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The ability to reliably detect enrofloxacin in animal-derived food products has important health implications. In the present study, a nanobody-horseradish peroxidase fusion specific for ENR was generated to enable a sensitive and rapid competitive ELISA suitable for detecting enrofloxacin in samples of milk and animal tissue. An enrofloxacin hapten generated via the glutaraldehyde method was initially used to immunize an adult Bactrian camel as a means of constructing a phage library. Enrofloxacin-specific nanobodies were then selected through three rounds of biopanning, and HRP-fused versions of these nanobodies were then expressed. Lastly, these nanobodies were used to develop a sensitive cELISA for enrofloxacin detection in milk and animal tissues, with the resultant assay exhibiting an IC 50 of 37.41 ng/mL and a linear detection range (IC 20 -IC 80 ) of 10.89 to 244.34 ng/mL. The limit of detection for this cELISA was 6.48 ng/mL, with 4.66 % cross-reactivity with ciprofloxacin, and recovery rates that ranged from84.99 % to 107.72 % together with an RSD below 10.70 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

A novel β-amyloid protein capillary microextraction column was designed and prepared using epitope molecular imprinting technology for specific recognition of trace β-amyloid proteins in complex biological matrices. Using N-terminal nonapeptide of β-amyloid protein as template molecule, choline chloride-MAA and N-hydroxymethyl acrylamide as functional monomers, ethylene glycol dimethacrylate as crosslinker, the imprinted capillary monolithic column was prepared by thermal polymerization in the acetonitrile-water system. The optimal preparation parameters were obtained with the ratio of template: functional monomer: crosslinker at 1:6:16 (mmol/mmol/mmol). The physical property evaluation through Scanning electron microscopy, Fourier transform infrared spectroscopy analysis, zeta potential analysis, particle size analysis, and Brunauer-emmett-teller showed that a porous imprinted monolithic column with a high specific surface area was successfully prepared. The static adsorption experiment showed that the theoretical maximum adsorption capacity was 0.060 μg/column and the optimal imprinting factor was 2.27. Under the optimal extraction conditions, the imprinted column exhibited good template selectivity and excellent robustness. Finally, the extraction efficiency of the capillary imprinted column in actual plasma was evaluated using ELISA method. For Aβ 40 and Aβ 42, the detection limits were 1.00 pg/mL and 0.67 pg/mL, the quantification limits were 3.00 pg/mL and 2.00 pg/mL, the detection ranges were 7.5-120.0 pg/mL and 5.0-80.0 pg/mL, respectively. After extraction of plasma samples, the measured concentrations of Aβ 40 and Aβ 42 by imprinted column were 157.31 pg/mL and 48.22 pg/mL, respectively, which were significantly higher than the measured concentrations by non-imprinted column of 89.60 pg/mL and 41.02 pg/mL. In summary, the epitope imprinted capillary monolithic column prepared in this study can effectively enrich and separate trace amounts of amyloid proteins in complex samples, and is expected to provide a new sample pretreatment method for clinical detection of amyloid proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Immunoprecipitation is among the most widely utilized methods in biomedical research, with applications that include the identification of antibody targets and associated proteins. The path to identifying these targets is not straightforward, however, and often requires the use of chemical cross-linking and/or gel electrophoresis to separate targets from an overabundance of immunoglobulin protein. Such experiments are labor intensive and often yield long lists of candidate antibody targets. Here, we describe an unbiased immunoprecipitation-to-mass spectrometry (IP-to-MS) method that relies on a novel protein tag to separate low abundance immunoprecipitated proteins from overwhelmingly abundant immunoglobulins. We demonstrate that the IP-to-MS serotyping workflow is highly reproducible and can be used for the identification of novel, patient-specific antigen targets in multiple disease states. Furthermore, we show that IP-to-MS may outperform conventional methods of antibody detection, including enzyme-linked immunosorbent assay, while also enabling patient stratification beyond what is possible with traditional approaches.

