Search

The concentration of Cys C in the patient's serum can reflect the level of glomerular filtration rate and indicate the occurrence of renal failure. The establishment of a simple and rapid analytical method to quantitatively monitor the concentration of Cys C in serum could help timely detection of renal failure. In this study, we have developed an Eu (III) chelate nanoparticles based lateral flow immunoassay to fulfill real-time monitoring of Cys C concentration in serum within 15 min. This method was performed as a sandwich immunoassay with a wide detection range (0.05-10 μg/mL) and a low limit of detection (24.54 ng/mL). The intra and inter-assay coefficients of variation were 8.31-8.61% and 8.92-9.95%, respectively. Furthermore, the application of this method was evaluated by comparing the determined results with those obtained by chemiluminescence immunoassay, exhibiting a satisfactory correlation (R

The level of anti-SARS-CoV-2 neutralizing antibodies (NAb) is an indispensable reference for evaluating the acquired protective immunity against SARS-CoV-2. Here, we established an ultrabright nanoparticles-based lateral flow immunoassay (LFIA) for one-step rapid semi-quantitative detection of anti-SARS-CoV-2 NAb in vaccinee's serum. Once embedded in polystyrene (PS) nanoparticles, the aggregation-induced emission (AIE) luminogen, AIE

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 sensitive homogeneous assays is a high-priority research target for clinical diagnostics. Quantum dots (QDs) present favorable photophysical properties, which implies their potential as an exceptional dye in fluorescence detection. QDs-based biosensors have been described in the literature; however, few of them have truly progressed to widespread clinical usage. In this work, a chemiluminescent homogeneous detecting biosensor is fabricated using QDs-doped polystyrene nanospheres to sensitively detect biomarkers in low-volume serum samples. Phthalocyanine-dyed and QDs-encapsulated carboxylate-functionalized polystyrene nanospheres with surface carboxyl groups (PPs and QPs, respectively) were fabricated and served as triggers and fluorescent probes, respectively, in this biosensing system. In this sandwich-format immunoassay, the PPs produced singlet oxygen once sensitized by 680 nm diode lasers, and the QPs, conjugated with antibodies, and then reacted with the singlet oxygen in the presence of specific antigens and emitted anti-Stokes fluorescence with wavelengths around 605 nm, as a result of fluorescence resonance energy transfer (FRET) within the QPs. We demonstrated the determination of carcinoembryonic antigen as a model protein target in 25 μL of serum samples with an unprecedented detection limit of 2.56 × 10-13 M (46 pg/mL) using this biosensor. Furthermore, excellent correlations ( R2 = 0.99718, n = 107) were obtained between utilizing this biosensor and commercialized chemiluminescence immunoassay kits in clinical serum detection. These results demonstrate that our flexible and reliable biosensor is suitable for direct integration into clinical diagnostics, and it is expected to be a promising diagnostic tool for early detection and screening tests as well as prognosis evaluation for patients.

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.

The outbreak of 2019 coronavirus disease (COVID-19) has been a challenge for hospital laboratories because of the huge number of samples that must be tested for the presence of the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Simple and rapid immunodiagnostic methods are urgently needed to identify positive cases. Here we report the development of a rapid and sensitive lateral flow immunoassay (LFIA) that uses lanthanide-doped polysterene nanoparticles (LNPs) to detect anti-SARV-CoV-2 IgG in human serum. A recombinant nucleocapsid phosphoprotein of SARS-CoV-2 was dispensed onto a nitrocellulose membrane to capture specific IgG. Mouse anti-human IgG antibody was labeled with self-assembled LNPs that served as a fluorescent reporter. A 100-μL aliquot of serum samples (1:1000 dilution) was used for this assay and the whole detection process took 10 min. The results of the validation experiment met the requirements for clinical diagnostic reagents. A value of 0.0666 was defined as the cutoff value by assaying 51 normal samples. We tested 7 samples that were positive by reverse-transcription (RT-)PCR and 12 that were negative but clinically suspicious for the presence of anti-SARS-CoV-2 IgG. One of the negative samples was determined to be SARS-CoV-2 IgG positive, while the results for the other samples were consistent with those obtained by RT-PCR. Thus, this assay can achieve rapid and sensitive detection of anti-SARS-CoV-2 IgG in human serum and allow positive identification in suspicious cases; it can also be useful for monitoring the progression COVID-19 and evaluating patients' response to treatment.

