Research progress in methods for detecting neutralizing antibodies against SARS-CoV-2.

The emergence of SARS-CoV-2 has seriously affected the lives of people worldwide. Clarifying the attenuation rule of SARS-CoV-2 neutralizing antibody (NAb) in vivo is the key to prevent reinfection and recurrence of virus. Currently, the commonly used methods for detecting NAb include virus neutralization tests, pseudovirus neutralization assays, lateral flow immunochromatography and enzyme-linked immunosorbent assays. The detection of NAb not only can be used to evaluate the level of immunity after vaccination or infection but also can provide important theoretical support for virus reinfection, recurrence and vaccine iteration. In this research, the related technologies of SARS-CoV-2 NAb detection were reviewed, aiming to provide better research ideas for SARS-CoV-2 epidemic prevention and control.

Highly effective stabilization of Cd and Cu in two different soils and improvement of soil properties by multiple-modified biochar.

Heavy metal contamination in soil has attracted great attention worldwide. In situ stabilization has been considered an effective way to remediate soils contaminated by heavy metals. In the present research, a multiple-modified biochar (BCM) was prepared to stabilize Cd and Cu contamination in two different soils: a farmland soil (JYS) and a vegetable soil (ZZS). The results showed that BCM was a porous-like flake material and that modification increased its specific surface area and surface functional groups. The incubation experiment indicated that BCM decreased diethylenetriaminepentaacetic (DTPA)-extractable Cd and Cu by 92.02% and 100.00% for JYS and 90.27% and 100.00% for ZZS, respectively. The toxicity characteristic leaching procedure (TCLP)-extractable Cd and Cu decreased 66.46% and 100.00% for JYS and 46.33% and 100.00% for ZZS, respectively. BCM also reduced the mobility of Cd and Cu in soil and transformed them to more stable fractions. In addition, the application of BCM significantly increased the soil dehydrogenase, organic matter content and available K (p < 0.05). These results indicate that BCM has great potential in the remediation of Cd- and Cu-contaminated soil.

Gypenosides improve diabetic cardiomyopathy by inhibiting ROS-mediated NLRP3 inflammasome activation.

NLRP3 inflammasome activation plays an important role in diabetic cardiomyopathy (DCM), which may relate to excessive production of reactive oxygen species (ROS). Gypenosides (Gps), the major ingredients of Gynostemma pentaphylla (Thunb.) Makino, have exerted the properties of anti-hyperglycaemia and anti-inflammation, but whether Gps improve myocardial damage and the mechanism remains unclear. Here, we found that high glucose (HG) induced myocardial damage by activating the NLRP3 inflammasome and then promoting IL-1ß and IL-18 secretion in H9C2 cells and NRVMs. Meanwhile, HG elevated the production of ROS, which was vital to NLRP3 inflammasome activation. Moreover, the ROS activated the NLRP3 inflammasome mainly by cytochrome c influx into the cytoplasm and binding to NLRP3. Inhibition of ROS and cytochrome c dramatically down-regulated NLRP3 inflammasome activation and improved the cardiomyocyte damage induced by HG, which was also detected in cells treated by Gps. Furthermore, Gps also reduced the levels of the C-reactive proteins (CRPs), IL-1ß and IL-18, inhibited NLRP3 inflammasome activation and consequently improved myocardial damage in vivo. These findings provide a mechanism that ROS induced by HG activates the NLRP3 inflammasome by cytochrome c binding to NLRP3 and that Gps may be potential and effective drugs for DCM via the inhibition of ROS-mediated NLRP3 inflammasome activation.

[Research advance of lateral flow assay labels].

Lateral flow assay is widely used in the point-of-care testing on-site and in-home testing with the advantage of being simple, rapid, sensitive and cost-effective. Proper labels are the key factors in lateral flow assay. Traditional labels include colloidal gold, selenium nanoparticle, and carbon nanoparticle, among which the colloidal gold is most commonly used. Lateral flow assay has been improved as a result of the discovery of new labels, such as quantum dots and nanozyme recently. Meanwhile, transformation of qualitative detection to quantitative detection is gradually realized. This article aims at introducing the most often used and the latest lateral flow assay labels, providing a basis theoretical investigation on screening proper labels for lateral flow assay researchers.

Precision detection of hepatocellular carcinoma-associated telomerase RNA with SA@Comb-HCR nanosystem.