Urine drug screening is carried out on numerous automated analysis platforms using enzyme-linked immunosorbent assays. While these methods are rapid, they often lack specificity. We report the case of a 5-year-old child treated for Dravet disease and hospitalized for clonic seizures. During her hospitalization, 3 urine samples were sent to the Toxicology laboratory to rule out any toxic origin to these seizures. The first two were positive for ecstasy, and negative for other drugs. For the last sample, all tests were negative. The presence of ecstasy in the first 2 samples was not confirmed by gas chromatography-mass spectrometry, which identified stiripentol in all 3 urines. Stiripentol is an antiepileptic drug that shares a 3,4-methylenedioxy group with MDMA. We determined that the antibody in the kit crossed 20% with stiripentol, and were able to define a stiripentol cut-off concentration of 2,500 ng/mL. We checked this cut-off by measuring stiripentol in the 3 urine samples, finding concentrations of 47,600 and 5,800 ng/mL for the first and second samples respectively, and 1,900 ng/mL for the last. These concentrations explain the false positives in the first two urine samples, and the negative result in the last. This work underlines the need to remain critical of the results of immuno-screening methods, and to confirm them with a reference method.

Neutralizing antibodies provide vital protection against foot-and-mouth disease virus (FMDV). The virus neutralization test (VNT) is a gold standard method for the detection of neutralizing antibodies. However, its application is limited due to the requirement for live virus and unsuitability for large-scale serological surveillance. In this study, a porcine broadly neutralizing monoclonal antibody (PO18-10) against FMDV was obtained from the heterologous sequentially vaccinated pig using single-B-cell antibody technology. A competitive enzyme-linked immunosorbent assay (C-ELISA) for detecting neutralizing antibodies against FMDV serotype O was developed using biotinylated PO18-10 as a detector antibody. The sensitivity and specificity of the assay were 100% and 99.55%, respectively, and the positive/negative coincidence rate with VNT was 94%, suggesting that C-ELISA based on natural host-derived monoclonal antibody (mAb) could be a promising tool to detect neutralizing antibodies against FMDV serotype O and evaluate the vaccine efficacy.IMPORTANCEFoot-and-mouth disease virus (FMDV) serotype O is one of the most prevalent serotypes in the world. The neutralizing antibody titers in primo-vaccinated animals are directly related to their level of protection against a virus challenge. The development of a safe, rapid, and accurate method for the detection of the neutralizing antibody is essential for the control and eradication of FMD. In this study, an inter-serotype broadly neutralizing monoclonal antibody PO18-10 was successfully produced using single-B-cell antibody technology from sequentially vaccinated pigs. A competitive ELISA based on this natural host-derived mAb for the detection of neutralizing antibodies against FMDV serotype O was developed and validated. The assay demonstrates high sensitivity, specificity, and coincidence rate with VNT, making it an alternative tool for confirming FMDV infection and evaluating the vaccine efficacy., Competing Interests: The authors declare no conflict of interest.

