Search Results (905)

Due to the severe hazard of aflatoxins (AFs) to humans, it is of great significance to detect the key aflatoxins, aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁), in food and feed in simple, rapid, and semi-quantitative ways. The hybridoma clone 3A1 was prepared in this study, and anti-AFB₁ monoclonal antibody (mAb) with high specificity and affinity (9.38 × 10⁸ L/mol) from 3A1 was purified. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) demonstrated that the linear detection range for AFB₁ was 0.029–1.526 ng/mL with a limits of determination (LOD) of 0.023 ng/mL. A latex microsphere-based immunochromatographic test strip (LM-ICTS) was constructed based on 3A1, which showed that the strip could detect AFB₁ (LOD: lower than 1.79 ng/mL) and AFG₁ (LOD: lower than 8.08 ng/mL), and the linear detection ranges for AFB₁ and AFG₁ are 1.79–48.46 ng/mL and 8.08–107.40 ng/mL, respectively. The average recoveries of intra-assay and inter-assay for peanuts were (98.4 ± 4.7)% and (92.6 ± 7.6)%, and the average coefficient of variation (CVs) were 4.38% and 8.15%, respectively. For sunflower seeds, the intra-assay and inter-assay recoveries were (94.4 ± 7.2)% and (89.2 ± 4.3)%, and the average CVs were 6.6% and 4.9%, respectively. In summary, the developed LM-ICTS exhibited excellent sensitivity and specificity, which provided a rapidly stable on-site detection choice for AFB₁ and AFG₁ to contaminated agricultural samples, including grain and feed. Full article

Background/Objectives: Hypercholesterolemia remains a major risk factor for cardiovascular disease and a leading cause of global mortality. Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of low-density lipoprotein receptors (LDLR), thereby reducing LDL-cholesterol (LDL-C) clearance. While monoclonal antibodies (mAbs) targeting PCSK9 are effective, their short half-life requires frequent dosing and incurs high treatment costs. This study evaluates a novel peptide-based Anti-PCSK9 product aimed at providing sustained LDL-C reduction. Methods: A novel PCSK9 based-peptide conjugated to diphtheria toxoid (DT) was evaluated in various preclinical models: high-fat diet-fed C57BL/6 mice, APOB100/hCETP transgenic mice, BALB/c mice and normocholesterolemic non-human primates. Immunogenicity (Anti-PCSK9 antibody titers, binding affinity by SPR), pharmacodynamics (LDL-C levels, inhibition of PCSK9-LDLR interaction) and safety were assessed. Toxicity was evaluated in rodents, rabbits and dogs through clinical monitoring, histopathology, organ function and safety pharmacology studies. Results: The Anti-PCSK9 product induced robust and long-lasting immune response in all models antibody titers in BALB/c mice peaked by week 6 and persisted for 12 months. LDL-C reductions of 44% in APOB100/hCETP mice and 37% in C57BL/6 mice correlated with high antibody titers and strong PCSK9-binding affinities (85 and 49 RU), leading to 59% and 58% inhibition of PCSK9-LDLR interaction, respectively. Non-human primates showed sustained responses. No systemic toxicity was observed; injection-site reactions were mild and reversible. No adverse effects were detected on cardiovascular, neurological, or respiratory systems. Conclusions: This peptide-based Anti-PCSK9 therapy offers sustained efficacy and safety, representing a promising long-acting alternative for managing hypercholesterolemia. Full article

