Determination of echinococcosis IgG antibodies using magnetic bead-based chemiluminescence immunoassay.

Echinococcosis is a kind of parasitic disease shared by humans and animals. The aim of this study was to establish a new detection method for echinococcosis screening using magnetic bead-based chemiluminescence immunoassay (CLIA). A magnetic bead-based CLIA to determine anti-echinococcosis IgG antibodies was optimized and established. The sensitivity, accuracy, precision and recovery rate were evaluated using the national reference serum, and the reference interval, specificity and comparison assays were performed using the clinical negative/positive echinococcosis serum samples. This study established a new CLIA to determine anti-echinococcosis IgG antibodies. The sensitivity of this CLIA method was higher than that of the registered ELISA kit and the national standard, the conformance rate of the negative/positive references was 100% (8/8), the CVs of the sensitivity reference were all below 5%, and the CVs of the precision reference were 5.7%. There was no obvious cross-reactivity with the common parasitic disease-positive serum and serum interferents. Clinical sample testing found that the cutoff value of this CLIA was 5537.15 (RLU), and there was no significant difference between the CLIA method and the registered ELISA kit. This study established a fully automated CLIA method with high sensitivity, specificity, accuracy, precision, recovery rate, and satisfactory clinical testing performance, which may provide a new choice for echinococcosis screening.

A New Method for Early Screening of Gastric Cancer (G17 and CA724 Dual-Labeled Time-Resolved Fluorescence Immunoassay).

Gastric carcinoma (GC) is one of the most common malignancies in the world with the great early screening challenges. The study is aimed at establishing a new detection method for early screening GC using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of gastrin-17 (G-17) and carbohydrate antigen 724 (CA724) in serum. Time-resolved analyzer measured the fluorescence intensity. The standards of G-17/CA724 were used for drawing the standard curve, which is used to calculate the concentration of G-17 and CA724 in serum sample. The sensitivity for G-17 was 0.54 pg/mL and for CA724 was 0.28 U/mL with a wide-range analyze concentration (0.1-1000) pg/mL or U/mL. The average recoveries ranged from 100.52% to 110.30% for G-17 and 103.02% to 116.00% for CA724. All CVs of the intra- and interassay were below 10% with high specificity. There was a high Pearson coefficient between this TRFIA method and the commercially available kits (Pearson r 0.9117 for G-17 and 0.9449 for CA724). Additionally, the cutoff value was 88.41 pg/mL and 5.47 U/mL for CA724 in health subjects. This study established a TRFIA method for simultaneous detection of the concentrations of G-17 and CA724 in serum, which provide a new method for sensitive, accurate, and specific early screening of gastric cancer.

Determination of human COVID-19 total antibodies in serum using a time-resolved fluorescence immunoassay.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading rapidly around the world. Antibody detection plays an important role in the diagnosis of COVID-19. Here, we established a new time-resolved fluorescence immunoassay (TRFIA) to determine COVID-19 total antibodies. A double-antigen sandwich TRFIA was optimized and established: recombinant nucleocapsid phosphoprotein (N protein) and spike protein (S protein) of COVID-19 immobilized on 96-well plates captured human COVID-19 antibodies and then banded together with the N/S proteins labeled with europium(III) (Eu3+ ) chelates, and finally, time-resolved fluorometry was used to measure the fluorescence values. We successfully established a TRFIA method for the detection of human COVID-19 total antibodies, and the cutoff value was 2.02. There was no cross-reactivity with the negative reference of the National Reference Panel for IgM and IgG antibodies to COVID-19. The CV of the precision assay was 3.19%, and the assay could be stored stably for 15 days at 37°C. Compared with that of the colloidal gold method and chemiluminescence method, the sensitivity of the TRFIA method was higher, and the false positive/negative rate was lower. This established TRFIA has high sensitivity, accuracy, and specificity, which indicates that this method provides a new detection method for the high-throughput routine diagnosis of COVID-19.

A New Method for Detection African Swine Fever Virus: Time-resolved Fluorescence Immunoassay.

African swine fever (ASF) has severely influenced the swine industry of the whole world. Fast and accurate African swine fever virus (ASFV) antigen detection is very important for ASF prevention. This study aims to establish a new detection method for detection ASFV antigen using time-resolved fluorescence immunoassay (TRFIA) in the nose and mouth discharge. A double antibody sandwich TRFIA method was optimized and established. Recombinant P30 recombinant antigen was captured by its antibodies immobilized on 96-well plate, and then banded together with another detection antibodies labeled with Europium(III) (Eu3+) chelates, finally time-resolved analyzer measured the fluorescence intensity. The performance of this TRFIA (sensitivity, specificity and accuracy) was evaluated using the clinical samples and compared with the nucleic acid testing method. The sensitivity of this TRFIA was 0.015 ng/mL (dynamic range 0.24-500 ng/mL) with high specificity. The recovery ranged from 92.00 to 103.62 %, the inter-assay CVs ranged from 5.50 to 11.96 %, and the intra-assay CVs was between 5.20 and 10.53 %. Additionally, the cutoff value was 0.016. TRFIA took only 45 min to generate results, and its detection capability comparable to the nucleic acid detection. This study developed a TRFIA method that could be used for qualitative/quantitative detection of ASFV antigen in pigs nasal discharge, which has high sensitivity, specificity and accuracy. This TRFIA provides a new method for rapidly screening ASFV infection in pigs industry.