Immuno-positron emission tomography (i-PET) is a non-invasive imaging technique that combines the specificity of monoclonal antibodies with the sensitivity of positron emission tomography to visualize and quantify the distribution of target antigens in vivo, providing detailed spatial information about the presence and localization of specific biomarkers. Due to the crucial role that cytotoxic T cells play in antitumor responses, a new tracer consisting of a humanized anti-CD8 nanobody labeled with fluorine-18 was developed to inform immuno-oncology treatments and support medical decision-making. Nanobodies are single-domain antibodies with low molecular weight and fast peripheral blood clearance, both of which are advantageous properties for same-day imaging. However, these unique characteristics pose bioanalytical challenges when developing clinical pharmacokinetic (PK) assays, including the need for high assay sensitivity. This manuscript focuses on overcoming bioanalytical challenges related to sensitivity and matrix interference during the development of a method to quantify this novel anti-CD8 nanobody tracer in human serum. Out of the three immunoassay platforms evaluated (ELISA, SMCxPRO™ and Gyrolab®), a Gyrolab method was ultimately selected due to its superior sensitivity, equal detectability of both conjugated and unconjugated forms of the nanobody and its ability to minimize matrix interference. By selecting the right assay format, along with the appropriate critical reagents for capture and detection, matrix interference was diminished. This novel PK method was successfully qualified demonstrating acceptable performance across all parameters. The acquired bioanalytical insights gained could be applied to nanobody-derived conjugates or other modalities that require high sensitivity in the clinical settings., Competing Interests: Declarations. Conflict of Interest: All authors are employees of Genentech Inc., a member of the Roche group and Roche shareholders., (© 2025. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Detection of Salmonella in food is topical due to known cases of salmonellosis epidemics. Immunochemical methods including ELISA are widely used for Salmonella detection. Traditionally, commercial ELISA kits are based on sandwich technique and detect lipopolysaccharide (LPS), which is considered to be the component of the outer membrane of Gram-negative bacteria. Our aim was elaboration of competitive ELISA test for Salmonella detection in food with improved parameters. It was shown that in the Salmonella culture after the standard sample preparation procedure LPS is present mainly outside cells as a component of outer membrane vesicles. Improved sample preparation procedure includes separation of bacteria from the medium and analysis of the medium, which increases analytical sensitivity. Immobilization of the bovine serum albumin (BSA)-LPS conjugate in microplate wells allows to obtain a more homogeneous coating than immobilization of LPS itself. Thus, we have developed test system for Salmonella detection in food by competitive ELISA of LPS secreted into the culture medium with the immobilized BSA-LPS conjugate and monoclonal antibodies (mAb) to LPS core in the liquid phase. New competitive ELISA test is high sensitive, give reproducible results, allows the detection of any Salmonella serotype and is important for the protection of human health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Effective detection technologies in food safety with the merits of portable and on-site detection potential are always in pressing demand. Herein, we developed a nanopore-assisted Enzyme-linked immunosorbent assay (NELISA) platform, which innovatively introduced hairpin DNA (HP DNA) probes as reaction substrates. This innovation of substrates effectively avoided the inherent limitations of colorimetric signals (i.e., low sensitivity and inaccurate results) and greatly improved the sensitivity and accuracy of NELISA platform. The alkaline phosphatase (ALP)-modified detection antibody (ALP-Ab2) can specifically bind to ricin and hydrolyze the phosphate groups modified on the HP DNA probes. Nanopore recordings demonstrated that two states of probes produced highly distinguishable nanopore events, enabling the qualitative and quantitative detection of ricin. This NELISA platform fully combined the specificity of ELISA with the ultra-sensitivity, and unique single-molecule fingerprint recognition of nanopore, showing a great on-site detection potential. This method achieved the ultrasensitive and reliable detection of ricin down to 2.46 fg/mL, which enhanced the detection sensitivity by at least 10 6 -fold compared to traditional ELISA. Furthermore, the proposed method was capable of accurately detecting ricin in real food samples with satisfactory recoveries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Bovine viral diarrhea virus (BVDV) is a significant immunosuppressive pathogen that has a major impact on the global cattle industry. Research efforts are currently focused on the envelope glycoprotein E2 of BVDV to improve immune responses. However, the full-length E2 protein is not ideal as an immune antigen and diagnostic tool, leading to