Development and evaluation of time-resolved fluorescent immunochromatographic assay for quantitative detection of SARS-CoV-2 spike antigen.

BACKGROUND: The spread of COVID-19 worldwide caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has necessitated efficient, sensitive diagnostic methods to identify infected people. We report on the development of a rapid 15-minute time-resolved fluorescent (TRF) lateral flow immunochromatographic assay for the quantitative detection of the SARS-CoV-2 spike protein receptor-binding domain (S1-RBD). OBJECTIVES: Our objective was to develop an efficient method of detecting SARS-CoV-2 within 15 min of sample collection. METHODS: We constructed and evaluated a portable, disposable lateral flow device, which detected the S1-RBD protein directly in nasopharyngeal swab samples. The device emits a fluorescent signal in the presence of S1-RBD, which can be captured by an automated TRF instrument. RESULTS: The TRF lateral flow assay signal was linear from 0 to 20 ng/ml and demonstrated high accuracy and reproducibility. When evaluated with clinical nasopharyngeal swabs, the assay was performed at >80% sensitivity, >84% specificity, and > 82% accuracy for detection of the S1-RBD antigen. CONCLUSION: The new S1-RBD antigen test is a rapid (15 min), sensitive, and specific assay that requires minimal sample preparation. Critically, the assay correlated closely with PCR-based methodology in nasopharyngeal swab samples, showing that the detected S1-RBD antigen levels correlate with SARS-CoV-2 virus load. Therefore, the new TRF lateral flow test for S1-RBD has potential application in point-of-care settings.

Application of multiplex immunoassay technology to investigations of ocular disease.

Eye-derived fluids, including tears, aqueous humour and vitreous humour often contain molecular signatures of ocular disease states. These signatures can be composed of cytokines, chemokines, growth factors, proteases and soluble receptors. However, the small quantities (<10 µl) of these fluids severely limit the detection of these proteins by traditional enzyme-linked immunosorbent assay or Western blot. To maximise the amount of information generated from the analysis of these specimens, many researchers have employed multiplex immunoassay technologies for profiling the expression or modification of multiple proteins from minute sample volumes.

Cytokines in cancer drug resistance: Cues to new therapeutic strategies.

The development of oncoprotein-targeted anticancer drugs is an invaluable weapon in the war against cancer. However, cancers do not give up without a fight. They may develop multiple mechanisms of drug resistance, including apoptosis inhibition, drug expulsion, and increased proliferation that reduce the effectiveness of the drug. The collective work of researchers has highlighted the role of cytokines in the mechanisms of cancer drug resistance, as well as in cancer cell progression. Furthermore, recent studies have described how specific cytokines secreted by cancer stromal cells confer resistance to chemotherapeutic treatments. In order to gain a better understanding of mechanism of cancer drug resistance and a prediction of treatment outcome, it is imperative that correlations are established between global cytokine profiles and cancer drug resistance. Here we discuss the recent discoveries in this field of research and discuss their implications for the future development of effective anti-cancer medicines.

Antibody arrays in biomarker discovery.

All of life is regulated by complex and organized chemical reactions that help dictate when to grow, to move, to reproduce, and to die. When these processes go awry, or are interrupted by pathological agents, diseases such as cancer, autoimmunity, or infections can result. Cytokines, chemokines, growth factors, adipokines, and other chemical moieties make up a vast subset of these chemical reactions that are altered in disease states, and monitoring changes in these molecules could provide for the identification of disease biomarkers. From the first identification of carcinoembryonic antigen, to the discovery of prostate-specific antigen, to numerous others described within, biomarkers of disease are detectable in a plethora of sample types. The growing number of biomarkers for infection, autoimmunity, and cancer allow for increasingly early detection, to identification of novel drug targets, to prognostic indicators of disease outcome. However, more and more studies are finding that a single cytokine or growth factor is insufficient as a true disease biomarker and that a more global perspective is needed to understand true disease biology. Such a broad view requires a multiplexed platform for chemical detection, and antibody arrays meet and exceed this need by performing this detection in a high-throughput fashion. Herein, we will discuss how antibody arrays have evolved, and how they have helped direct new drug target design, helped identify therapeutic disease markers, and helped in earlier disease detection. From asthma to renal disease, and neurological dysfunction to immunologic disorders, antibody arrays afford a bright future for new biomarkers discovery.

Tumor-induced perturbations of cytokines and immune cell networks.

Until recently, the intrinsically high level of cross-talk between immune cells, the complexity of immune cell development, and the pleiotropic nature of cytokine signaling have hampered progress in understanding the mechanisms of immunosuppression by which tumor cells circumvent native and adaptive immune responses. One technology that has helped to shed light on this complex signaling network is the cytokine antibody array, which facilitates simultaneous screening of dozens to hundreds of secreted signal proteins in complex biological samples. The combined applications of traditional methods of molecular and cell biology with the high-content, high-throughput screening capabilities of cytokine antibody arrays and other multiplexed immunoassays have revealed a complex mechanism that involves multiple cytokine signals contributed not just by tumor cells but by stromal cells and a wide spectrum of immune cell types. This review will summarize the interactions among cancerous and immune cell types, as well as the key cytokine signals that are required for tumors to survive immunoediting in a dormant state or to grow and spread by escaping it. Additionally, it will present examples of how probing secreted cell-cell signal networks in the tumor microenvironment (TME) with cytokine screens have contributed to our current understanding of these processes and discuss the implications of this understanding to antitumor therapies.