Objective To evaluate the analytical performance of our previously developed chemiluminescence immunoassay (CLIA) kit for the detection of procalcitonin (PCT) and compare with the results obtained using the Vidas B.R.A.H.M.S. PCT™ test (PCT-V). Design and methods Our laboratory previously designed a novel CLIA kit and supporting instrument (AE-180) for the detection of PCT. We analyzed the clinical performance of this system, including the imprecision, limit of detection, and linearity of analyses of 305 serum specimens. The results were compared with measurements of the same serum samples obtained with PCT-V. Results The limit of detection and blank of our kit were 0.0075 and 0.0039 ng/mL, respectively. The intra- and inter-assay coefficient of variation of the kit were both between 0.8% and 3.9%. The equation of linearity was found to be y = 1.03× + 0.06 (r = 0.99) for concentrations in the range of 0.01–110 ng/mL. The correlation coefficient with the results of PCT-V was 0.995, and the equation obtained for Passing and Bablok regression analysis was 1.061 for our CLIA PCT kit and − 0.003 for PCT-V. Our kit slightly overestimated the concentration according to comparison with PCT-V results. Conclusion The kit that was previously developed in our laboratory for the measurement of serum PCT concentration using CLIA technology shows excellent performance, just that the functional sensitivity is not as good as the PCT-V; therefore, we suggest that this kit is suitable for clinical use.

A sensitive, rapid and homogeneous reaction measurement method for quantitation of human epididymis protein 4 (HE4) in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) was described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one biotinylated monoclonal antibody combining on the surface of streptavidin coated donor beads, and "sandwiched" by another monoclonal antibody coated on acceptor beads. The coefficient variations of the method were lower than 10%, and the recoveries were in the range of 90-110% for serum samples. A value of 0.88pmol/l was identified as the minimum detectable dose of the present method for HE4. Compared with the results from electrochemiluminescence immunoassay kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. The present assay demonstrated high sensitivity, wider effective detection range and excellent reproducibility for quantitation of HE4 can be useful for early screening and prognosis evaluation of patients with ovarian cancer.

Enzyme-linked immunosorbent assays (ELISA) specific for anti-HSV glycoprotein G (gG) are most commonly used in the clinical diagnosis of HSV infection. But most of them are qualitative and with narrow detection ranges. A novel time-resolved fluoroimmunoassay (TRFIA) methodology was developed for the quantitative determination of HSV IgG in human serum. The assay was based on an indirect immunoassay format, and performed in 96-well microtiter plates. HSV-1 and HSV-2 were used as the coating antigens. Eu3+-labeled goat anti-(human IgG) polyclonal antibodies were used as tracers. The fluorescence intensity of each well was measured and serum HSV IgG levels quantified against a calibration curve. The detection range of the novel TRFIA was between 5 and 500 AU/mL. Assay sensitivity was 0.568 AU/mL. The intra- and inter-assay coefficients of variation were 0.59–3.63% and 3.65–6.81%, respectively. Analytical recovery, dilution tests and serum panel tests were performed using TRFIA and the results proved satisfactory. There were no statistically significant differences in sensitivity and specificity between the TRFIA and commercial ELISAs. An effective, sensitive and accurate quantitative HSV type 1 and type 2 IgG TRFIA was successfully developed and provided diagnostic value in clinical use. Copyright © 2014 John Wiley & Sons, Ltd.

