Virus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

新型冠状病毒SARS-CoV-2在全球蔓延，给全球公共卫生带来严重威胁。快速研制疫苗、抗体和治疗药物成为科学界面临的重大挑战。由于SARS-CoV-2的高度传染性，采用病毒感染模型进行中和抗体及小分子抑制剂的药效评估需要在高等级生物安全实验室中进行，且常需要数天时间才能完成检测，限制了抗体和药物筛选的效率。发展快速、可视、不依赖于活病毒的新冠病毒入胞检测探针和细胞模型，对于加速新冠病毒抗体和药物的研究有重要意义。夏宁邵教授团队通过CHO真核表达系统高效表达制备出C端融合抗酸荧光蛋白Gamillus的重组新冠病毒spike蛋白STG。STG经SEC分子筛和冷冻电镜确认呈现与天然病毒刺突高度相似的三聚体结构，且与ACE2有很高的亲和力（18.2nM）。STG具备良好的细胞相容性和荧光性质，研究者进一步开发了可定量测定感染恢复期血清、疫苗免疫血清中和抗体（入胞阻断抗体）水平的CSBT检测方法。除了抗体检测评估方面的应用外，该研究发展的探针和模型还可用于筛选分析抑制新冠病毒入胞及胞内转运的小分子化合物。 我校博士后张雅丽，博士生王邵娟、巫洋涛，博士后侯汪衡、袁伦志和深圳市第三人民医院沈晨光博士为共同第一作者。厦门大学夏宁邵教授、袁权教授、程通教授为该论文共同通讯作者。The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system,a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed.In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.This study was supported by National Natural Science Foundation of China (81993149041 for N.X.; 81902057 for Y.Z.; 81871316 and U1905205 for Q.Y.), the National Science and Technology Major Project of Infectious Diseases (No. 2017ZX10304402‐002‐003 for T.C. and No. 2017ZX10202203‐009 for Q.Y.), the National Science and Technology Major Projects for Major New Drugs Innovation and Development (No. 2018ZX09711003‐005‐003 for T.C.), the Science and Technology Major Project of Fujian (2020YZ014001), the Science and Technology Major Project of Xiamen (3502Z2020YJ01), and the Guangdong Basic and Applied Basic Research Foundation (2020A1515010368 for C.S.). 该研究得到了国家自然科学基金、传染病防治国家科技重大专项、福建省应急科技攻关项目和厦门应急科技攻关项目的支持

Development of an ELISA and Immunochromatographic Strip for Highly Sensitive Detection of Microcystin-LR

A monoclonal antibody for microcystin–leucine–arginine (MC-LR) was produced by cell fusion. The immunogen was synthesized in two steps. First, ovalbumin/ bovine serum albumin was conjugated with 6-acetylthiohexanoic acid using a carbodiimide EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)/ NHS (N-hydroxysulfosuccinimide) reaction. After dialysis, the protein was reacted with MC-LR based on a free radical reaction under basic solution conditions. The protein conjugate was used for immunization based on low volume. The antibodies were identified by indirect competitive (ic)ELISA and were subjected to tap water and lake water analysis. The concentration causing 50% inhibition of binding of MC-LR (IC50) by the competitive indirect ELISA was 0.27 ng/mL. Cross-reactivity to the MC-RR, MC-YR and MC-WR was good. The tap water and lake water matrices had no effect on the detection limit. The analytical recovery of MC-LR in the water samples in the icELISA was 94%–110%. Based on this antibody, an immunochromatographic biosensor was developed with a cut-off value of 1 ng/mL, which could satisfy the requirement of the World Health Organization for MC-LR detection in drinking water. This biosensor could be therefore be used as a fast screening tool in the field detection of MC-LR

Fabrication of Superhydrophobic Coating Based on Waterborne Silicone-Modified Polyurethane Dispersion and Silica Nanoparticles

In this work, eco-friendly superhydrophobic coatings were prepared by dispersing hydrophobic silica nanoparticles and a waterborne silicone-modified polyurethane dispersion into an ethanol solution, which was free of fluorine and volatile toxic solvents. The effects of the silica content on the hydrophobicity and scratch resistance of the hydrophobic surfaces were investigated by WCA measurements and a sandpaper abrasion test, respectively. The experimental results indicated that when the silica content exceeded 30% by mass, the silica/silicone-modified polyurethane coatings had superhydrophobicity. Meanwhile, the superhydrophobic coatings with a silica content of 30% by mass simultaneously had the optimal mechanical stability. We studied the morphology and roughness of the hydrophobic surfaces with different silica content and attempted to briefly explain the influence mechanism of silica content. Furthermore, anti-icing and oil–water separation experiments were carried out on the superhydrophobic coatings, which exhibited good anti-icing performance and high separation efficiency. The eco-friendly superhydrophobic coating is expected to be applied in the fields of oil–water separation, anti-icing, and self-cleaning, etc

