Establishment of fluorescence multi-flow cytometric immunoassay for the simultaneous quantitative detection of six allergen-sIgE antibodies.

BACKGROUND: The in vitro specific IgE (sIgE) assays now commonly used in clinical laboratories are not only time-consuming and expensive but also require many serum samples. To address these limitations, a novel fluorescent microsphere-based multiplex flow cytometric immunoassay was developed. This innovative assay enables rapid and simultaneous quantitative detection of multiple allergen-sIgE antibodies. OBJECTIVE: To establish a new method for the simultaneous quantitative detection of 6 allergen-sIgE antibodies based on fluorescence multiplex flow cytometry. METHODS: Six different encoded fluorescent microspheres were selected to covalently couple 6 allergens, and their antigen-coupling activities were verified. After optimizing the multiplexing procedure and reaction conditions, including the concentration of microspheres encapsulated by allergens, reaction temperature, and reaction time, standard curves were established to quantify the 6 allergen-sIgE, and their performance was evaluated according to clinical guidelines. RESULTS: The chosen analytical mode was optimized for the detection of the 6 allergens-sIgE for 70 minutes. The established coefficients of variation for multiplex flow cytometry reproducibility and intermediate precision were less than 10%. Linear regression analysis showed a highly significant quantitative correlation between the results of the multiple analyses of Dermatophagoides pteronyssinus, Dermatophagoides farinae, Artemisia, and cat hair allergens and ImmunoCAP (Thermo Fisher Scientific): the r2 values ranged from 0.85 to 0.97 (P < .0001). In addition, there was a high correlation between the results of the multiplex analysis of dog hair allergens and the capture enzyme-linked immunosorbent assay (r2 = 0.92, P < .0001). CONCLUSION: A high-throughput system called multiplex flow cytometry has been developed for the simultaneous detection of 6 inhalant allergens. The method has the advantage of being rapid and using less serum. Furthermore, it has the potential to be expanded to include other allergens and biologic agents.

Establishment of a homogeneous chemiluminescence immunoassay for cat dander sIgE antibody detection.

BACKGROUND: In vitro specific IgE (sIgE) testing has become an important tool for the diagnosis of IgE-mediated allergic diseases. Current methods used to detect allergen sIgE are time consuming and/or expensive. Therefore, a new method was developed for rapid quantitative detection of cat dander-sIgE antibody based on homogeneous chemiluminescence immunoassay. METHODS: Selection of chemibeads with different chemical groups, and the best Light-initiated chemiluminescence assay (LiCA) analytical mode for cat dander-sIgE detection. To validate and eliminate the interference of IgE on the detection of cat dander-sIgE, concentration of biotinylated anti-human IgE antibody was optimized. For quantification of cat dander-sIgE, a calibration curve was established, and the performance of the assay was evaluated according to clinical guidelines. RESULTS: Indirect LiCA is the best mode of analysis and biotinylated anti-human IgE antibody at a dilution ratio of 1:250 minimizes IgE interference. The coefficient of variation of the developed LiCA was 1.49% to 4.66%, with an intermediate precision of 6.90% to 8.21%. The LoB, LoD, and LoQ of the assay were 0.023 kUA/L, 0.056 kUA/L and 0.185 kUA/L. The coefficient of correlation (r) between LiCA and ImmounoCAP was 0.9478. CONCLUSIONS: A cat dander-sIgE quantitation assay based on homogeneous chemiluminescence immunoassay was established, which could be a new reliable analytical tool for the determination of cat dander-sIgE.

Performance evaluation of a laboratory-developed light-initiated chemiluminescence assay for quantification of egg white-specific IgE.

BACKGROUND: Specific IgE (sIgE) testing has become one of the most important tools for diagnosing IgE-mediated food allergy. Enzyme-linked immunosorbent assay (ELISA) and dot-enzyme-linked immunosorbent assay (Dot-ELISA) have been used to measure sIgE in clinical widely. Light-initiated chemiluminescence assay (LICA) is a new method for measuring allergen-sIgE. We aimed to establish a LICA method for quantitative detection of egg white-sIgE and evaluate its performances. METHODS: The best chemibeads coupling method in detecting egg white-sIgE was selected, and a LICA method for quantitative detection of egg white-sIgE was established. The precision study was performed according to Clinical and Laboratory Standards Institute (CLSI) EP5-A2. Detection capability which contains limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) was evaluated according to National Health Commission of the People's Republic of China (NHC) WS/T 514-2017. Linear range was evaluated according to CLSI EP6-A. All data were analyzed using SPSS software. RESULTS: Precision contains repeatability and intermediate precision. The CV of repeatability ranged from 2.72% to 7.29%, and the CV of intermediate precision ranged from 4.93% to 8.64%. The LoB, LoD, and LoQ of the assay were 0.000 kUA/L, 0.053 kUA/L, and 0.076 kUA/L. The assay linear range was 0.076-34.125 kUA /L (r = 0.9979 ≥ 0.9900). CONCLUSION: This laboratory-developed LICA method can detect egg white-sIgE, and performance meets clinical requirements. This method shows rapid turnaround cycles and high sensitivity. It can be used as an alternative method for clinical detection of egg white-sIgE.

Quantitative Detection of House Dust Mites-Specific IgE by Light-Initiated Chemiluminescence Assay.

