Search Results (49)

Background/Objectives: Colorectal cancer (CRC) arises from the epithelial lining of the colon or rectum, often following a progression from benign adenomatous polyps to malignant carcinoma. Screening modalities such as colonoscopy, faecal immunochemical tests (FIT), and FIT-DNA are critical for early detection and prevention, but non-invasive methods lack sensitivity to polyps and early CRC. Chromosome conformations (CCs) are potent epigenetic regulators of gene expression. We have previously developed an epigenetic assay, EpiSwitch^®®, that employs an algorithmic-based CCs analysis. Using EpiSwitch^®® technology, we have shown the presence of cancer-specific CCs in peripheral blood mononuclear cells (PBMCs) and primary tumours of patients with melanoma and prostate cancer. EpiSwitch^®®-based commercial tests are now available to diagnose prostate cancer with 94% accuracy (PSE test) and response to immune checkpoint inhibitors across 14 cancers with 85% accuracy (CiRT test). Methods/Results/Conclusions: Using blood samples collected from n = 171 patients with CRC, n = 44 patients with colorectal polyps and n = 110 patients with a ‘clear’ colonoscopy we performed whole Genome DNA screening for CCs correlating to CRC diagnosis. Our findings suggest the presence of two eight-marker CC signatures (EpiSwitch^®® NST) in whole blood that allow diagnosis of CRC and precancerous polyps, respectively. Independent validation cohort testing demonstrated high accuracy in identifying colorectal polyps and early versus late stages of CRC with an exceptionally high sensitivity of 79–90% and a high positive prediction value of 60–84%. Linking the top diagnostic CCs to nearby genes, we have built pathways maps that likely underline processes contributing to the pathology of polyp and CRC progression, including TGFβ, cMYC, Rho GTPase, ROS, TNFa/NFκB, and APC. Full article

The safety of titanium dioxide (TiO₂), widely used in foods and personal care products, has been of ongoing concern. Significant toxicity of TiO₂ has been reported, suggesting a risk to human health. To evaluate its potential epigenotoxicity, the effect of exposure to a TiO₂ product to which humans could be exposed on DNA methylation, a primary epigenetic mechanism, was investigated using two human cell lines (Caco-2 (colorectal) and HepG2 (liver)) relevant to human exposure. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Gene promoter methylation was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNA methyltransferases, MBD2, and URHF1 was quantified by qRT-PCR. A decrease in global DNA methylation was observed in both cell lines. Across the cell lines, seven genes (BNIP3, DNAJC15, GADD45G, GDF15, INSIG1, SCARA3, and TP53) were identified in which promoters were methylated. Changes in promoter methylation were associated with gene expression. Results also revealed aberrant expression of regulatory genes, DNA methyltransferases, MBD2, and UHRF1. Findings from the study clearly demonstrate the impact of TiO₂ exposure on DNA methylation in two cell types, supporting the potential involvement of this epigenetic mechanism in its biological responses. Hence, epigenetic studies are critical for complete assessment of potential risk from exposure. Full article

NSCLC is a very aggressive solid tumor, with a poor prognosis due to post-surgical recurrence. Analysis of the specific tumor and immune signatures of NSCLC samples is a critical step in prognostic evaluation and management decisions for patients after surgery. Routine histological assays have some limitations. Therefore, new diagnostic tools with the capability to quickly recognize NSCLC subtypes and correctly identify various markers are needed. We developed a technique for ex vivo isolation of cancer and immune cells from surgical tumor and lung tissue samples of patients with NSCLC (adenocarcinomas and squamous cell carcinomas) and their examination on ex vivo cell preparations and, parallelly, on histological sections after Romanovsky–Giemsa and immunofluorescent/immunochemical staining for cancer-specific and immune-related markers. As a result, PD-L1 expression was detected for some patients only by ex vivo analysis. Immune cell profiling in the tumor microenvironment revealed significant differences in the immunological landscapes between the patients’ tumors, with smokers’ macrophages with simultaneous expression of pro- and anti-inflammatory cytokines, neutrophils, and eosinophils being the dominant populations. The proposed ex vivo analysis may be used as an additional diagnostic tool for quick examination of cancer and immune cells in whole tumor samples and to avoid false negatives in histological assays. Full article