the exploration of alternative strategies. In this study, we optimized the E2 gene using IDEB and ExpOptimizer software, then expressed the E2 gene using both baculovirus and E. coli expression systems. Subsequently, we assessed the immunogenicity of the purified E2 protein in mice and its application in indirect ELISA assays. Our findings showed that the Bac-E2 protein produced by the baculovirus system induced higher levels of antibody production and splenic lymphocyte proliferation in mice compared to the E. coli system. Moreover, the indirect ELISA assay developed using Bac-E2 protein exhibited superior specificity, sensitivity, and accuracy in comparison to the E. coli-expressed E2 ELISA. Overall, our study demonstrates that the optimized E2 protein generated through a baculovirus expression system elicits robust humoral and cellular immune responses in mice, making it a promising candidate for vaccine development. Furthermore, the optimized E2 protein ELISA assay shows enhanced sensitivity and accuracy, suggesting its potential as a valuable diagnostic antigen., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The MICA and MICB molecules, expressed on the cell membrane in response to cellular stress or cancer transformation, pose significant challenges for immunoassays. They exhibit high sequence and structural similarity, alongside considerable allelic polymorphism, with 291 and 53 known protein sequences, respectively. Some researchers treat MICA and MICB as a unified target because of their structural and functional similarities, while others distinguish between them. However, which approach is superior and under what circumstances remains unknown. Moreover, information about assays' reactivity with MICA and MICB allelic variants is often missing. In this study, we developed 10 monoclonal antibodies (mAbs) and two sandwich ELISAs for the detection and quantification of these molecules. We assembled a panel of recombinant proteins representing the diversity of MICA and MICB in the European population and profiled the reactivities of the mAbs and ELISAs. The performance of these sandwich ELISAs was evaluated using samples from prostate cancer patients and pregnant women experiencing premature rupture of membranes. Our study assessed the impact of MICA and MICB polymorphism on their detection and quantification by immunological methods, providing evidence to support differential or non-differential approaches for their detection., (© 2025 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The enzyme-linked immunosorbent assay (ELISA) detects antigen-antibody interactions by using enzyme-labelled conjugates and enzyme substrates that generate colour changes. This review aims to provide an overview of ELISA, its various types, and its applications in detecting metabolites in biological fluids. The article discusses the history of the assay, its underlying principles and procedures, common ELISA protocols, and the most accurate and reliable techniques for measuring peptide molecules in biological fluids. Additionally, we emphasize best laboratory practices to achieve consistent, high-quality results and outline the essential materials for setting up an ELISA laboratory, drawing from our over 30 years of experience in the field., Competing Interests: Declaration of conflicting interestThe authors declare there are no conflicts of interest.

African swine fever (ASF), which is caused by the African swine fever virus (ASFV). There are currently no effective vaccines or therapies for ASF; rapid diagnosis has become an important way to control the disease. The p11.5 and p14.5 proteins are structural proteins of ASFV that have immunogenicity and potential as diagnostic antigens. In this study, p11.5 and p14.5 proteins were used as diagnostic antigens in an indirect ELISA examine to detect ASFV antibodies. The p11.5 and p14.5-iELISA methods showed no cross-reaction with other swine virus positive sera. The lowest detection limit of positive serum was 1: 6400, and the coefficients of variation (CV) of intra - and inter-assay were less than 7%. By comparing 71 serum samples, the coincidence rate of p11.5 and p14.5-iELISA with commercial ELISA kits was 91.55%. Taken together, these results showed that there is a reliable method of detecting ASFV antibodies in clinical pig serum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Dengue, a vector-borne disease, affects nearly 400 million people annually. Although commercially available dengue NS1 antigen-based ELISA kits are simple and rapid, they are expensive as monoclonal antibodies are used in these tests, and also, they have short expiry dates. As an alternative, the polyclonal antibodies generated against dengue NS1 antigen from the individuals who recovered from the dengue infection (human polyclonal antibodies against dengue; HuPA-D) can be explored for the detection of NS1 antigen in the dengue virus (DENV) infected patients' sera. In this study, blood samples were collected from the dengue-recovered patients after obtaining the IHEC approval. The anti-NS1 HuPA-D (IgG) was purified using NAb™ Spin Column kit and tested on SDS-PAGE. HuPA-D ELISA was developed to test the sensitivity and specificity of the antibodies