Sensitive, precise and rapid detection tests are needed in the quality control of rabies vaccine for rabies virus nucleoprotein. Previous studies for quantitation of rabies virus nucleoprotein focused on enzyme-linked immunosorbent assay (ELISA). A novel immunoassay for rapid determination of rabies virus nucleoprotein in rabies vaccine was first established by time-resolved fluoroimmunoassay (TRFIA). Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating in the wells and "sandwiched" by another monoclonal antibody labeled with europium chelates. The immunocomplex was retained after washing, and then adopted treatment with enhancement solution; fluorescence was then measured according to the number of europiumions dissociated. Levels of the rabies virus nucleoprotein were measured in a linear range (5-2500 mEU/mL) with a lower limit of quantitation (0.95 mEU/mL) under optimal conditions. The repeatability, recovery, and linearity of the immunoassay were demonstrated to be acceptable. The correlation coefficient of nucleoprotein values obtained by novel TRFIA method and ELISA method was 0.981. These results showed good correlation and confirmed that this sensitive, precise and rapid TRFIA was feasible and could be more suitable for the quality control in the process of rabies vaccine production than ELISA.

Objectives Diabetes mellitus is a chronic disease affecting millions of people globally and resulting in significant death rates each year. A fast, inexpensive alternative to traditional testing and monitoring techniques is desirable, since secretion of insulin and C-peptide is impaired in diabetes mellitus. Design and methods A highly sensitive immunoassay was developed for the simultaneous measurement of C-peptide and insulin levels in human serum, utilizing dual-label time-resolved fluoroimmunoassay (TRFIA) and magnetic particle technologies. This assay was characteristic for a single-step sandwich-type immunoassay, wherein antibody-coated magnetic particles were used as the solid phase and Eu 3 + and Sm 3 + chelate labels were used for detection. Results Antibody-coated magnetic particles in a TRFIA format performed well in addressing a number of quantitative needs. Conclusions The results of this assay correlated well with commercial chemiluminescence assays and provided a number a advantages, including reduced sample volume, reduced reagent and personnel costs and reduced assay time, while maintaining the required clinical sensitivity.

A highly sensitive, rapid and novel simultaneous measurement method for cytokeratin 19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA) in human serum by magnetic nanoparticle-based dual-label time-resolved fluoroimmunoassay was developed. On the basis of a sandwich-type immunoassay format, analytes in the samples were captured by antibodies coated onto the surface of the magnetic beads and sandwiched by other antibodies labeled with europium and samarium chelates. The lower limit of quantitation of the present method for CYFRA 21-1 was 0.77 ng mL−1 and CEA was 0.85 ng mL−1. The coefficient variations of the method were less than 7%, and the recoveries were in the range of 90–110% for serum samples. The concentrations of CYFRA 21-1 and CEA serum samples determined by the present method were compared with those obtained by the chemiluminescence immunoassay. A good correlation was obtained with the correlation coefficients of 0.961 for CYFRA 21-1 and 0.938 for CEA. This novel method demonstrated high sensitivity, wide effective detection range and excellent reproducibility for the simultaneous determination of CYFRA 21-1 and CEA, which can be useful for the early screening and prognosis evaluation of patients with lung cancer.

Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

Measurement of the free β subunit of human chorionic gonadotropin (free β-hCG) in serum is useful for prenatal screening. Concentrations of free β-hCG vary in different races. Conventional assays used for such measurements have limitations. We applied the AlphaLISA to measure levels of free β-hCG in maternal serum during 8-20 weeks of gestation in women from southern China. Two anti-free β-hCG antibodies were used: one was coated on AlphaLISA acceptor beads and the one was biotinylated. The assay also contained donor beads coated with streptavidin. The AlphaLISA assay detection limit was 0.11 ng/mL, and the analytical range was 0.11-200 ng/mL. The intra- and interassay coefficients of variation were 1.32%-2.50% and 3.44%-5.45%, respectively. The correlation with commercial Eu(3+)-labeled free β-HCG-TRFIA assay was good (y = 1.045x + 1.580, r(2) = 0.978). Median levels of free β-hCG in maternal serum at 8-20 weeks gestation were higher in women from southern China compared with those reported in women from other countries. The AlphaLISA for free β-HCG could become the assay of choice for applications in clinical diagnostics. The established median value of free β-HCG is helpful in clinical diagnosis specific for southern Chinese women.

