) was 1.07 ng/L. The IC-TRFIA showed strong cross-reactivity (CRs) of 36.2% for norvancomycin, and less than 0.1% for polymyxin B, kanamycin and ampicillin. It was found that the IC-TRFIA for Van spiked in different milk powder samples determination were with good accuracy, stability and repeatability, and its recoveries are 83.0–97.9%, also with its coefficient of variations (CVs) are 3.0–12.9% in intra-assay and inter-assay. The results showed that the established IC-TRFIA was promising for ultrasensitive determination of Van in food samples. Highlights Prepared high-quality Van-KLH/BSA conjugates. Obtained high activity and purify anti-Van polyclonal antibodies. Prepared high-quality Eu3+-labelled goat anti-rabbit IgG monoclonal antibody. Firstly established an IC-TRFIA for Van based on the anti-Van polyclonal antibodies and Eu3+-labelled goat anti-rabbit IgG monoclonal antibody. The IC-TRFIA can be used for ultrasensitive determination of Van in milk powder samples.

Monoclonal antibody production and the development of a quantitative time-resolved fluoroimmunoassay for rifaximin in milk

ABSTRACT Rifaximin (RIF) is an important drug in clinical veterinary, and its residue in milk might pose a potential threat to human health. To monitor RIF residue in milk, a europium nanospheres-based time-resolved fluorescence immunoassay (TRFIA) was developed. The TRFIA is based on the competitive reaction between RIF-ovalbumin (OVA) and the target RIF in milk for binding to the Eu-Nanospheres-mAbs. The limit of detection (LOD) and limit of quantity (LOQ) were 3.05 and 6.63 ng/mL, respectively. The recoveries of the TRFIA to detect RIF in spiked milk samples ranged from 73.7% to 103.9%, and the interday and intraday variation were respectively less than 12.1% and 10.5%. A quantitative comparison of TRFIA and UPLC-MS/MS method exhibited a significant correlation (R 2 = 0.9725) at the six various spiked levels. These results indicated that the TRFIA we developed was applicable for the detection of large numbers of milk samples.

Measurement of urinary matrix metalloproteinase-7 for early diagnosis of acute kidney injury based on an ultrasensitive immunomagnetic microparticle-based time-resolved fluoroimmunoassay

The morbidity and mortality associated with acute kidney injury (AKI) remain obstinately high. Early diagnosis is urgently required and should be pursued in at-risk populations. Recently, a newly validated biomarker, matrix metalloproteinase-7 (MMP-7), was reported as a novel indicator for early AKI prediction and a noninvasive surrogate biomarker of kidney function. Monitoring urinary MMP-7 (uMMP-7) levels fills the gaps in early diagnosis of AKI at early onset. However, the lack of available reagents for its rapid detection limits its use. Herein, we established an ultrasensitive and rapid immunomagnetic microparticles-based time-resolved fluoroimmunoassay to measure urinary MMP-7 in AKI patients. The assay time is 30 min. The calibration curve showed high linear correlation (r = 0.9998) with a linearity of detection of 0.063–150 ng mL−1 and lower limit of detection of 0.039 ng mL−1. The coefficient variation of the intra- and inter-assay lower than 5.17%, and the analytical recovery was 99.06%–105.60%. Testing of clinical samples using the proposed assay and a DUOSET@ ELISA kit showed good correlations in the comparison of uMMP-7 levels (r = 0.9541) and uMMP-7/uCreatinine (r = 0.9595). The proposed assay has satisfactory analytical performance and may serve as a promising tool for early diagnosis of AKI.

A New Serological Method for the Differential Diagnosis of Hashimoto's Thyroiditis and Graves' Disease