Accurate intracellular visualization of human telomerase RNA (hTR) is imperative for early diagnosis and treatment monitoring of hepatocellular carcinoma (HCC). While isothermal amplification-based DNA cascade strategies are promising, challenges persist in achieving great intake efficiency of detection probes within tumor cells and enhancing intracellular reaction efficiency. This study introduces a SA@Comb-HCR nanosystem, a highly effective approach for in situ hTR detection in HCC cells. Sodium alginate-coated liposomes ensures efficient nanoprobe delivery, which are then combined with proximity effect-inspired signal amplification. The coating of sodium alginate facilitates receptor-mediated endocytosis, prevents serum protein adhesion, and mitigates cationic liposome cytotoxicity. The designed Comb-like consolidated hairpin probe enhances the concentration of the local reactant, resulting in cascade amplification upon hTR activation. This technique achieves precision detection of intracellularly overexpressed hTR in HCC cells with a remarkable detection limit of 0.7 pM. This approach holds great promise for advancing targeted and sensitive early clinical diagnosis of HCC.

Reversal effect of arsenic sensitivity in human leukemia cell line K562 and K562/ADM using realgar transforming solution.

The success of arsenic trioxide (ATO) in treatment of acute promyelocytic leukemia (APL) attracts a great deal of attention to researchers to explore its activity of anti-leukemia. However, ATO has unavailable effect on chronic myeloid leukemia (CML), especially multidrug resistant (MDR)-CML, unless using high concentration. Realgar (As(4)S(4)) has been employed in Chinese traditional medicine for 1500 years. Research evidences confirmed realgar has similar effect on treating with APL as ATO, but the problem of large dose and long period in the CML/MDR-CML treatment still exist. By using a microbial leaching process with Acidithiobacillus ferrooxidans, we obtained realgar transforming solution (RTS) which showed significantly higher extent in inhibiting CML cell line K562 and MDR-CML cell line K562/ADM, and then trigger apoptosis. Both K562 and K562/ADM showed arsenic-dose-dependent effect on RTS. Interestingly, the overexpression of MDR1 mRNA and P-glucoprotein (P-gp) in K562/ADM cells were down-regulated by RTS, where there are no obvious effects on ATO and realgar and arsenic can be subsequently accumulated in K562/ADM cells efficiently. The intracellular accumulation of arsenic in K562/ADM cells treated with RTS for 4 h was 2-fold and 16-folds higher than those treated with realgar or ATO. Meanwhile, Western blot analysis of AQP9, the main transporter of arsenic, was increased by RTS treatment particularly in K562/ADM. Thus, these results suggested that the effect from a certain arsenical or a variety of arsenicals in RTS might be a promising candidate both for treating CML/MDR-CML alone and as combinations with currently used anti-CML/MDR-CML drug, although arsenical forms in RTS are undefined.

Development of an Effective Neutralizing Antibody Assay for SARS-CoV-2 Diagnosis.

Introduction: Neutralizing antibodies (NAbs) are essential for preventing reinfection with SARS-CoV-2 and the recurrence of COVID-19; nonetheless, the formation of NAbs following vaccination and infection remains enigmatic due to the lack of a practical and effective NAb assay in routine laboratory settings. In this study, we developed a convenient lateral flow assay for the rapid and precise measurement of serum NAb levels within 20 minutes. Methods: Receptor-binding domain-fragment crystallizable (RBD-Fc) and angiotensin-converting enzyme 2-histidine tag (ACE2-His) were expressed by the eukaryotic expression systems of Spodoptera frugiperda clone 9 and human embryonic kidney 293T, respectively. Then, colloidal gold was synthesized and conjugated with ACE2. After optimizing various operating parameters, an NAb lateral flow assay was constructed. Subsequently, its detection limit, specificity, and stability were systematically evaluated, and clinical samples were analyzed to validate its clinical feasibility. Results: RBD-Fc and ACE2-His were obtained with 94.01% and 90.05% purity, respectively. The synthesized colloidal gold had a uniform distribution with an average diameter of 24.15 ± 2.56 nm. With a detection limit of 2 µg/mL, the proposed assay demonstrated a sensitivity of 97.80% and a specificity of 100% in 684 uninfected clinical samples. By evaluating 356 specimens from infected individuals, we observed that the overall concordance rate between the proposed assay and conventional enzyme-linked immunosorbent assay was 95.22%, and we noticed that 16.57% (59/356) of individuals still did not produce NAbs after infection (both by ELISA and the proposed assay). All the above tests by this assay can obtain results within 20 minutes by the naked eye without any additional instruments or equipment. Conclusion: The proposed assay can expediently and reliably detect anti-SARS-CoV-2 NAbs after infection, and the results provide valuable data to facilitate effective prevention and control of SARS-CoV-2. Clinical trial registration: Serum and blood samples were used under approval from the Biomedical Research Ethics Subcommittee of Henan University, and the clinical trial registration number was HUSOM-2022-052. We confirm that this study complies with the Declaration of Helsinki.

