Rong- Liang

Simultaneous quantitation of cytokeratin-19 fragment and carcinoembryonic antigen in human serum via quantum dot-doped nanoparticles

A novel quantum dot-doped polystyrene nanoparticles-based lateral flow test strips (QPs-LFTS) system was developed to simultaneously detect a cytokeratin-19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA) in human serum to aid the diagnosis and prognosis of lung cancer. Quantum dot-doped carboxylate-functionalized polystyrene nanoparticles (QPs) were prepared and introduced as fluorescent reporters in QPs-LFTS. The detection was based on a sandwich immunoassay and performed on lateral flow test strips, with an assay time of 15min. The strips were read by a fluorescence strip reader to obtain the fluorescence peak heights of the test lines (HT) and the control line (HC). The ratio of HT/HC was used for quantitation. The QPs showed excellent photoproperties and good performance. Under optimal conditions, the QPs-LFTS system exhibited a wide linear range for CYFRA 21-1 (1.3-480ng/mL) and CEA (2.8-680ng/mL). The detection limits for CYFRA 21-1 and CEA were 0.16 and 0.35ng/mL, respectively. The recovery and reproducibility of the method were satisfactory. Furthermore, excellent correlations (n =120, R2 =0.9862, P<0.0001 for CYFRA 21-1; n =70, R2 =0.9509, P<0.0001 for CEA) were obtained between the QPs-LFTS and commercially available chemiluminescence immunoassay kits in clinical serum testing. The results indicate that this developed test system is highly efficient and is expected to be useful for early screening and prognosis evaluation for lung cancer patients.

A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection.

Objective The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure. Methods Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction. Results A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B. Conclusion A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.

Europium (III) chelate microparticle-based lateral flow immunoassay strips for rapid and quantitative detection of antibody to hepatitis B core antigen

Quantitative hepatitis B core antigen (anti-HBc) measurements could play an important role in evaluating therapeutic outcomes and optimizing the antiviral therapy of chronic hepatitis B infection. In this study, we have developed a simple and rapid fluorescence point-of-care test based on a lateral flow immunoassay (LFIA) method integrated with Eu (III) chelate microparticles to quantitatively determine anti-HBc concentrations in serum. This assay is based on a direct competitive immunoassay performed on lateral flow test strips with an assay time of 15 min. The Eu (III) chelate microparticle-based LFIA assay could quantitatively detect anti-HBc levels with a limit of detection of 0.31 IU mL−1, and exhibited a wide linear range (0.63–640 IU mL−1). The intra- and inter-assay coefficients of variation for anti-HBc were both less than 10% and a satisfactory dilution test and accuracy were demonstrated. There were no statistically significant differences in sensitivity or specificity in serum samples between the Eu (III) chelate microparticle-based LFIA strips and the Abbott Architect kit. A simple, rapid and effective quantitative detection of anti-HBc was possible using the Eu (III) chelate microparticle-based LFIA strips. The strips will provide diagnostic value for clinical application.

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.

NLRP3 inflammasomes in macrophages drive colorectal cancer metastasis to the liver

Inflammation and immunity are important determinants of cancer initiation, promotion, and progression to cancer equilibrium or suppression. The NOD-like receptor family pyrin domain containing 3 (NLRP3) is an oligomeric intracellular immune receptor, and the main component of inflammasome. As a widely distributed effector of innate immunity, NLRP3 inflammasome affects development of many cancer types, but its exact role in colorectal cancer (CRC) is controversial. We found that cells with the macrophage (MΦ) marker CD68 and strong NLRP3 expression densely surrounded CRC tissue. The NLRP3 inflammasome was activated in MΦs by MΦ-CRC cell crosstalk; it resulted in faster migration of CRC cells, whereas blocking NLRP3 signaling suppressed CRC cell migration in vitro, and metastatic ability in vivo. NLRP3 signaling activation in MΦs can contribute to CRC cell migration and invasion.

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.

Detection of Janus-activated kinase-1 and its interacting proteins by the method of luminescent oxygen channeling

Janus-activated kinase-1 (JAK1) plays an important role in many signaling pathways, including the JAK–STAT and SOCS pathways. The activation of the JAK1–STAT3 pathway is a key role in tumor inflammation and immunity by promoting inflammation. The detection of its interacting proteins is of great significance in many diseases. Although luminescent oxygen channeling is commonly used in immunoassay technologies, its use in the detection of JAK1 and interacting proteins has not been reported to date. We constructed eukaryotic overexpression plasmids pENTER–JAK1-His and pENTER–STAT3-His and, following single transfection and co-transfection, measured their expression by western blotting. Co-immunoprecipitation and luminescent oxygen channeling were then used to identify the protein interactions. JAK1 and STAT3 proteins were shown to be successfully overexpressed, and co-immunoprecipitation identified their interaction in vitro. We also detected protein interactions between JAK1 and interleukin 4 receptor (IL4R), JAK1 and growth hormone receptor (GHR). In conclusion, we verified that JAK1 and STAT3 interact in vitro using co-immunoprecipitation, and demonstrated for the first time the potential application of luminescent oxygen channeling for detecting JAK1 and its interacting proteins. Also we think this assay could help other researchers find unreported proteins that also have interactions in vitro.