Identification of non-neutralizing anti-factor X autoantibodies in three Japanese cases of autoimmune acquired factor X deficiency.

INTRODUCTION: Autoimmune factor X (FX or F10) deficiency (AiF10D) is an extremely rare acquired haemorrhagic disorder characterized by a severe reduction in FX activity due to autoantibodies against FX. AIM: Anti-FX autoantibodies were investigated in four patients with suspected AiF10D, and their properties were analysed. METHODS AND RESULTS: Anti-FX auto antibodies in plasma were detected by ELISA with three of four cases. One case of anti-FX autoantibody negativity was later diagnosed as AL-amyloidosis. IgG1 and IgG3 coexisted in all anti-FX autoantibodies of the three patients with AiF10D (cases X1, X2, and X3). Western blot analysis showed that the antibodies were bound to the FX light chain for cases X2 and X3, but the binding was weak for case X1. When the fusion proteins of a secretory luciferase with full-length FX or its γ-carboxylated glutamic acid (Gla) domain were added to the plasma of the three patients, both fusion proteins were immunoprecipitated as antigen-antibody complexes. Contrarily, the latter fusion protein produced in the presence of warfarin demonstrated a decrease in the collection rate, suggesting that their autoantibodies recognized the light chain and regions containing Gla residues. Since all three patients were essentially negative for FX inhibitors, it was concluded that the anti-FX autoantibodies for these cases were predominantly non-neutralizing. The concentration of the FX antigen also significantly reduced in these patients, suggesting that anti-FX autoantibodies promote the clearance of FX. CONCLUSION: Immunological anti-FX autoantibody detection is highly recommended to ensure that AiF10D cases are not overlooked, and to start necessary immunosuppressive therapies.

Rapid and sensitive detection of SARS-CoV-2 based on a phage-displayed scFv antibody fusion with alkaline phosphatase and NanoLuc luciferase.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses.

Affinity-Based Analysis Methods for the Detection of Aminoglycoside Antibiotic Residues in Animal-Derived Foods: A Review.

With the increasingly serious problem of aminoglycoside antibiotic residues, it is imperative to develop rapid, sensitive and efficient detection methods. This article reviews the detection methods of aminoglycoside antibiotics in animal-derived foods, including enzyme-linked immunosorbent assay, fluorescent immunoassay, chemical immunoassay, affinity sensing assay, lateral flow immunochromatography and molecular imprinted immunoassay. After evaluating the performance of these methods, the advantages and disadvantages were analyzed and compared. Furthermore, development prospects and research trends were proposed and summarized. This review can serve as a basis for further research and provide helpful references and new insights for the analysis of aminoglycoside residues. Accordingly, the in-depth investigation and analysis will certainly make great contributions to food safety, public hygiene and human health.

Advancements in the Worldwide Detection of Severe Fever with Thrombocytopenia Syndrome Virus Infection from 2009 to 2023.

Severe fever with thrombocytopenia syndrome (SFTS) is a growing concern as an emerging tick-borne infectious disease originating from the SFTS virus (SFTSV), a recent addition to the Phlebovirus genus under the family of bunyaviruses. SFTS is typically identified by symptoms such as fever, thrombocytopenia, leukopenia, and gastrointestinal problems, accompanied by a potentially high case fatality rate. Thus, early and accurate diagnosis is essential for effective treatment and disease management. This review delves into the existing methodologies for SFTS detection, including pathogenic, molecular, and immunological technologies.

COVID-19 diagnostics: Molecular biology to nanomaterials.

The SARS-CoV-2 pandemic has claimed around 6.4 million lives worldwide. The disease symptoms range from mild flu-like infection to life-threatening complications. The widespread infection demands rapid, simple, and accurate diagnosis. Currently used methods include molecular biology-based approaches that consist of conventional amplification by RT-PCR, isothermal amplification-based techniques such as RT-LAMP, and gene editing tools like CRISPR-Cas. Other methods include immunological detection including ELISA, lateral flow immunoassay, chemiluminescence, etc. Radiological-based approaches are also being used. Despite good analytical performance of these current methods, there is an unmet need for less costly and simpler tests that may be performed at point of care. Accordingly, nanomaterial-based testing has been extensively pursued. In this review, we discuss the currently used diagnostic techniques for SARS-CoV-2, their usefulness, and limitations. In addition, nanoparticle-based approaches have been highlighted as another potential means of detection. The review provides a deep insight into the current diagnostic methods and future trends to combat this deadly menace.

Production and application of monoclonal antibodies against ORF66 of cyprinid herpesvirus 2.

Cyprinid herpesvirus 2(CyHV-2)is the main pathogen causing haematopoietic necrosis disease of goldfish (Carassius auratus auratus) and gibel carp (Carassius auratus gibelio), which has caused huge economic losses to aquaculture industry of goldfish and gibel carp around the world. Currently, various detection methods based on nucleic acids have been established for the detection of CyHV-2. However, there is still a lack of rapid and effective immunological detection technology. In this study, anti-CyHV-2 ORF66 monoclonal antibodies (MAbs) were prepared to use the recombinant ORF66 protein as the antigen. Firstly, the open reading frame of CyHV-2 ORF66 was cloned into the pET-28a vector and expressed in Escherichia coli. Three MAbs (2F11, 2G8, and 3D6) against recombinant ORF66 protein were developed by immunization of Balb/C mice. Among them, MAb-2F11 belonged to the IgG2b isotype, 2G8 and 3D6 belonged to the IgG1 isotype. Western blotting analysis was performed to assess the ability of the MAbs to bind to the ORF66 recombinant protein and CyHV-2 nucleocapsid protein ORF66. In addition, the MAb-2F11 was used to detect the virus particles that infected in cell line and tissues of gibel carp virus infection by immunological methods. These results indicated that the anti-CyHV-2 ORF66 MAb-2F11 prepared in this study could not only detect the presence of the virus but also provide a research tool for further studying the role of ORF66 in the process of CyHV-2 infection.