Substrate-directed divergent synthesis of fused indole polycycles through Rh(II)-catalyzed cascade reactions of bis(diazo)indolin-2-ones.

Herein, we report a substrate-directed diverse synthetic strategy toward two kinds of structurally intriguing fused indole polycycles through Rh(II)-catalyzed cascade reactions of bis(diazo)indolin-2-ones with enaminones. The subtle structural changes in enaminones lead to different reactivities. Cyclic enaminones produce indolo[2,3-b]indoles, whereas acyclic ones afford oxazolo[3,2-a]indoles.

Development of a Point-of-Care Test Based on Selenium Nanoparticles for Heart-Type Fatty Acid-Binding Proteins in Human Plasma and Blood.

Purpose: A rapid, convenient, cost-effective in-home test method for identifying heart-type fatty acid-binding protein (H-FABP) in plasma and blood by a lateral-flow immunoassay (LFIA) based on selenium nanoparticles (SeNPs) was developed. Methods: SeNPs were synthesized by using L-ascorbic acid to reduce seleninic acid at room temperature and conjugated with an anti-H-FABP monoclonal antibody. The limit of detection, specificity, and stability were measured, and clinical samples were analyzed. Results: The SeNPs were spherical with a diameter of 39.48 ± 3.72 nm and were conjugated successfully with an anti-H-FABP antibody, resulting in a total diameter of 46.52 ± 2.95 nm. The kit was designed for the determination of H-FABP in plasma specimens and whole blood specimens. The limit of detection was 1 ng/mL in plasma and blood, and the results could be determined within 10 min. No cross-reaction occurred with cardiac troponin I, creatine kinase-MB or myoglobin. The kits were stored at 40 °C for up to 30 days without significant loss of activity. The sensitivity was determined to be 100%, the specificity 96.67%, and the overall coincidence rate 97.83%. Conclusion: This SeNP assay kit can conveniently, rapidly, and sensitively detect H-FABP in plasma or blood with a readout of a simple color change visible to the naked eye with no special device, and can be used as an auxiliary means for the early screening of AMI. Clinical Trial Registration: Plasma and blood samples were used under approval from the Experimental Animal Ethics committee of the Joint National Laboratory for Antibody Drug Engineering, Henan University. The clinical trial registration number was HUSOM-2019-047.

Rapid Detection of Anti-SARS-CoV-2 Antibody Using a Selenium Nanoparticle-Based Lateral Flow Immunoassay.

Coronavirus disease 2019 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is highly transmissible. Early and rapid testing is necessary to effectively prevent and control the outbreak. Detection of SARS-CoV-2 antibodies with lateral flow immunoassay can achieve this goal. In this study, SARS-CoV-2 nucleoprotein (NP) was expressed and purified. We used the selenium nanoparticle as the labeling probe coupled with the NP to prepare an antibody (IgM and IgG) detection kit. The detection limit, cross reaction, sensitivity and specificity of the kit is verified. Separate detection of IgM and IgG, such as in this assay, was performed in order to reduce mutual interference and improve the accuracy of the test results.The final purity of NP was 91.83%. Selenium nanoparticle and NP successfully combined with stable effect. The LOD of the kit was 20 ng/mL for anti-NP IgG and 60 ng/mL for anti-NP IgM, respectively. The kit does not cross reaction with RF. The sensitivity of the kit was 94.74% and the specificity was 96.23%. The assay kit does not require any special device for reading the results and the readout is a simple color change that can be evaluated with the naked eye. This kit is suitable for rapid and real-time detection of the SARS-CoV-2 antibody IgG and IgM.

Rapid Detection of Ractopamine and Salbutamol in Swine Urine by Immunochromatography Based on Selenium Nanoparticles.