A Rapid and Semi-Quantitative Gold Nanoparticles Based Strip Sensor for Polymyxin B Sulfate Residues

Increasing attention is now being directed to the utilization of polymyxin B (PMB) as a last-line treatment for life-threatening infections caused by multidrug resistant Gram-negative bacteria. Unfortunately, polymyxins resistance is also increasingly reported, leaving a serious threat to human health. Therefore, the establishment of rapid detection methods for PMB residues is highly essential to ensure public health. In this study, two monoclonal antibodies (mAb; 2A2 and 3C6) were obtained using PMB-bovine serum albumin as the immunogen and PMB-ovalbumin as the coating antigen, which were prepared with N-(γ-maleimidobutyryloxy) succinimide ester and glutaraldehyde as cross-linking agents, respectively. Through an indirect competitive enzyme-linked immunosorbent assay, resultant two mAbs were compared and the results indicated that 3C6 showed higher sensitivity with a half maximum inhibition concentration of 13.13 ng/mL. Based on 3C6, a gold nanoparticles (AuNPs)-based immunochromatographic test (ICT) strip was then established, the mechanism of which is that free PMB competes with the fixed coating antigen to combine with mAb labeled by AuNPs. Using ICT strip to detect milk and animal feed samples revealed the visible detection limits were 25 ng/mL and 500 μg/kg, respectively and the cutoff limits were 100 ng/mL and 1000 μg/kg, respectively. The ICT strip provides results within 15 min, facilitating rapid and semi-quantitative analysis of PMB residues in milk and animal feed

Development of an Immunochromatographic Strip Test for Rapid Detection of Ciprofloxacin in Milk Samples

A rapid, simple, and sensitive immunochromatographic test strip has been developed for testing residues of ciprofloxacin (CIP). A specific and sensitive monoclonal antibody (mAb) for CIP was generated by immunizing BALB/c mice with well-characterized CIP-Keyhole limpet haemocyanin. Under the optimized conditions, the cut-off limits of test strips for CIP were found to be 5 ng/mL in phosphate-buffered saline and 2.5 ng/mL in milk samples. Each test can be evaluated within 3 min. The cross-reactivities of the CIP test strip to enrofloxacin (ENR), norfloxacin (NOR), nadifloxacin (NDF), danofloxacin (DANO), pefloxacin (PEX), lomefloxacin (LOME), enoxacin (ENO), and sarafloxacin (SAR) were 71.4%, 71.4%, 66%, 50%, 33%, 20%, 12.5%, and 6.25%, respectively. The data indicate that the method is sensitive, specific, and has the advantages of simplicity and speed, therefore, this test strip is a useful screening method for the detection of CIP residues in milk samples

Detection of β-Lactamase Residues in Milk by Sandwich ELISA

β-Lactamase residues in milk represent a public health risk. The cylinder plate detection method, which is based on bacterial growth, is laborious and time consuming. In this study, 15 monoclonal antibodies (mAbs) were selected against Temoneira (TEM) 1 β-lactamase. A sandwich enzyme-linked immunosorbent assay (ELISA) based on an optimum mAb pair was developed and validated for the detection of β-lactamase. The limit of detection and linear dynamic range of the method were 4.17 ng/mL and 5.5–100 ng/mL, respectively. β-Lactamase recovery in pure milk was 96.82–103.13%. The intra- and inter-assay coefficients of variation were 6.21–7.38% and 12.96–13.74%, respectively. Our developed sandwich ELISA can be used as a rapid detection method of β-lactamase in milk

Three-dimensional study of grain scale tensile twinning activity in magnesium: a combination of microstructure characterization and mechanical modeling

Tensile twinning is a main deformation mode in hexagonal close packed structure metals, so it is important to comprehensively understand twinning mechanisms which are not fully disclosed using 2D or small volume 3D characterization techniques. A large area 3D electron backscatter diffraction (EBSD) measurement and crystal plasticity modeling were carried out to investigate the tensile twinning behaviors in a magnesium (Mg) alloy. The results showed that tensile twinning activity was underestimated using conventional 2D EBSD scans. When compressed to yield point, the examined twin frequency with 2D was lower than that using 3D EBSD. The effects of Schmid factor (SF) on twinning were investigated. Almost all high Schmid factor (SF>0.35) grains were twinned. A surprising high twin frequency of 82% in middle SF (0.35>=SF>=0.15) grains was observed, which was unexpected since the middle SF grains were believed to be unfavorable for twinning. The twin frequency in low SF (SF<0.15) grains was slightly increased from 2D to 3D EBSD due to the small volume of twins. The shear stress maintained a high level and was homogeneously distributed in high SF grains, facilitating twin nucleation and growth. The shear stress was distributed heterogeneously within the middle SF grains, and twins were nucleated within areas with positive shear stress. The shear stress in low SF grains was not favorable for twinning and twins occurred in the vicinity of stress accumulation. Twinning activities in the same grain varied on different layers. It was attributed to the stress fluctuation derived from grain environment changes