Glial fibrillary acidic protein (GFAP) has been shown to be a reliable biomarker for detecting neurological disorders. Recently, we developed the Lumipulse G GFAP plasma assay, which is a commercially available tool. Compared to existing assays, the LUMIPLSE G platform offers the high-throughput, rapid, and fully automated quantification of biomarkers, enabling more standardized and accessible clinical study. In this study, we evaluated this assay using cerebrospinal fluid (CSF) samples. Assessing GFAP in CSF may provide more direct insights into central nervous system pathology than plasma and could improve the characterization of Alzheimer’s disease (AD) stages and support treatment monitoring. The LUMIPULSE G system is a chemiluminescent enzyme immunoassay (CLEIA) platform equipped with full automation, utilizing specialized cartridges to process samples within 30 min. The assay, which employs a pair of proprietary monoclonal antibodies targeting GFAP, was evaluated for clinical performance using 30 CSF samples from patients diagnosed with AD, patients with mild cognitive impairment (MCI), and cognitively unimpaired (CU) individuals, with 10 samples from each group. In addition, levels of β-amyloid 1–40 (Aβ40), β-amyloid 1–42 (Aβ42), and pTau181 were simultaneously measured. The Lumipulse G GFAP assay significantly differentiated (p < 0.05) between the amyloid accumulation and non-amyloid accumulation groups, as classified based on the CSF Aβ test. Furthermore, GFAP showed a moderate correlation with pTau181 (r = 0.588), as determined based on Spearman’s rank correlation coefficient. Moreover, receiver operating characteristic (ROC) analysis was performed to determine the performance of GFAP in distinguishing amyloid-positive and amyloid-negative subjects, with an area under the curve (AUC) of 0.72 (0.50–0.93). When stratified by CSF pTau181 positivity, GFAP demonstrated an improved diagnostic accuracy, achieving an AUC of 0.86 (95% CI: 0.68–1.00). This study demonstrates that the Lumipulse G GFAP assay, when applied to CSF samples, has the potential to differentiate AD from non-AD cases, particularly suggesting its utility in detecting tau-related pathology. While GFAP has previously been established as a biomarker for AD, our findings highlight that combining GFAP with other biomarkers such as Aβ40, Aβ42, and pTau181 may enhance the understanding of AD pathogenesis, disease staging, and possibly treatment responses. These findings suggest that GFAP may serve as a complementary biomarker reflecting astroglial reactivity associated with tau positivity, alongside established biomarkers such as Aβ40, Aβ42, and pTau181. However, since GFAP levels may also be elevated in other neurological disorders beyond AD, further investigation into these conditions is required. Full article

African swine fever (ASF) is a highly pathogenic and hemorrhagic swine infectious disease caused by the African swine fever virus (ASFV). It encodes over 150 proteins, among which the CD2v protein plays multiple roles throughout the infection process. Single B-cell antibody technology is a cutting-edge method for preparing monoclonal antibodies (mAbs), which has the advantages of rapid, efficient, and high yield in antibody production, while possessing natural conformations. In this study, by cloning and expressing antibody genes in vitro, 14 murine-derived mAbs were prepared using recombinant CD2v proteins as immunogenic sources, which brings sufficient enrichment and selectivity for the development of antibodies based on the single B-cell antibody technique. All 14 mAbs demonstrated reactivity with CD2v protein by indirect ELISA, whereas 8 mAbs successfully detected CD2v in ASFV-infected PAM cells by IFA, indicating the tested mAbs can effectively recognize and bind to ASFV CD2v. Finally, a blocking ELISA method for detecting CD2v antibodies using CD2v mAb C89 was established, which holds significant potential for broad application in the serological diagnosis of ASFV with determination of the CD2v-blocking ELISA specificity, sensitivity, reproducibility, and compliance rate. It could be used for the rapid clinical detection of ASFV CD2v protein to provide a powerful tool for the monitoring of epidemics. Full article