PURPOSE: The purpose of this study was to establish a lateral flow immunoassay using selenium nanoparticles (Se-NPs) as a probe to detect ractopamine (RAC) and salbutamol (SAL) in swine urine. METHODS: SDS and PEG were used as templates to prepare Se-NPs; anti-RAC monoclonal antibodies or anti-SAL monoclonal antibodies were labelled with Se-NPs; and rapid detection kits were prepared. The sensitivity, specificity, and stability were measured, and actual samples were analysed. RESULTS: The Se-NPs were spherical with a diameter of 40.63 ± 5.91 nm, and were conjugated successfully with an anti-RAC antibody to give a total diameter of 82.33 ± 17.91 nm. The detection limit of a RAC kit in swine urine was 1 ng/mL, and that of a SAL kit was 3 ng/mL. Both procedures could be completed within 5 minutes. No cross-reaction occurred with clenbuterol, bambuterol and phenylethanolamine A. Samples were tested consistently across different batches of kits for swine urine. The results of the kits were identical to those of actual clinical samples analysed by ELISA, and the coincidence rate was 100%. CONCLUSION: The assay kit does not require any special device for reading the results, and the readout is a simple colour change that can be evaluated with the naked eye. It is easy to operate, sensitive, specific, and stable This kit is suitable for the rapid and real-time detection of RAC and SAL residues in swine urine samples. CLINICAL TRIAL REGISTRATION: Swine urines samples were used under approval from the Experimental Animal Ethics committee of the Joint National Laboratory for Antibody Drug Engineering, Henan University.

Rapid detection of human heart-type fatty acid-binding protein in human plasma and blood using a colloidal gold-based lateral flow immunoassay.

The incidence of acute myocardial infarction (AMI) is currently increasing. Early detection is important for the treatment and prognosis of patients with AMI. Heart-type fatty acid-binding protein (H-FABP) may be used as an early marker of AMI due to its high sensitivity, specificity and prognostic value. Therefore, in the present study, H-FABP was used as a biomarker in a double-antibody sandwich method and colloidal gold-based lateral flow immunoassay to develop a rapid detection kit for H-FABP with a processing time of only 5 min. The sensitivity of the kit in plasma and whole blood was 1 ng/ml and this method had good specificity, exhibiting no cross-reaction with cardiac troponin I, myoglobin or creatine kinase-Mb. The kits had good shelf life and stability, as they were able to be stored at 40˚C for 30 days. A total of 12 clinical samples were collected for detection and the coincidence rate with the ELISA method was up to 91.67%. Therefore, the present study provided a simple, rapid and economical early-detection in-home testing kit.

A point-of-care selenium nanoparticle-based test for the combined detection of anti-SARS-CoV-2 IgM and IgG in human serum and blood.

COVID-19 is a widespread and highly contagious disease in the human population. COVID-19 is caused by SARS-CoV-2 infection. There is still a great demand for point-of-care tests for detection, epidemic prevention and epidemiological investigation, both now and after the epidemic. We present a lateral flow immunoassay kit based on a selenium nanoparticle-modified SARS-CoV-2 nucleoprotein, which detects anti-SARS-CoV-2 IgM and anti-SARS-CoV-2 IgG in human serum, and the results can be read by the naked eye in 10 minutes. We expressed and purified the SARS-CoV-2 nucleoprotein in HEK293 cells, with a purity of 98.14% and a concentration of 5 mg mL-1. Selenium nanoparticles were synthesized by l-ascorbic acid reduction of seleninic acid at room temperature. After conjugation with the nucleoprotein, a lateral flow kit was successfully prepared. The IgM and IgG detection limits of the lateral flow kit reached 20 ng mL-1 and 5 ng mL-1, respectively, in human serum. A clinical study sample comprising 90 COVID-19-diagnosed patients and 263 non-infected controls was used to demonstrate a sensitivity and specificity of 93.33% and 97.34%, respectively, based on RT-PCR and clinical results. No cross-reactions with rheumatoid factor and positive serum for anti-nuclear antibodies, influenza A, and influenza B were observed. Moreover, the lateral flow kit remained stable after storage for 30 days at 37 °C. Our results demonstrate that the selenium nanoparticle lateral flow kit can conveniently, rapidly, and sensitively detect anti-SARS-CoV-2 IgM and IgG in human serum and blood; it can also be suitable for the epidemiological investigation of COVID-19.

1,25(OH)2 D3 alleviates DSS-induced ulcerative colitis via inhibiting NLRP3 inflammasome activation.

1,25-dihydroxyvitamin D3 (1,25(OH)2 D3, VitD3) is the major active ingredient of vitamin D and has anti-inflammatory activity; however, the mechanism for this remains poorly understood. In this study, we found that VitD3 was able to abolish NOD-like receptor protein 3 (NLRP3) inflammasome activation and subsequently inhibit caspase-1 activation and IL-1ß secretion via the vitamin D receptor (VDR). Furthermore, VitD3 specifically prevented NLRP3-mediated apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) oligomerization. In additional to this, NLRP3 binding to NIMA-related kinase 7 (NEK7) was also inhibited. Notably, VitD3 inhibited autophagy, leading to the inhibition of the NLRP3 inflammasome. Uncoupling protein 2-reactive oxygen species signaling may be involved in inflammasome suppression by VitD3. Importantly, VitD3 had both preventive and therapeutic effects on mouse model of ulcerative colitis, via inhibition of NLRP3 inflammasome activation. Our results reveal a mechanism through which VitD3 represses inflammation and prevents the relevant diseases, and suggest a potential clinical use of VitD3 in autoimmune syndromes or other NLRP3 inflammasome-driven inflammatory diseases.

Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats.

Myocardial infarction (MI) is a leading cause of death worldwide for which there is no cure. Although cardiac cell death is a well-recognized pathological mechanism of MI, therapeutic blockade of cell death to treat MI is not straightforward. Death receptor 5 (DR5) and its ligand TRAIL [tumor necrosis factor (TNF)-related apoptosis-inducing ligand] are up-regulated in MI, but their roles in pathological remodeling are unknown. Here, we report that blocking TRAIL with a soluble DR5 immunoglobulin fusion protein diminished MI by preventing cardiac cell death and inflammation in rats, pigs, and monkeys. Mechanistically, TRAIL induced the death of cardiomyocytes and recruited and activated leukocytes, directly and indirectly causing cardiac injury. Transcriptome profiling revealed increased expression of inflammatory cytokines in infarcted heart tissue, which was markedly reduced by TRAIL blockade. Together, our findings indicate that TRAIL mediates MI directly by targeting cardiomyocytes and indirectly by affecting myeloid cells, supporting TRAIL blockade as a potential therapeutic strategy for treating MI.

Inclusion of membrane-anchored LTB or flagellin protein in H5N1 virus-like particles enhances protective responses following intramuscular and oral immunization of mice.

We previously demonstrated that intramuscular immunization with virus-like particles (VLPs) composed of the haemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of A/meerkat/Shanghai/SH-1/2012 (clade 2.3.2.1) protected mice from lethal challenge with viruses from other H5 HPAI clades. The inclusion of additional proteins that can serve as immunological adjuvants in VLPs may enhance adaptive immune responses following vaccination, and oral vaccines may represent the safest choice. Here, we report the generation of H5N1 VLPs composed of the viral HA, NA, and M1 proteins and membrane-anchored forms of the Escherichia coli heat-labile enterotoxin B subunit protein (LTB) or the Toll-like receptor 5 ligand flagellin (Flic). Mice intramuscularly or orally immunized with VLPs containing LTB or Flic generated greater humoural and cellular immune responses than those administered H5N1 VLPs without LTB or Flic. Intramuscular immunization with VLPs protected mice from lethal challenge with homologous or heterologous H5N1 viruses irrespective of whether the VLPs additionally included LTB or Flic. In contrast, oral immunization of mice with LTB- or Flic-VLPs conferred substantial protection against lethal challenge with both homologous and heterologous H5N1 influenza viruses, whereas mice immunized orally with VLPs lacking LTB and Flic universally succumbed to infection. Mice immunized orally with LTB- or Flic-VLPs showed 10-fold higher virus-specific IgG titres than mice immunized with H5N1-VLPs lacking LTB or Flic. Collectively, these results indicate that the inclusion of immunostimulatory proteins, such as LTB and Flic, in VLP-based vaccines may represent a promising new approach for the control of current H5N1 HPAI outbreaks by eliciting higher humoural and cellular immune responses and conferring improved cross-clade protection.

Lateral flow test strip based on colloidal selenium immunoassay for rapid detection of melamine in milk, milk powder, and animal feed.

Although high melamine (MEL) intake has been proven to cause serious health problems, MEL is sometimes illegally added to milk products and animal feed, arousing serious food safety concerns. A satisfactory method of detecting MEL in onsite or in-home testing is in urgent need of development. This work aimed to explore a rapid, convenient, and cost-effective method of identifying MEL in milk products or other food by colloidal selenium-based lateral flow immunoassay. Colloidal selenium was synthesized by L-ascorbic acid to reduce seleninic acid at room temperature. After conjugation with a monoclonal antibody anti-MEL, a test strip was successfully prepared. The detection limit of the test strip reached 150 µg/kg, 1,000 µg/kg, and 800 µg/kg in liquid milk, milk powder, and animal feed, respectively. No cross-reactions with homologues cyanuric acid, cyanurodiamide, or ammelide were found. Moreover, the MEL test strip can remain stable after storage for 1 year at room temperature. Our results demonstrate that the colloidal selenium MEL test strip can detect MEL in adulterated milk products or animal feed conveniently, rapidly, and sensitively. In contrast with a colloidal gold MEL test strip, the colloidal selenium MEL test strip was easy to prepare and more cost-efficient.