Efficacy of the InvictDetectTM Immunostrip® to Taxonomically Identify the Red Imported Fire Ant, Solenopsis invicta, Using a Single Worker Ant.

The early detection and identification of the red imported fire ant Solenopsis invicta are crucial to intercepting and preventing it from becoming established in new areas. Unfortunately, the visual identification of fire ants to species is difficult and ant samples must often be couriered to an expert for positive identification, which can delay control interventions. A lateral flow immunoassay that provides a rapid and portable method for the identification of S. invicta ants was developed and commercialized, and it is available from Agdia, Inc. under the trade name InvictDetectTM. While the test was 100% accurate when using the recommended minimum sample of three ant workers, InvictDetectTM was field tested for the first time while using homogenates prepared from single S. invicta workers to determine the effectiveness of the method under these non-recommended conditions. Disregarding social form, the false negative rate was 25.5% for an initial single worker ant test and 10% after a repeat test was performed. The InvictDetectTM false negative response was independent of worker weight. Though InvictDetectTM requires a minimum of three worker ants, the test improves upon current identification methods because it can be conducted in the field, be completed in 10-30 min, and requires no special training or expertise.

Metal-organic frameworks for virus detection.

Virus severely endangers human life and health, and the detection of viruses is essential for the prevention and treatment of associated diseases. Metal-organic framework (MOF), a novel hybrid porous material which is bridged by the metal clusters and organic linkers, has become a promising biosensor platform for virus detection due to its outstanding properties including high surface area, adjustable pore size, easy modification, etc. However, the MOF-based sensing platforms for virus detection are rarely summarized. This review systematically divided the detection platforms into nucleic acid and immunological (antigen and antibody) detection, and the underlying sensing mechanisms were interpreted. The nucleic acid sensing was discussed based on the properties of MOF (such as metal ion, functional group, geometry structure, size, porosity, stability, etc.), revealing the relationship between the sensing performance and properties of MOF. Moreover, antibodies sensing based on the fluorescence detection and antigens sensing based on molecular imprinting or electrochemical immunoassay were highlighted. Furthermore, the remaining challenges and future development of MOF for virus detection were further discussed and proposed. This review will provide valuable references for the construction of sophisticated sensing platform for the detection of viruses, especially the 2019 coronavirus.

New system for the detection of Legionella pneumophila in water samples.

Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved, but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis. This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.

Microfluidic devices for sample preparation and rapid detection of foodborne pathogens.

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line.

A new spot quality control for protein macroarray based on immunological detection.

Protein macroarray is a new, simple and multiple biochemistry detection system, in which the test spots are more than 1mm diameter and results directly visible and instrumentation is not necessary. This technology, however, possesses recognized problems with spot quality and uniformity, issues that can limit its application. Previous methods have been developed for spot quality control, but they are complicated or require specific instrumentation. Therefore, we have developed a spot quality control buffer supplement with Ponceau S (SQCB-PS) as a direct and visible means of monitoring spot quality on nitrocellulose membrane prior to hybridization. In this report, 1% (w/v) Ponceau S and 10% (v/v) glycerol in spotting buffer were found to be optimal for spot quality control and food-borne pathogens multiple detection. Furthermore, the sensitivity and specificity of the protein macroarray were not compromised by spotting buffer with Ponceau S. Under optimal conditions, this visible spot quality control method makes the detection more reliable, and should enhance the wider use of the technique.

Recommended Immunological Assays to Screen for Ricin-Containing Samples.

Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories' capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods "dangerous" samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin.

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples.

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A-G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as "category A" bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.

Development of a novel anti ESAT-6 monoclonal antibody for screening of Mycobacterium tuberculosis.

Tuberculosis (TB) is a major global public health problem with an estimated one-third of the world's population infected with Mycobacterium tuberculosis, thus necessitating fast and accurate diagnosis of TB. However, the diagnosis of latent infection remains difficult due to the lack of a simple, reliable test for M. tuberculosis infection. The objective of the current study was to generate and characterize a novel monoclonal antibody (mAb) that specifically target the early secretory antigenic target 6 (ESAT-6) protein for tuberculosis (TB) immunological diagnosis. The BALB/c mice were immunized by a peptide with 13 amino acids (amino acid residues 24 to 36) of ESAT-6 protein. Stable hybridomas cell lines were established and mAb was identified by Western Blot, immunoprecipitation (IP) and Enzyme linked immunosorbent assay (ELISA). In addition, this mouse mAb was used to coat plates, and biotin-labelled polyclonal antibodies were used to detect the antigens. Finally, the antibody was verified in 280 patient sputum culture supernatants and pleural effusion aspirates. The positive detection rate of the generated ESAT-6 mAb was 91% in the clinical diagnosis of tuberculosis pleural effusion, and this mAb had a sensitivity of 92.4% and a specificity of 100% in all M. tuberculosis cases. Our data indicated that the mAb generated in this study can potentially serve as a tool in the laboratory diagnosis of TB.