Background/Objectived: Daratumumab is an anti-CD38 monoclonal antibody used in the treatment of multiple myeloma. Its use interferes with the indirect antiglobulin test (IAT). Treatment of reagent red blood cells (RBCs) with dithiothreitol (DTT) is one of the most validated techniques to resolve this interference. The objective of this study is to evaluate the rate of alloimmunization in transfused patients receiving daratumumab and the occurrence of hemolytic transfusion reactions. Materials and Methods: We conducted a single-center, retrospective, descriptive analysis of all patients treated with daratumumab at our institution from October 2016 to April 2024. For daratumumab-treated patients requiring RBC transfusions, an IAT with DTT-pretreated RBCs (DTT-IAT) was performed using the automated Orthovision system. Transfusion was administered only with a previous negative DTT-IAT while respecting Rh and Kell phenotyping. We assessed the transfusion profile of our patient cohort, including their rates of alloimmunization before and after daratumumab initiation, as well as the incidence of hemolytic complications. Additionally, a literature review was performed on reported alloimmunization rates in daratumumab-treated patients. Results: Among all patients, 106 received RBC and/or platelet transfusions after starting daratumumab. Four had known pre-existing alloantibodies. None developed new alloantibodies or experienced hemolytic complications while receiving anti-CD38 therapy. There were four cases of false-positive DTT-IAT due to residual drug interference or technical variability, in which no alloantibodies or adverse transfusion reactions were detected. Conclusions: Patients receiving daratumumab exhibit a low risk of alloimmunization. This may be partly explained by adherence to Rh and Kell phenotyping and daratumumab’s immunosuppressive effects on alloantibody production. These results support the conclusion that an extended red blood cell phenotype or genotype before starting daratumumab could be omitted if a fast and reliable technique for pretransfusion testing (such as automated DTT-IAT) is available 24 h. Full article

Background and Objectives: Over the past few years, there has been a significant shift in focus from developing better diagnostic tools to detecting Alzheimer’s disease (AD) earlier and initiating treatment interventions. This review will explore four main objectives: (a) the role of biomarkers in enhancing the diagnostic accuracy of AD, highlighting the major strides that have been made in recent years; (b) the role of neuropsychological testing in identifying biomarkers of AD, including the relationship between cognitive performance and neuroimaging biomarkers; (c) the amyloid hypothesis and possible molecular mechanisms of AD; and (d) the innovative AD therapeutics and the challenges and limitations of AD research. Materials and Methods: We have searched PubMed and Scopus databases for peer-reviewed research articles published in English (preclinical and clinical studies as well as relevant reviews and meta-analyses) investigating the molecular mechanisms, biomarkers, and treatments of AD. Results: Genome-wide association studies (GWASs) discovered 37 loci associated with AD risk. Core 1 biomarkers (α-amyloid Aβ42, phosphorylated tau, and amyloid PET) detect early AD phases, identifying both symptomatic and asymptomatic individuals, while core 2 biomarkers inform the short-term progression risk in individuals without symptoms. The recurrent failures of Aβ-targeted clinical studies undermine the amyloid cascade hypothesis and the objectives of AD medication development. The molecular mechanisms of AD include the accumulation of amyloid plaques and tau protein, vascular dysfunction, neuroinflammation, oxidative stress, and lipid metabolism dysregulation. Significant advancements in drug delivery technologies, such as focused Low-Ultrasound Stem, T cells, exosomes, nanoparticles, transferin, nicotinic and acetylcholine receptors, and glutathione transporters, are aimed at overcoming the BBB to enhance treatment efficacy for AD. Aducanumab and Lecanemab are IgG1 monoclonal antibodies that retard the progression of AD. BACE inhibitors have been explored as a therapeutic strategy for AD. Gene therapies targeting APOE using the CRISPR/Cas9 genome-editing system are another therapeutic avenue. Conclusions: Classic neurodegenerative biomarkers have emerged as powerful tools for enhancing the diagnostic accuracy of AD. Despite the supporting evidence, the amyloid hypothesis has several unresolved issues. Novel monoclonal antibodies may halt the AD course. Advances in delivery systems across the BBB are promising for the efficacy of AD treatments. Full article