OBJECTIVE: To establish a new method for quantitative detection of house dust mite (HDM)-sIgE based on light-initiated chemiluminescence assay (LiCA). METHODS: The assay was established after optimizing the reaction conditions, and the assay performance was evaluated according to the clinical guidelines. Further, the results of LiCA were compared with those from the ELISA and ImmunoCAP methods. RESULTS: Coefficients of variation for repeatability ranged from 4.22% to 7.69%, and intermediate precision from 8.38% to 10.34%. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) were 0.066 kUA/L, 0.165 kUA/L, and 0.171 kUA/L, respectively. The coefficient of correlation (r) between the results of LiCA and ELISA was 0.9263, and the r between the results of LiCA and ImmunoCAP was 0.8870. CONCLUSIONS: A HDM-sIgE quantitation assay based on LiCA was established, which could be used as a new reliable analytical tool for the determination of HDM-sIgE.

A Highly Sensitive Dual-Drive Microfluidic Device for Multiplexed Detection of Respiratory Virus Antigens.

Conventional microfluidic systems that rely on capillary force have a fixed structure and limited sensitivity, which cannot meet the demands of clinical applications. Herein, we propose a dual-drive microfluidic device for sensitive and flexible detection of multiple pathogenic microorganisms antigens/antibodies. The device comprises a portable microfluidic analyzer and a dual-drive microfluidic chip. Along with capillary force, a second active driving force is provided by a removable self-driving valve in the waste chamber. The interval between these two driving forces can be adjusted to control the reaction time in the microchannel, optimizing the formation of antigen-antibody complexes and enhancing sensitivity. Moreover, the material used in the self-driving valve can be changed to adjust the active force strength needed for different tests. The device offers quantitative analysis for respiratory syncytial virus antigen and SARS-CoV-2 antigen using a 35 µL sample, delivering results within 5 min. The detection limits of the system were 1.121 ng/mL and 0.447 ng/mL for respiratory syncytial virus recombinant fusion protein and SARS-CoV-2 recombinant nucleoprotein, respectively. Although the dual-drive microfluidic device has been used for immunoassay for respiratory syncytial virus and SARS-CoV-2 in this study, it can be easily adapted to other immunoassay applications by changing the critical reagents.

Distribution Characteristics and Restoration Application of Vegetation in Chengcun Bay Surrounding Areas of Yangjiang City.

In recent years, global warming and sea level rise have further aggravated the risk of coastal erosion. Coastal vegetation plays an important role in resisting storm surges and alleviating coastal erosion. Therefore, screening plant species for the purpose of constructing ecological seawalls to protect or repair damaged coastal zones has become a hot issue. In this paper, a field survey was conducted to investigate the vegetation in Chengcun Bay surrounding areas of Yangjiang City by combining a line survey and sample plot survey. By understanding the vegetation types, distribution and community structure in the bay's surrounding areas and analyzing the restricting environmental factors of those plants, we put forward some countermeasures for coastal vegetation restoration in difficult site conditions from the aspects of plant species selection, vegetation configuration and restoration technology, so as to provide reference for ecological vegetation restoration in similar locations.

The Effect of Domestic Sewage Treatment on Islands Using Ecological Treatment Processes: A Case Study of Haimen Island, Fujian Province.

Islands are characterized by a lack of land and freshwater resources, public finances, and technical personnel. As a result, domestic sewage may not be effectively treated, which can lead to major pollution on islands and in the surrounding sea areas. In this study, a pilot treatment of domestic sewage was conducted using an ecological treatment process (i.e., a constructed wetland and ecological pond) in an abandoned pond located on Haimen Island, Fujian Province, China. The pollutant indicators were monitored to analyze this treatment method at different treatment stages. The results showed that the combination of multiple ecological treatment processes had favorable treatment effects on various pollutants in the sewage. The treatment rates of the chemical oxygen demand (CODCr) and suspended solids (SS) surpassed 88%. The treatment rate of the biochemical oxygen demand (BOD5), ammonia nitrogen (NH3-N), total nitrogen (TN), total phosphorus (TP), and fecal coliform surpassed 93%, and all the indicators met or were close to the level I B emission standards for urban sewage treatment plants. Different treatment stages have different treatment effects on different pollutants. The constructed wetland played an important role in sewage treatment through plant absorption, substrate adsorption, sedimentation, and microbial decomposition, particularly for the TP, CODCr, and BOD5. In contrast, algal photosynthesis in the ecological pond produced a large amount of dissolved oxygen, and the treatment effect was highest for the TN and NH3-N. The treatment effects on the fecal coliform in the constructed wetland and ecological pond were very significant. Ecological treatment processes based on the combination of a constructed wetland and ecological pond have favorable treatment effects, low construction and maintenance costs, and pollution-free conditions, which are suitable for application in island areas.

Analysis of the characteristics of Eco-Exergy-based indices and diversity indices in the Zhoushan archipelago, China.

Anthropogenic disturbance of coastal ecosystems has increased the need for comprehensive methods and indicators to assess the health status of marine ecosystems. Biotic indices are important as they indicate different ecological states. In this study, Eco-Exergy-based indices (Exergy and Structural Exergy) and diversity indices (Shannon-Wiener index and Margalef index) were applied in the seas around the Zhoushan archipelago, and correlation analysis between biotic indices and environmental factors was used to test the robustness of these indices in capturing the environment changes, aimed to provide reference in selecting the indices for assessing marine eco-environment quality. Results showed that the benthic indices of Exergy, Shannon-Wiener, and Margalef increased with the distance from the mainland. The Eco-Exergy-based indices and Margalef index were significantly, negatively correlated with dissolved inorganic nitrogen and reactive phosphate, and significantly, positively correlated with the salinity of seawater. Overall, the marine ecosystem was more stable further away from the coast, where salinity was higher, and pollutants such as nutrients in seawater were lower. In addition, biodiversity and Eco-Exergy was also higher. Generally, Eco-Exergy-based indices were observed to be good indicators for detecting the environment changes in the study area.