Simple analytical devices suitable for the analysis of various biochemical and immunechemical markers are highly desirable and can provide laboratory diagnoses outside standard hospitals. This study focuses on constructing an easily reproducible do-it-yourself ELISA plate reader biosensor device, assembled from generally available and inexpensive parts. The colorimetric biosensor was based on standard 96-well microplates, 3D-printed parts, and a smartphone camera as a detector was utilized here as a tool to replace the ELISA method, and its function was illustrated in the assay of TNFα as a model immunochemical marker. The assay provided a limit of detection of 19 pg/mL when the B channel of the RGB color model was used for calibration. The assay was well correlated with the ELISA method, and no significant matrix effect was observed for standard biological samples or interference of proteins expected in a sample. The results of this study will inform the development of simple analytical devices easily reproducible by 3D printing and found on generally available electronics. Full article

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (K_d), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC₅₀ approaches the K_d value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to K_d. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols. Full article

Oxidative stress is defined by an imbalance between the generation of reactive oxygen species and the biological system’s ability to neutralize them. This condition is commonly linked to various pathological conditions [¹]. Superoxide dismutase (SOD) is a widely used enzyme to assess oxidative stress, and various techniques have been developed for its detection in biological samples such as blood, urine, and saliva [²]. Surface-enhanced photoluminescence (PL) is a particularly sensitive method, offering minimal interference from the sample matrix [³]. In this work, silver nanostructured surfaces were implemented as substrates for the immunochemical determination of SOD in synthetic saliva through PL. The substrates were prepared using a single-step metal-assisted chemical etching method (MACE), resulting in the formation of silicon nanowires decorated with silver dendrites of approximately 1.5 μm in height [⁴]. For SOD detection, a three-step competitive immunoassay configuration was followed. Briefly, SOD was immobilized onto the substrates and then the functionalized substrates were incubated with mixtures of SOD with anti-SOD primary antibody, prepared either in assay buffer or synthetic saliva. Then, a solution of biotinylated anti-species specific antibody was added, followed by a reaction with streptavidin labelled with the fluorescent dye Rhodamine Red-X, and the signal was determined through an in-house developed optical set-up. The developed method presents similar or slightly lower sensitivity (detection limit 0.05 μg/mL) compared to the literature; however, it does not require labor-intensive sample pretreatment steps [⁵,⁶]. The aforementioned findings demonstrate the capability of the developed method to detect superoxide dismutase in natural saliva, in order to evaluate the oxidative stress status of an organism. Full article

Oxidative stress refers to the overproduction of reactive oxygen species and is often associated with numerous pathological conditions. Superoxide dismutase (SOD) is a widely used enzyme for evaluating oxidative stress, with numerous methods being developed for its detection in biological specimens like blood, urine, and saliva. In this study, a simple metal-assisted chemical etching method was employed for the fabrication of nanostructured silicon surfaces decorated with either silver dendrites or silver aggregates. Those surfaces were used as substrates for the immunochemical determination of SOD in synthetic saliva through surface-enhanced Raman spectroscopy (SERS) and surface-enhanced fluorescence (SEF). The immunoassay was based on a 3-step competitive assay format, which included, after the immunoreaction with the specific anti-SOD antibody, a reaction with a biotinylated secondary antibody and streptavidin. Streptavidin labeled with peroxidase was used in combination with a precipitating tetramethylbenzidine substrate for detection through SERS, whereas for SEF measurements, streptavidin labeled with the fluorescent dye Rhodamine Red-X was utilized. Both immunoassays were sensitive, with a detection limit of 0.01 μg/mL and a linear dynamic range from 0.03 to 3.3 μg/mL, enabling the evaluation of the oxidative stress status of an organism. Full article