Solid organ transplant recipients (SOTRs) are at high risk of severe coronavirus disease 2019 (COVID-19), therefore early treatment of mild infections is crucial to prevent increased morbidity and mortality. The effectiveness of early treatment in SOTRs has yet to be fully characterized due to the emergence of new SARS-CoV-2 variants and to COVID-19 vaccination implementation. The aim of this single-center retrospective study is to evaluate the outcomes, safety and impact on SARS-CoV-2 viral load kinetics of COVID-19 early treatment in SOTRs. The study includes 80 SOTRs with a laboratory-confirmed diagnosis of symptomatic SARS-CoV-2 infection enrolled between January and October 2022 and treated with either monoclonal antibodies or antivirals. All patients received COVID-19 vaccination and 68.8% of them showed detectable levels of anti-spike (S) antibodies. The occurrence of clinical events (hospitalization, intensive care unit admission, or death) was assessed within 30 days after treatment initiation. The quantification of SARS-CoV-2 viral load were performed at baseline and at day-7. The rate of hospitalization was 2.5% [0.3–9%] and no deaths occurred. All patients completed treatment with no serious adverse events. Median viral load decrease was 0.48 [0.26–0.69] log₂ cycle threshold (ct) values, with no significant differences between SOTRs treated with monoclonal antibodies and those treated with antivirals. Viral load decrease was significantly associated with positive anti-s serology at baseline ($\beta$ = 0.196, p = 0.01), number of days between symptom onset and treatment ($\beta$ = 0.05, p = 0.03) and the number of comorbidities ($\beta$ = −0.05, p = 0.03). We provide evidence of real-world effectiveness of early therapy in SOTRs infected with SARS-CoV-2 and demonstrate the relevant role of humoral response to vaccination in enhancing early viral load decay during treatment. Full article

Background/Objectives: This study investigates the potential of Anisakis sp. as a novel source of α-Gal (Galα1-3Galβ1-4GlcNAc-R) epitopes capable of inducing allergic sensitization in humans. While α-Gal is classically associated with delayed IgE-mediated hypersensitivity following tick bites, emerging evidence suggests that parasitic helminths such as Anisakis sp. may also express α-Gal-containing glycoconjugates, offering an alternative sensitization pathway. Methods: Protein extracts from Anisakis sp. third-stage larvae and mammalian tissues (beef, pork) were analyzed by SDS-PAGE and Western blot using a monoclonal anti-α-Gal antibody (clone M86), and α-Gal epitopes were detected by ELISA. Sera from urticaria patients, stratified by Anisakis sp. sensitization status, were evaluated for anti-α-Gal IgG, IgE, and IgG4 antibodies. Inhibition assays assessed cross-reactivity. Results: Results confirmed the presence of α-Gal epitopes on Anisakis sp. proteins, with prominent bands at ~250 kDa and 65 kDa. Urticaria patients sensitized to Anisakis sp. exhibited significantly elevated anti-α-Gal antibody levels compared to controls. Inhibition ELISA demonstrated substantial reduction in antibody binding with Anisakis sp. extracts, indicating shared antigenic determinants with mammalian α-Gal. Conclusions: These findings establish Anisakis sp. as a source of α-Gal-containing glycoproteins capable of eliciting specific antibody responses in humans, highlighting a potential parasitic route for α-Gal sensitization. Full article

Protein polarimetry has been evaluated as a simple and straightforward technique to detect the cryptic denaturation of exemplary proteins. The general rules of rotation vs. amino acid and structural composition and the respective knowledge gaps were reviewed, and the specific rotation of cystine was determined in 4 M NaCl solution as [α]_D²⁰ = –302.5°. The specific rotations at 589 nm and 436 nm and the ratio were measured for several model proteins, some purified plasma-derived proteins and for three monoclonal antibodies. The immunoglobulin G concentrates all showed a narrow ratio range likely characteristic for this protein class. Heat denaturation experiments were conducted at temperatures between 50 and 85 °C both for short-time (10 min) and for prolonged periods of heat exposure (up to 210 min). Denaturation by heat resulted not only in the known levorotatory shift, but also in a shift in the specific rotation ratio. The stabilizing effect of fatty acids in bovine serum could be demonstrated by this parameter. Polarimetry thus appears to be a particularly sensitive and simple method for the characterization of the identity and the thermal stability of proteins and should therefore be added again as a complimentary method to the toolbox of protein chemistry. Full article

