[Preparation of Bamboo-based N, P Co-doped Activated Carbon and Its Lanthanum Ion Adsorption Performance].

Using diammonium hydrogen phosphate as an activator and N and P source and and bamboo chips as the carbon source, N, P co-doped activated carbon was prepared by one-step pyrolysis and used to efficiently remove La3+ in aqueous solutions. The effects of activation temperature and pH value on the adsorption performance of La3+ were analyzed, and the activation and adsorption mechanisms were explored using TG-IR, SEM-EDX, pore structure, XPS, and hydrophilicity. The results showed that diammonium hydrogen phosphate easily decomposed at a high temperature to produce ammonia and phosphoric acid, which activated the material and promoted the increase in the specific surface area and pore volume of the activated carbon. As an N and P source, the addition of diammonium hydrogen phosphate successfully achieved the N, P co-doping of activated carbon, and the introduction of N- and P-containing functional groups was the key to enhance the adsorption of La3+. Among them, graphitic nitrogen could provide interactions between La3+-π bonds, and C-P=O and C/P-O-P could provide active sites for the adsorption of La3+ through complexation and electrostatic interaction. The adsorption of La3+ on N, P co-doped activated carbons was endothermic and spontaneous, and the adsorption process conformed to the Langmuir isotherm and secondary kinetic model. Under the process conditions of an activation temperature of 900℃ and pH=6, the adsorption capacity of the N, P co-doped activated carbon was as high as 55.18 mg·g-1, which was 2.53 times higher than that of the undoped sample, and its adsorption selectivity for La3+ in the La3+/Na+and La3+/Ca2+ coexistence systems reached 93.49% and 82.49%, respectively. Additionally, the removal efficiency remained above 54% after five successive adsorption-desorption cycle experiments.

Establishment of Magnetic Microparticles-Assisted Time-Resolved Fluoroimmunoassay for Determinating Biomarker Models 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.

Dual-labeled time-resolved immunofluorometric assay for the determination of IgM antibodies to rubella virus and cytomegalovirus in human serum.

OBJECTIVES: This study established a novel time-resolved fluorescence immunoassay (TRFIA) that allows the simultaneous determination of rubella virus (RV) IgM and cytomegalovirus (CMV) IgM in human serum. DESIGN AND METHODS: Lanthanum elements labeled antibody and streptavidin-biotin system were used in the "capture sandwich" format simultaneously. RESULTS: The working range of TRFIA for RV IgM was 2-80 AU/mL and for CMV IgM was 5-400 AU/mL. Intra- and inter-assay coefficient of variation (CV) for RV IgM and CMV IgM were both less than 10% and recoveries were from 90% to 110%. No significant statistical difference in sensitivity or specificity was observed between dual-TRFIA and commercial chemiluminescent immunoassays (CLIA) in serum samples. CONCLUSION: The novel dual-TRFIA for RV IgM and CMV IgM detection might have valuable clinical application, with satisfactory sensitivity, specificity and accuracy.

Development of a time-resolved fluorescence immunoassay for herpes simplex virus type 1 and type 2 IgG antibodies.

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. Eu(3+)-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.

Development of an improved time-resolved fluoroimmunoassay for simultaneous quantification of C-peptide and insulin in human serum.

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 rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum.

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.

Magnetic particle-based time-resolved fluoroimmunoassay for the simultaneous determination of α-fetoprotein and the free ß-subunit of human chorionic gonadotropin.

In this paper, a novel time-resolved fluoroimmunoassay (TRFIA) protocol using magnetic particles for the simultaneous determination of α-fetoprotein (AFP) and the free ß-subunit of human chorionic gonadotropin (free ß-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 ß-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 ß-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.

AlphaLISA for the determination of median levels of the free ß subunit of human chorionic gonadotropin in the serum of pregnant women.