The protein profile of Bothrops rhombeatus venom was compared to Bothrops asper and Bothrops atrox, and the effectiveness of antivenoms from the National Institute of Health of Colombia (INS) and Antivipmyn-Tri (AVP-T) of Mexico were analyzed. Protein profiles were studied with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high-performance liquid chromatography (RP-HPLC). The neutralizing potency and the level of immunochemical recognition of the antivenoms to the venoms were determined using Western blot, affinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Bands of phospholipase A2 (PLA2), metalloproteinases (svMPs) I, II, and III as well as serine proteinases (SPs) in the venom of B. rhombeatus were recognized by SDS-PAGE. With Western blot, both antivenoms showed immunochemical recognition towards PLA2 and svMP. INS showed 94% binding to B. rhombeatus venom and 92% to B. asper while AVP-T showed 90.4% binding to B. rhombeatus venom and 96.6% to B. asper. Both antivenoms showed binding to PLA2 and svMP, with greater specificity of AVP-T towards B. rhombeatus. Antivenom neutralizing capacity was calculated by species and mL of antivenom, finding the following for INS: B. asper 6.6 mgV/mL, B. atrox 5.5 mgV/mL, and B. rhombeatus 1.3 mgV/mL. Meanwhile, for AVP-T, the following neutralizing capacities were found: B. asper 2.7 mgV/mL, B. atrox 2.1 mgV/mL, and B. rhombeatus 1.4 mgV/mL. These results show that both antivenoms presented similarity between calculated neutralizing capacities in our trial, reported in a product summary for the public for the B. asper species; however, this does not apply to the other species tested in this trial. Full article

In this study, we characterized recombinant hemagglutinin (HA) of influenza A (H5N8) virus produced in Chinese hamster ovary cells (CHO-K1s). Immunochemical analysis showed that the recombinant hemagglutinin was recognized by the serum of ferrets infected with influenza A (H5N8) virus, indicating that its antigenic properties were retained. Two groups of Balb/c mice were immunized with intramuscular injection of recombinant hemagglutinin or propiolactone inactivated A/Astrakhan/3212/2020 (H5N8) influenza virus. The results demonstrated that both immunogens induced a specific antibody response as determined by ELISA. Virus neutralization assay revealed that sera of immunized animals were able to neutralize A/turkey/Stavropol/320-01/2020 (H5N8) influenza virus—the average neutralizing titer was 2560. Immunization with both recombinant HA/H5 hemagglutinin and inactivated virus gave 100% protection against lethal H5N8 virus challenge. This study shows that recombinant HA (H5N8) protein may be a useful antigen candidate for developing subunit vaccines against influenza A (H5N8) virus with suitable immunogenicity and protective efficacy. Full article

Nanostructured noble metal surfaces enhance the photoluminescence emitted by fluorescent molecules, permitting the development of highly sensitive fluorescence immunoassays. To this end, surfaces with silicon nanowires decorated with silver nanoparticles in the form of dendrites or aggregates were evaluated as substrates for the immunochemical detection of two ovarian cancer indicators, carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4). The substrates were prepared by metal-enhanced chemical etching of silicon wafers to create, in one step, silicon nanowires and silver nanoparticles on top of them. For both analytes, non-competitive immunoassays were developed using pairs of highly specific monoclonal antibodies, one for analyte capture on the substrate and the other for detection. In order to facilitate the identification of the immunocomplexes through a reaction with streptavidin labeled with Rhodamine Red-X, the detection antibodies were biotinylated. An in-house-developed optical set-up was used for photoluminescence signal measurements after assay completion. The detection limits achieved were 2.5 U/mL and 3.12 pM for CA125 and HE4, respectively, with linear dynamic ranges extending up to 500 U/mL for CA125 and up to 500 pM for HE4, covering the concentration ranges of both healthy and ovarian cancer patients. Thus, the proposed method could be implemented for the early diagnosis and/or prognosis and monitoring of ovarian cancer. Full article

Methylglyoxal (MG) is a precursor for advanced glycation end-products (AGEs), which have a significant role in diabetes. The present study is designed to probe the immunological response of native and glycated low-density lipoprotein (LDL) in experimental animals. The second part of this study is to probe glycoxidative lesion detection in low-density lipoproteins (LDL) in diabetes subjects with varying disease duration. The neo-epitopes attributed to glycation-induced glycoxidative lesion of LDL in DM patients’ plasma were, analyzed by binding of native and MG-modified LDL immunized animal sera antibodies using an immunochemical assay. The plasma purified human LDL glycation with MG, which instigated modification in LDL. Further, the NewZealand-White rabbits were infused with unmodified natural LDL (N-LDL) and MG-glycatedLDL to probe its immunogenicity. The glycoxidative lesion detection in LDL of DM with disease duration (D.D.) of 5–15 years and D.D. > 15 years was found to be significantly higher as compared to normal healthy subjects (NHS) LDL. The findings support the notion that prolonged duration of diabetes can cause structural alteration in LDL protein molecules, rendering them highly immunogenic in nature. The presence of LDL lesions specific to MG-associated glycoxidation would further help in assessing the progression of diabetes mellitus. Full article