Background: Although Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) have overlapping symptoms, the treatment of these conditions is different, making the differential diagnosis of these disorders crucial for optimal patient outcomes. Therefore, we developed and evaluated a novel serological method for the differential diagnosis of HT and GD. Methods: We developed a highly sensitive, time-resolved fluoroimmunoassay for measuring serum concentrations of anti-phospholipase A2 receptor (PLA2R)-IgG antibody; other thyroid health indicators such as thyroid-stimulating hormone (TSH), free T3, free T4; and antibodies against thyroglobulin (TGAb), thyroperoxidase (TPOAb), and TSH receptor (TRAb). We analyzed the differences in the concentrations of these indicators among healthy controls, patients with HT, and patients with GD. Serum anti-PLA2R-IgG, TGAb, TPOAb, TRAb, and other indicator concentrations were used to differentially diagnose HT and GD. Results: In the control, GD, and HT groups, the average concentrations (±standard deviation) of anti-PLA2R-IgG were 0.49 ± 0.24 mg/L, 0.47 ± 0.11 mg/L, and 3.23 ± 1.89 mg/L, respectively, and the percentages of patients above the threshold of 1.1 mg/L were 0%, 0%, and 95% respectively. The positive results obtained in both the TPOAb and anti-PLA2R-IgG tests were good diagnostic indicators of HT, with a sensitivity of 90% and specificity of 100%. Conclusions: The serum anti-PLA2R-IgG antibody concentration was significantly elevated in the sera of HT patients compared with that in healthy controls and GD patients. Measurement of serum anti-PLA2R-IgG concentrations in conjunction with the TPOAb test provides a reliable method for the differential diagnosis of HT and GD. Funding Statement: Funding was provided by the Clinical Medical Science fund of Jiangsu Province (No. BL2014022), the Directive Planning Project of Wuxi Science and Technology Bureau(No. CSE31N1302), the Key Subject of Wuxi’s Hospital Administration Center (No. YGZXM14013), Wuxi Key Medical Talents (zdrc006), Jiangsu Province Science and Education Project (CXTDB2017016), Wuxi Technology Development Fund (Medical and public health project) (WX18IIAN027) and Medical and Public Health Project of Wuxi Sci-Tech Development Fund (WX18IIAN647). Declaration of Interests: The authors declare they have no competing financial interests. Ethics Approval Statement: The institutional review board of Jiangyuan Hospital and Wuxi People’s Hospital approved the study, and written informed consent was obtained from all the study subjects. Patients treated at Jiangyuan Hospital (China) and Wuxi People’s Hospital (China) between January 2015 and May 2017 were selected as the subjects for this study.

Establishment of Eu3+ /Sm3+ dual-label time-resolved fluoroimmunoassay for measurement of antibodies to hepatitis C virus and its clinical application

Objective To establish a time-resolved fluoroimmunoassay (TRFIA) system for simultaneous measurement of immunoglobulin (Ig)M and IgG antibodies to HCV. Methods Recombinant HCV antigen was fixed on microtiter plates to detect serum HCV antibodies. Eu3+ -labeled anti-human IgM and Sm3+ -labeled anti-human IgG were prepared. HCV-IgM and HCV-IgG TRFIA were established using indirect assay and further optimized and evaluated. The one-sided 95th-percentile was used to calculate the cut-off (CO) values. Results Defining 1 sample/CO (S/CO) as measuring unit, the detection limit was 0.06 S/CO for HCV-IgM and 0.15 S/CO for HCV-IgG. When diluted a strong-positive specimen from 1∶12.5 to 1∶51 200, there was a good liner range within 1∶12.5 to 1∶12 800 for HCV-IgM and 1∶25 to 1∶6 400 for HCV-IgG. The average intra-assay CV of HCV-IgM and HCV-IgG were 3.37% and 3.66%, respectively, and the average inter-assay CV were 6.52% and 6.75%, respectively. Compared with enzyme-linked immunosorbent assay (ELISA) kits, the positive conformity rate, the negative conformity rate and total conformity rate were 100.0%(20/20), 90.0%(18/20), 95.0%(38/40) for HCV-IgM TRFIA, and were 100.0%(20/20), 95.0%(19/20), 97.5%(39/40) for HCV-IgG TRFIA, respectively. Additionally, the established HCV-IgM and HCV-IgG assay kits presented good stability with a decline in the value of fluorescence of 11.1% and 9.5% respectively after being stored at 37 ℃ for 7 d. Conclusions The established HCV-IgM and HCV-IgG TRFIA could simultaneously measure HCV-IgM and HCV-IgG antibodies at one step. The method has wider detectable range and may be a more sensitive, stable, and reliable method for diagnosing HCV infection. Key words: Hepatitis C antibodies; Fluoroimmunoassay; Europium; Samarium

PLA2R: The Same Target Antigen of Idiopathic Membranous Nephropathy and Hashimoto's Thyroiditis?