Measurement of the free ß subunit of human chorionic gonadotropin (free ß-hCG) in serum is useful for prenatal screening. Concentrations of free ß-hCG vary in different races. Conventional assays used for such measurements have limitations. We applied the AlphaLISA to measure levels of free ß-hCG in maternal serum during 8-20 weeks of gestation in women from southern China. Two anti-free ß-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 ß-HCG-TRFIA assay was good (y = 1.045x + 1.580, r(2) = 0.978). Median levels of free ß-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 ß-HCG could become the assay of choice for applications in clinical diagnostics. The established median value of free ß-HCG is helpful in clinical diagnosis specific for southern Chinese women.

Development of a dual-label time-resolved fluoroimmunoassay for the detection of α-fetoprotein and hepatitis B virus surface antigen.

Time-resolved fluorometry of lanthanide chelates is one of the most useful non-isotopic detection techniques and has been used in numerous applications in biomedical science. We developed a time-resolved fluoroimmunoassay (TRFIA) to quantify α-fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg) in human serum. Based on a two-site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co-coated in 96 microtitration wells and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu(3+)- and Sm(3+)-McAbs were added and fluorescence signals of Sm(3+) and Eu(3+) tracers were collected. Detection limits of AFP and HBsAg were 0.09 mIU/L and 0.01 µg/L, respectively. Measurement ranges of AFP-TRFIA and HBsAg-TRFIA were 1-1000 mIU/L and 0.2-150 µg/L, respectively. Intra- and inter-assay coefficients of variation of AFP-TRFIA were 3.3-4.1% and 5.7-7.2% and for HBsAg-TRFIA were 2.9-3.9% and 4.9-6.8%, respectively. Linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. For the endurance test, Eu-labeled McAbs were stable for at least one year at -20°C and the results of the TRFIA with the same reagents were also reproducible after one year. The availability of a highly sensitive, reliable and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg, thereby providing diagnostic value in various clinical conditions and could be applied for clinical use.

Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein.

Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605 nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Förster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1Å. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was logY=3.65786+0.43863·logX (R=0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4 ng mL(-1). By assaying test samples against the standard curve, the coefficient of variations was <5%, indicating that QDs were suitable for this homogenous time-resolved fluoroimmunoassay. This work extended the potential applications of QDs in future homogeneous analytical bioassays. In the coming research, hepatitis B surface antigen, another primary marker for hepatocellular carcinoma, will be studied for practical detection using a QD-based homogenous multiplex fluoroimmunoassay.

Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum.

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.

[Development of a time-resolved fluoroimmunoassay for detecting S1 subunit of pertussis toxin and its application].

OBJECTIVE: To develop a time-resolved fluoroimmunoassay (TRFIA) for detection of pertussis toxin (PT) S1 subunit for quality control of human PT vaccine. METHODS: A double antibody sandwich one-step method was used to establish the TRFIA for detecting PT S1 subunit in the vaccine. RESULTS: The sensitivity of c peptide analysis reached 2.5 ng/ml without cross-reactions with other antigens. This assay could be used in detecting S1 subunit in the vaccine. CONCLUSION: The TRFIA for detecting PT S1 subunit is simple, sensitive and rapid for quality control of the PT vaccine.

[Development of an amplified luminescent proximity homogeneous immunoassay kit for detecting human hepatitis B virus e antibody].

OBJECTIVE: To develop an amplified luminescent proximity homogeneous immunoassay (AlphaLISA) kit for the detection of human hepatitis B virus e antibody (HBeAb). METHODS: The neutralizing and competitive inhibition method was used to develop the AlphaLISA kit for detection of serum HBeAb. RESULTS: The working range of the kit was 0.003-16 NCU/ml with a sensitivity up to 0.003 NCU/ml. The intra- and inter-assay coefficient of variation was 5.3% and 6.8%, respectively. The kit showed no cross-reaction with HBcAb, and comparison of the detection results with those of a commercially available Elecsys HBeAb kit (Roche) for 136 samples showed a correlation coefficient of 0.961. CONCLUSION: The AlphaLISA kit for HBeAb detection meets the clinical requirements for detection HBeAb in human serum.