Spatiotemporal Resolved Live Cell Membrane Tracking through Photo-click Reactions Enriched in Lipid Phase.

A set of photo-switchable monopeptides derived from cis-ß-dibenzodiazocine-l-alanine (cis-DBDAA) have been designed and synthesized, which are capable of photo-click reacting with diaryltetrazoles or diarylsydnones in a hydrophobic phospholipid bilayer environment. The DBDAA monopeptides include both a hydrophobic tail on C-terminal, providing high affinity toward lipid membrane, and a modularized functional moiety on N-terminal, enabling rapid optimization of the self-assembly strength to form multifunctional supramolecules. With the cis-DBDAA monopeptides photo-switched into trans-configuration, we were able to disrupt the supramolecular assembly through an efficient photo-click reaction across the lipid bilayer of liposomes. We reveal that the performance of the photo-click reactions between the monopeptides and photo-generated nitrile imine intermediates is significantly enhanced by enrichment of both reactants in the hydrophobic membrane lamel of liposomes. Enrichment of the DBDAA monopeptide in lipid phase serves as a convenient method to introduce bioorthogonal chemical handles on live cell membranes, which enables fluorescence labelling of single cell's membrane with high spatiotemporal resolution to facilitate the studies on cell membrane dynamics.

Determination of parvovirus antigen in the vaccine using time-resolved fluorescence immunoassay.

As a highly contagious and potentially fatal disease of dogs, canine parvovirus type 2 (CPV-2) usually causes severe myocarditis and gastroenteritis, while vaccine injection has greatly reduced the incidence of CPV-2 diseases. However, there is currently a lack of simple and effective method for quantitative detection of CPV-2 in vaccine. Therefore, this study aims to prepare an accurate method to determine the CPV-2 antigen (CPV-2-Ag) in vaccine. Here, a sandwich time-resolved fluorescence immunoassay (TRFIA) was established and optimized. Anti-CPV-2 antibodies were immobilized on 96-well plates to capture CPV-2-Ag, and then bound together with the detection antibodies labeled with Europium(III) (Eu3+ ) chelates; finally, time-resolved fluorometry was employed to measure the fluorescence intensity. Vaccination was performed to evaluate the relationship between CPV-2-Ag concentration and antibody titer. The sensitivity is 1.15 mEU/mL (LogY = 1.524 + 0.8667 × LogX, R2  = 0.9933), and the average recovery is among 91.00% to 106.39% without cross-reactions with the other canine viral antigen. The correlation between ELISA assay and this method is up to 0.9861. And, there is high correlation between the CPV-2-Ag concentration and antibody titers (R2  = 0.9234). This immunoassay established has high sensitivity, accuracy, and specificity, which indicate that this method could be suitable for quantitative detection of CPV-2-Ag in vaccine evaluation.

Photoisomerization-enhanced 1,3-dipolar cycloaddition of carbon-bridged octocyclic azobenzene with photo-released nitrile imine for peptide stapling and imaging in live cells.

A photo-induced 1,3-dipolar cycloaddition between nitrile imine and highly ring-strained N[double bond, length as m-dash]N double bond as a dipolarophile was discovered. The photo-isomerization of carbon-bridged octocyclic azobenzene (CBOA) into its trans-configuration accelerates the ligation reaction at a very rapid rate (28 400 M-1 s-1). The CBOA-based photo-click reaction was proved to be bioorthogonal. In addition, the NoxaB peptide was successfully cross-linked by a CBOA stapler which plays a dual role: photo-control of the conformation of the peptide and photo-conjugation of probes in live cells.

The {V4Nb6O30} cluster: a new type of vanadoniobate anion structure.

A novel {V(4)Nb(6)O(30)} addenda: Through the successful introduction of a vanadium atom into the polyoxoniobate system, three novel compounds containing the same unprecedented mixed addenda vanadoniobate cluster {V(4)Nb(6)O(30)} were obtained. The incorporation of suitable atoms like V is an intriguing subject providing vast possibilities to enrich polyoxoniobate chemistry.

An unprecedented vanadoniobate cluster with 'trans-vanadium' bicapped Keggin-type {VNb12O40(VO)2}.

The first organic-inorganic hybrid vanadoniobate cluster compounds with a bicapped Keggin-type {VNb(12)O(40)(VO)(2)} core have been synthesized and characterized.