A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and "sandwiched" by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. Levels of the model analyte, carcinoembryonic antigen (CEA), were determined in a linear range (1-1000 ng mL(-1)) with a limit of detection of 0.5 ng mL(-1) under optimal conditions. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. To evaluate this novel assay for clinical applications, 239 serum samples were evaluated. Compared with the conventional TRFIA and chemiluminescence immunoassay (CLIA), the correlation coefficients of the developed immunoassay were 0.985 and 0.975, respectively. These results showed good correlation and confirmed that our method is feasible and could be used for the clinical determination of CEA (or other tumor antigens) in human serum.

In order to early screen and detect suspected biomarkers from pathogens and the human body itself, tracers or reaction strategies that can act as signal enhancers have been proposed forth at purpose. In this paper, we discussed the applicability of magnetic microparticles-assisted time-resolved fluoroimmunoassay (MMPs-TRFIA) for sensitive determination of potential analytes. Hepatitis B e antigen, antibody to hepatitis B surface antigen and free triiodothyronine were used as biomarker models to explore the reliability of the method. By coupling with bioprobes, MMPs were used as immunoassay carriers to capture target molecules. Under optimal condition, assay performance, including accuracy, precision and specificity, was outstanding and demonstrated satisfactory. To further evaluate the performance of the MMPs-TRFIA in patients, a total of 728 serum samples from hospital were analyzed for three biomarkers in parallel with the proposed method and chemiluminescence immunoassay kit commercially available. Fairly good agreements are obtained between the two methods via data analysis. Not only that but the reliability of MMPs-TRFIA has also been illustrated by three different reaction models. It is confirmed that the novel method modified with MMPs has been established and showed great potential applications in both biological detection and clinical diagnosis, including big molecule protein and low molecular weight haptens.

A sensitive, rapid and novel measurement method for cytokeratin 19 fragment (CYFRA 21–1) in human serum by magnetic particle-based time-resolved fluoroimmunoassay (TRFIA) is described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating onto the surface of magnetic beads and “sandwiched” by another monoclonal antibody labeled with europium chelates. The coefficient variations of the method were lower than 7 %, and the recoveries were in the range of 90–110 % for serum samples. The lower limit of quantitation of the present method for CYFRA 21–1 was 0.78 ng/ml. The correlation coefficient of CYFRA 21–1 values obtained by our novel TRFIA and CLIA was 0.980. The present novel TRFIA demonstrated high sensitivity, wider effective detection range and excellent reproducibility for determination of CYFRA 21–1 can be useful for early screening and prognosis evaluation of patients with non-small cell lung cancer.

In this paper, a novel time-resolved fluoroimmunoassay (TRFIA) protocol using magnetic particles for the simultaneous determination of α-fetoprotein (AFP) and the free β-subunit of human chorionic gonadotropin (free β-hCG) in human serum is described. The new approach uses magnetic particles as an immobilization matrix and means of separation, while the luminescent europium and samarium chelates are used as probes. The proposed method was evaluated via a single-step, sandwich-type TRFIA immunoassay of AFP and free β-hCG as model analytes in serum. With the advantages of magnetic particles, the TRFIA immunoassay exhibited a wide dynamic range for AFP of 0.1-750 ng mL(-1), with a lower detection limit of 0.05 ng mL(-1). The dynamic range for free β-hCG was 0.16-450 ng mL(-1), with a lower detection limit of 0.08 ng mL(-1). Satisfactory specificity, reproducibility, and recovery of the immunoassay were demonstrated. Good correlations were obtained in the analysis of 446 human serum samples between the proposed method and a commercial TRFIA kit. These results demonstrate the feasibility and potential of the new method as a rapid and highly sensitive immunoassay that could be developed into a platform for multi-analyte determinations in clinical practice.