Dengue, Zika, yellow fever, chikungunya, and Mayaro arboviruses represent an increasing threat to public health because of the serious infections they cause annually in many countries. Serological diagnosis of these viruses is challenging, making the development of new diagnostic strategies imperative. In this study, we investigated the effectiveness of gold nanorods (GNRs) functionalized with specific anti-dengue and anti-orthoflavivirus antibodies in detecting viral particles. GNRs were created with a length-to-width ratio of up to 5.5, a size of 71.4 ± 6.5 nm, and a light absorption peak at 927 nm, and they were treated with 4 mM polyethyleneimine. These GNRs were attached to a small amount of monoclonal antibodies that target flaviviruses, and the viral particles were detected by measuring the localized surface plasmon resonance using an UV-Vis/NIR spectrometer. The tests found Orthoflavivirus dengue and Orthoflavivirus zikaense in diluted human serum and ground-up mosquitoes, with the lowest detectable amount being 100 PFU/mL. The GNRs described in this study can be used to enhance flavivirus diagnostic tests or to develop new, faster, and more accurate diagnostic techniques. Additionally, the functionalized GNRs presented here are promising for supporting virological surveillance studies in mosquitoes. Our findings highlight a fast and highly sensitive method for detecting Orthoflavivirus in both human and mosquito samples, with a detection limit as low as 100 PFU/mL. Full article

The history of the rabies virus dates back four millennia, with the virus being considered by many to be the first known transmitted between animals and humans. In Brazil, rabies virus variants associated with terrestrial wild animals, marmosets, and different bat species have been identified. In this study, bat samples from different regions of São Paulo State, in Southeast Brazil, were analyzed to identify their genetic variability and patterns. A total of 51 samples were collected over ten years (1999–2009) and submitted to the immunofluorescent technique using monoclonal antibodies for antigenic profile detection (the diagnostic routine used in Latin American countries) and genetic evolution analysis through maximum likelihood approaches. Three antigenic profiles were detected: one related to the rabies virus maintained by hematophagous bat populations (AgV3), part of the monoclonal antibody panel used, and two other profiles not included in the panel (called NC1 and NC2). These antigenic profiles were genetically distributed in five groups. Group I was related to hematophagous bats (AgV3), Groups II and III were related to insectivorous bats (NC1) and Groups IV and V were also related to insectivorous bats (NC2). The results presented herein show that genetic lineages previously restricted to the northwest region of São Paulo State are now found in other state regions, highlighting the need for a comprehensive genetic study of bat rabies covering geographic and temporal space, through expanded genomic analysis using a standard genomic fragment. Full article

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

Background/Objectives: Vedolizumab is a gut-selective anti-integrin monoclonal antibody approved for the treatment of inflammatory bowel disease (IBD). While clinical trials have demonstrated a favorable safety profile, real-world studies are essential for identifying rare adverse events (AEs) and evaluating post-marketing safety. This study assessed vedolizumab’s safety in a real-world cohort and supported the detection of potential safety signals. Methods: A retrospective chart review was conducted on adult IBD patients treated with vedolizumab at a tertiary center in the Republic of Serbia between October 2021 and August 2022. Data included demographics, AEs, and newly reported extraintestinal manifestations (EIMs). Exposure-adjusted incidence rates were calculated per 100 patient-years (PYs). Disproportionality analysis using the FDA Adverse Event Reporting System (FAERS) was performed to identify safety signals, employing reporting odds ratios (RORs) and proportional reporting ratios (PRRs) for AEs also observed in the cohort. Prior IBD therapies and reasons for discontinuation were evaluated. Results: A total of 107 patients (42.1% Crohn’s disease, 57.9% ulcerative colitis) were included, with a median vedolizumab exposure of 605 days. There were 92 AEs (56.51/100 PYs), most frequently infections (23.95/100 PYs), gastrointestinal disorders (4.30/100 PYs), and skin disorders (4.30/100 PYs). The most frequently reported preferred terms (PTs) included COVID-19, COVID-19 pneumonia, nephrolithiasis, and nasopharyngitis. Arthralgia (12.90/100 PYs) was the most frequent newly reported EIM. No discontinuations due to vedolizumab AEs occurred. FAERS analysis revealed potential signals for events not listed in prescribing information but observed in the cohort: nephrolithiasis, abdominal pain, diarrhea, malaise, cholangitis, gastrointestinal infection, blood pressure decreased, weight decreased, female genital tract fistula, respiratory symptom, and appendicectomy. Most patients had received three prior therapies, often stopping one due to AEs. Conclusions: Vedolizumab demonstrated a favorable safety profile in the IBD cohort. However, FAERS-identified signals, such as nephrolithiasis, gastrointestinal infections, and decreased blood pressure, warrant further investigation in larger, more diverse populations. Full article

