Guan-Feng Lin

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 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.

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.

Simultaneous determination of the cytokeratin 19 fragment and carcinoembryonic antigen in human serum by magnetic nanoparticle-based dual-label time-resolved fluoroimmunoassay

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.

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.

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

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 Eu3+-labeled free β-HCG-TRFIA assay was good (y = 1.045x + 1.580, r2 = 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.

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.

Simple and rapid monitoring of doxorubicin using streptavidin-modified microparticle-based time-resolved fluorescence immunoassay

Developing a simple analytical method suitable for therapeutic drug monitoring in a clinical setting is key to establishing guidelines on accurate dose administration and the advancement of precision medicine. We devised a simple rapid analytical method through the combination of streptavidin-modified microparticles and a time-resolved fluorescence immunoassay for therapeutic drug monitoring. The analytical performance of this method was investigated and validated using clinical samples. By determination of doxorubicin concentration, the proposed assay has shown a satisfactory linear range of detection (3.8–3000 ng mL−1) with a limit of detection of 3.8 ng mL−1 and an IC50 of 903.9 ng mL−1. The intra and inter-assay coefficients of variation were 4.12–5.72% and 5.48–6.91%, respectively, and the recovery was acceptable. The applicability of the proposed assay was assessed by comparing the determined results with those measured by LC-MS/MS, presenting a satisfactory correlation (R2 = 0.9868). The proposed assay, which shows satisfactory analytical performance, has great potential for application in the field of TDM in the future.

A time-resolved fluoroimmunoassay to assay the rabies virus glycoprotein: application for estimation of human rabies vaccine potency.

Replacement of the in vivo rabies vaccine potency test (NIH test) by in vitro methods had been discussed by several researcher including WHO expert working groups. In this paper, a time-resolved fluoroimmunoassay (TRFIA) for the assay of rabies virus glycoprotein in rabies vaccine was first established to estimate the rabies vaccine potency by using specific monoclonal antibody that only recognized the native, trimeric and immunogenic form of rabies virus glycoprotein. Potency of the rabies virus glycoprotein was assayed with satisfactory performance under optimal conditions, and the method demonstrated satisfactory results when applied in practical samples. The correlation coefficient of potency values obtained from the present TRFIA and ELISA was 0.912, and 0.903 for those from the present TRFIA and NIH test. These preliminary results confirmed that this TRFIA can replace ELISA with higher performance, and could be a promising replacement of the NIH test. Based upon these results, the present TRFIA seemed to be a convenient tool for evaluating rabies vaccine potency and its products at different stages accordingly.

Anthropometric research of congenital auricular deformities for newborns

Abstract Objective: This study aimed to get the data of Neonatal auricle for helping pediatricians and obstetricians to make definite diagnosis and individualized treatment for newborn congenital auricular deformities in China. Methods: A total of 1500 newborns’ ears were evaluated in this study. Six surface measurements were performed directly on the auricles of the subjects as follows: physiognomic ear length and breadth, morphological ear length and breadth, cephalo-auricular distance, and cephalo otic angle. Results: The incidence of neonatal auricular deformities in the Pearl River Delta area was 57.47% and the self-healing rate was 31.61% by the 30-day follow-up. There were significant differences between different types of auricular deformities (protruding ear, cup ear, lop ear, Stahl’s ear, conchal crus, helical rim deformity, and composite deformity) and normal morphological differences of the auricle. Conclusions: Our findings suggested that individual differences in ear morphology are large and there were some differences between measurement data in our study and EarWell. Application of the EarWell system may lead to localized skin excoriations or breakdown as a result of mismatch with the ear. Therefore, early use of personalized ear molds produced by three-dimensional printing to determine if more deformed auricles may be corrected.