This overview concerns recent patents and patented technologies in relation to the development of hydrogel-based biosensors, published until 2022. As a result, 257 patent documents and 145 simple patent families have been searched through different specialized patent databases. Furthermore, the patent classification confirmed that the most claimed inventions concern chemical analysis of biological material and biospecific binding assay materials with an insoluble carrier for immobilizing immunochemicals. Overall, the research, development, and innovation concerning hydrogel-based biosensors are based on improvements in the synthesis of hydrogels, biomolecule immobilization and detection, as well as microelectronic device integration and microfabrication techniques. A collection of recent patented technologies is proposed at the end. In this respect, it aimed to demonstrate the potential trends and challenges in relation to the development of hydrogel-based biosensors. Full article

Lateral flow immunoassay (LFIA) has found a broad application for testing in point-of-care (POC) settings. LFIA is performed using test strips—fully integrated multimembrane assemblies containing all reagents for assay performance. Migration of liquid sample along the test strip initiates the formation of labeled immunocomplexes, which are detected visually or instrumentally. The tradeoff of LFIA’s rapidity and user-friendliness is its relatively low sensitivity (high limit of detection), which restricts its applicability for detecting low-abundant targets. An increase in LFIA’s sensitivity has attracted many efforts and is often considered one of the primary directions in developing immunochemical POC assays. Post-assay enhancements based on chemical reactions facilitate high sensitivity. In this critical review, we explain the performance of post-assay chemical enhancements, discuss their advantages, limitations, compared limit of detection (LOD) improvements, and required time for the enhancement procedures. We raise concerns about the performance of enhanced LFIA and discuss the bottlenecks in the existing experiments. Finally, we suggest the experimental workflow for step-by-step development and validation of enhanced LFIA. This review summarizes the state-of-art of LFIA with chemical enhancement, offers ways to overcome existing limitations, and discusses future outlooks for highly sensitive testing in POC conditions. Full article

Cow milk is more allergenic than milk from other species, and therefore the adulteration of ewe or goat milk with cow milk can pose a serious threat to consumers. In this work, a silicon-based photonic immunosensor, which includes two U-shaped Mach–Zehnder Interferometers (MZIs), was employed for the detection of ewe and goat milk adulteration with cow milk through the immunochemical determination of the milk. The method was fast and sensitive with a detection limit of 0.04 μg/mL bovine k-casein (which corresponds to approximately 0.06% cow milk) in ewe or goat milk, respectively, and with a total assay time of 12 min. Full article

Background: Hepatocellular carcinoma (HCC) leads to 600,000 people’s deaths every year. The protein ubiquitin carboxyl-terminal hydrolase 15 (USP15) is a ubiquitin-specific protease. The role of USP15 in HCC is still unclear. Method: We studied the function of USP15 in HCC from the viewpoint of systems biology and investigated possible implications using experimental methods, such as real-time polymerase chain reaction (qPCR), Western blotting, clustered regularly interspaced short palindromic repeats (CRISPR), and next-generation sequencing (NGS). We investigated tissues samples of 102 patients who underwent liver resection between January 2006 and December 2010 at the Sir Run Run Shaw Hospital (SRRSH). Tissue samples were immunochemically stained; a trained pathologist then scored the tissue by visual inspection, and we compared the survival data of two groups of patients by means of Kaplan–Meier curves. We applied assays for cell migration, cell growth, and wound healing. We studied tumor formation in a mouse model. Results: HCC patients (n = 26) with high expression of USP15 had a higher survival rate than patients (n = 76) with low expression. We confirmed a suppressive role of USP15 in HCC using in vitro and in vivo tests. Based on publicly available data, we constructed a PPI network in which 143 genes were related to USP15 (HCC genes). We combined the 143 HCC genes with results of an experimental investigation to identify 225 pathways that may be related simultaneously to USP15 and HCC (tumor pathways). We found the 225 pathways enriched in the functional groups of cell proliferation and cell migration. The 225 pathways determined six clusters of pathways in which terms such as signal transduction, cell cycle, gene expression, and DNA repair related the expression of USP15 to tumorigenesis. Conclusion: USP15 may suppress tumorigenesis of HCC by regulating pathway clusters of signal transduction for gene expression, cell cycle, and DNA repair. For the first time, the tumorigenesis of HCC is studied from the viewpoint of the pathway cluster. Full article