Background: Autoimmune thyroid disease associated nephropathy was identified 40 years ago; however, the cause of the disease remains unknown. Methods: Phospholipase A2 receptor (PLA2R) antibodies was assayed quantitatively in the serum of patients with Graves' disease, Hashimoto's thyroiditis (HT), idiopathic membranous nephropathy (IMN) and normal controls by time-resolved fluoroimmunoassay. PLA2R antigens were immunohistochemically analyzed in the thyroid tissue with Graves' disease and HT, as well as the nephridial tissue with IMN. Findings: The concentration of phospholipase A2 receptor (PLA2R) antibodies in the serum of normal controls, patients with Nodular goiter, Graves' disease and HT, as well as patients with IMN was 1.13 ± 0.43 mg/L, 1.07 ± 0.22mg/L, 2.12 ± 2.11mg/L, 8.07 ± 4.74mg/L, and 15.91 ± 19.50mg/L, respectively. The concentration of PLA2R antibodies in the serum and its areas under the curve of ROC in HT and IMN cases increased significantly. The immunohistochemistry revealed that the tissues of the patients with HT were obviously stained, with a positive rate of 66.67%. Interpretation: PLA2R may be a pathogenic target antigen for HT, and the production of anti-PLA2R antibodies may cause autoimmune thyroid disease associated nephropathy. Funding Statement: Clinical Medical Science fund of Jiangsu Province (No. BL2014022), Jiangsu Maternal and Child Health Research Project (FRC201741), and Wuxi City Science and Education Project (ZDRC006) supported the study. Declaration of Interests: All Authors declare no competing financial interests. Ethics Approval Statement: Patients from Jiangsu Jiangyuan Hospital and the Wuxi People’s Hospital affiliated to Nanjing Medical University, were enrolled. The study protocol was approved by the medical ethics committee of Jiangsu Jiangyuan Hospital and Affiliated Wuxi People's Hospital of Nanjing Medical University.

The Use of Biochemical and Biophysical Markers in Early Screening for Preeclampsia in Mongolia.

Preeclampsia (PE) is a major cause of maternal and perinatal morbidity and mortality, particularly in developing countries. In Mongolia, preeclampsia and eclampsia have occurred among pregnancy complications at a rate of 25% in recent years. Recent studies in the literature have screened for preeclampsia by combining maternal factors with biomarkers. This study was conducted using prospective cohort research including 393 singleton pregnancies at 11–13+6 weeks. Maternal plasmas pregnancy-associated plasma protein-A (PAPP-A) and maternal serum placental growth factor (PlGF) were measured using Perkin Elmer time-resolved fluoroimmunoassay (DELFIA) kits, and the measurement of mean arterial pressure (MAP) was performed by automated devices and the uterine artery pulsatility index was measured by Doppler ultrasound. In the study population, there were 16.7% showing complicated preeclampsia. The receiver-operating characteristics (ROC) curve analysis showed a sensitivity of 71.21%, and a specificity of 75.54% when the mean arterial pressure cut-off was 89.5 mm; while a sensitivity of 33.36% and specificity of 77.68% were observed when the uterine artery mean pulsatility index (mPI) cut-off was 2.34; a sensitivity of 79.66% and specificity of 44.04% were observed when the PAPP-A cut-off was 529.1 mU/L; and a sensitivity of 74.58% and specificity of 46.6% were observed when the PlGF cut-off was 39.87 pg/mL. The detection rates following the combination of markers with the maternal history were as follows: 62.7% with mean arterial pressure, 69.5–82.9% with two markers 86.5% with three markers and 91.4% with four markers. In conclusion, the mean arterial pressure was highly sensitive and demonstrated its easy usage and cost-effectiveness as a predictive marker for the early screening of preeclampsia from other biomarkers.