Background: Lysosomal-associated membrane protein 1 (LAMP1), typically localized to the lysosomal membrane, is increasingly implicated as a marker of cancer aggressiveness and metastasis when expressed on the cell surface. This study aimed to develop a LAMP1-targeted antibody-based PET tracer and assess its efficacy in mouse models of human breast and colon adenocarcinoma. Methods: To determine the source of LAMP1 expression, we utilized human single-cell RNA sequencing and spatial transcriptomics, complemented by in-house flow cytometry on xenografted mouse models. Tissue microarrays of multiple epithelial cancers and normal tissue were stained for LAMP-1, and staining was quantified. An anti-LAMP1 monoclonal antibody was conjugated with desferrioxamine (DFO) and labeled with zirconium-89 (⁸⁹Zr). Human triple-negative breast cancer (MDA-MB-231) and colon cancer (Caco-2) cell lines were implanted in nude mice. PET/CT imaging was conducted at 24, 72, and 168 h post-intravenous injection of ⁸⁹Zr-DFO-anti-LAMP1 and ⁸⁹Zr-DFO-IgG (negative control), followed by organ-specific biodistribution analyses at the final imaging time point. Results: Integrated single-cell and spatial RNA sequencing demonstrated that LAMP1 expression was localized to myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs) in addition to the cancer cells. Tissue microarray showed significantly higher staining for LAMP-1 in tumor tissue compared to normal tissue (3986 ± 2635 vs. 1299 ± 1291, p < 0.001). Additionally, xenograft models showed a significantly higher contribution of cancer cells than the immune cells to cell surface LAMP1 expression. In vivo, PET imaging with ⁸⁹Zr-DFO-anti-LAMP1 PET/CT revealed detectable tumor uptake as early as 24 h post-injection. The ⁸⁹Zr-DFO-anti-LAMP1 tracer demonstrated significantly higher uptake than the control ⁸⁹Zr-DFO-IgG in both models across all time points (MDA-MB-231 SUV_max at 168 h: 12.9 ± 5.7 vs. 4.4 ± 2.4, p = 0.003; Caco-2 SUV_max at 168 h: 8.53 ± 3.03 vs. 3.38 ± 1.25, p < 0.01). Conclusions: Imaging of cell surface LAMP-1 in breast and colon adenocarcinoma is feasible by immuno-PET. LAMP-1 imaging can be expanded to adenocarcinomas of other origins, such as prostate and pancreas. Full article

The human Tau protein stands for one of the most conspicuous and crucial hallmarks of Alzheimer’s disease (AD) diagnosis, along with other tauopathies. However, the assay for direct detection of tiny Tau protein concentrations in human samples continues to pose a significant challenge for the early diagnosis of AD. Thus, an amplification-based strategy is required. In this proposed work, we established an impedimetric immunosensor to detect human Tau-441 protein in PBS buffer using a sandwich approach, wherein we employed two distinct monoclonal antibodies (HT7 and BT2) that specifically recognize the amino acids 159–198 of the target protein. Through this strategy, we were able to detect as low as 0.08 pg/mL. These findings were attributed to the use of a biotinylated antibody (BT2)-streptavidin complex, which facilitated the amplification of the normalized signal, resulting in a lower limit of detection in comparison to the directly based immunosensors. Subsequently, we investigated the designed immunosensor to assess the assay’s selectivity in the presence of different off-targets, and no cross-interaction was recorded. The outcomes of our study provide valuable new insights into the application of sandwich-based assay as a highly sensitive and selective immunosensor for the detection of small protein. Full article