Dual-antigen sandwich-type time-resolved fluoroimmunoassay for phospholipase A2 receptor antibody, a new marker of membranous nephropathy

Objective To establish a time-resolved fluoroimmunoassay (TRFIA) method for detecting M-type phospholipase A2 receptor (PLA2R) antibody and to investigate the diagnostic value of serum PLA2R antibody for idiopathic membranous nephropathy (IMN). Methods The microplates coated with recombined PLA2R antigen and Eu3+ -streptavidin labeled PLA2R antigen were used to establish a dual-antigen sandwich-type TRFIA for PLA2R antibody detection (anti-PLA2R-TRFIA). The serum concentrations of PLA2R antibody in 63 IMN patients (36 males, 27 females, age 25-75 years) and 90 healthy volunteers (30 males, 60 females, age 22-53 years) were quantitatively analyzed. Kruskal-Wallis H test and Mann-Whitney u test were used to analyze the data. Results The range of anti-PLA2R-TRFIA was 0-10 mg/L and the sensitivity was 5 μg/L, while ED20, ED50 and ED80 of the standard curve were (0.144±0.012), (0.707±0.029) and (3.466±0.098) mg/L, respectively. The CV of inter- and intra-assay were 4.7% and 5.1%, respectively. The average concentration of serum PLA2R antibody in healthy volunteers was 0.455(0.320, 0.593) mg/L, but in IMN patients it was 2.690(0.717, 7.750) mg/L (z=-3.688, P<0.05). Meanwhile, the serum levels of PLA2R antibody in IMN patients were significantly different between different stages and ages (χ2 values: 10.328, 9.716, both P<0.05). According to the receiver operating characteristic (ROC) curve, when the diagnostic cut-off was set at 0.80 mg/L for IMN detection, the sensitivity and specificity of anti-PLA2R-TRFIA were 73.0%(46/63) and 95.6%(86/90), respectively. Conclusions Anti-PLA2R-TRFIA has been well established. This quick and easy-performance method could increase the diagnostic accuracy for IMN. Key words: Glomerulonephritis, membranous; Receptors, phospholipase A2; Fluoroimmunoassay

Development of Time-Resolved Fluoroimmunoassay for Detection of Cylindrospermopsin Using Its Novel Monoclonal Antibodies

Cylindrospermopsin (CYN) is a cyanotoxin that is of particular concern for its potential toxicity to human and animal health and ecological consequences due to contamination of drinking water. The increasing emergence of CYN around the world has led to urgent development of rapid and high-throughput methods for its detection in water. In this study, a highly sensitive monoclonal antibody N8 was produced and characterized for CYN detection through the development of a direct competitive time-resolved fluorescence immunoassay (TRFIA). The newly developed TRFIA exhibited a typical sigmoidal response for CYN at concentrations of 0.01–100 ng mL−1, with a wide quantitative range between 0.1 and 50 ng mL−1. The detection limit of the method was calculated to be 0.02 ng mL−1, which is well below the guideline value of 1 μg L−1 and is sensitive enough to provide an early warning of the occurrence of CYN-producing cyanobacterial blooms. The newly developed TRFIA also displayed good precision and accuracy, as evidenced by low coefficients of variation (4.1–6.5%). Recoveries ranging from 92.6% to 108.8% were observed upon the analysis of CYN-spiked water samples. Moreover, comparison of the TRIFA with an ELISA kit through testing 76 water samples and 15 Cylindrospermopsis cultures yielded a correlation r2 value of 0.963, implying that the novel immunoassay was reliable for the detection of CYN in water and algal samples.

Establishment of a time-resolved fluoroimmunoassay in detection of human epidermal growth factor receptor 2

Objective To set up a time-resolved fluoroimmunoassay (TRFIA) method for human epidermal growth factor receptor 2 (HER2) detection and to evaluate its performance. Methods Each well of the 96-microwell plate was coated with monoclonal antibody of HER2(H7) and another monoclonal antibody of HER2(E5) was labeled by Eu3+ . The sensitivity, stability, specificity, measurement range and reference value of this method were tested. The correlation between chemiluminescence (CLIA) method and TRFIA method was analyzed. Results The sensitivity of HER2-TRFIA method was 0.214 ng/ml. The measurement range was 0.214-1 000 ng/ml. The mean within-run CV and mean between-run CV were 3.48% and 4.13%, respectively. HER2-TRFIA method had no cross-reaction with HER1 and its reference range was 0-13.20 ng/ml. The correlation coefficient between TRFIA and CLIA was 0.997. The same batch of reagents were found to be stable for more than 6 months at 4 ℃. Conclusions HER2-TRFIA method has high sensitivity, specificity, stability and wide detecting range. It might be suitable for clinical use. Key words: Receptor, epidermal growth factor; Breast neoplasms; Fluoroimmunoassay