using the recombinant NS1 antigens of dengue serotypes and flaviviruses. Further, the HuPA-D were used to detect the sera of the dengue patients. The assay was found to be sensitive to detect all the serotypes of recombinant dengue NS1 antigen and also NS1 antigen from the sera of DENV infected patients., Competing Interests: Declaration of competing interest There are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Calanthe mild mosaic virus (CalMMV) infecting orchids is an important potyvirus which is known to cause mild leaf mosaic and flower colour-breaking symptoms in Calanthe and other orchid plants. The present study reports the production of polyclonal antibodies against CalMMV using bacterially expressed recombinant coat protein as immunogen, which in turn would be useful in routine indexing and screening of orchid germplasm. The coat protein (CP) gene (~ 807 bp) of CalMMV isolated from infected orchid sample was cloned in expression vector, pET-28a ( +) that yielded ~ 31 kDa fusion protein with Histidine tag (His 6 BP). The expression of fusion CP was confirmed through SDS-PAGE and Western blotting. The His 6 BP-CalMMV-CP obtained in soluble state after purification was used to immunize New Zealand white rabbit for the production of polyclonal antibodies (PAb). The PAb produced against the purified fusion protein successfully detected CAlMMV in the orchid samples at a dilution of 1:2000 in direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). This study presents the first report of Histidine tag (His 6 BP) fusion CalMMV-CP-based antibody production and its successful application in the identification of the virus in orchid plants. Outcome of this study will be helpful in routine certification programmes, screening of orchid germplasm and production of CalMMV-free planting materials of orchids., Competing Interests: Declarations. Conflict of interest: All the authors declare that this is an innovative work that has not been published in other research journal and they have no conflict of interest between them or personal relationship that might alter the research work summarized in the manuscript. All the authors contributed outstandingly to the work. Ethical Approval: Not Applicable. Informed Consent: Not Applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Paratuberculosis (Johne's disease), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a common, economically-important and potentially zoonotic contagious disease of cattle, with worldwide distribution. Disease management relies on identification of animals which are at high-risk of being infected or infectious. The disease is chronic in nature, and infected animals may be infectious in the absence of overt clinical signs. Coupled with limited sensitivity of available diagnostic tests, this creates difficulties in identifying high-risk animals. In some disease-control programmes, dairy cows are classified with regards to risk according to the results of serial tests which quantify MAP antibodies in milk samples. Such classification systems are limited by the influence of non-disease factors on test results, dichotomisation of continuous results into "positive" or "negative" according to an imperfect threshold, and subjectivity in defining which patterns of serial test results indicate different risk-categories. An unsupervised learning (clustering) approach was applied to paratuberculosis test results and milk-recording data collated from 47 farms over an approximately ten-year period between 2010 and 2021. Paratuberculosis test results were first adjusted according to influential non-disease factors using linear models. Continuous-time hidden Markov models were fit to the adjusted test results. The final model revealed four distinct latent states (clusters). Examination of the distribution of adjusted test results associated with each latent state suggested that states were ordinal and aligned with disease progression. Model transition probabilities demonstrated that the probability of an animal progressing to the highest state was dependent on its current state. Of particular note was the existence of a latent state, characterised by paratuberculosis test results below the conventional test-positive threshold, which was associated with a relatively high probability of progression to the highest cluster. This research has led to objective classification of animals according to serial test results, and furthermore suggests the presence of groups of different disease risk amongst animals whose test results fall below the routinely used test-positive threshold. Identification of such groups could be used to better manage disease on farms, through implementation of management practices which limit disease transmission from high-risk animals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Aim: Diagnosis of pleural tuberculosis (TB) is challenging; thus, an efficient method is urgently needed., Methods: We developed a magnetic-bead-gold nanoparticle-PCR amplified immunoassay (MB-AuNP-I-PCR, liquid system) to detect the Mycobacterium tuberculosis MPT-64 protein in pleural TB patients. AuNPs functionalized with detection antibodies/oligonucleotides were characterized by UV-vis spectroscopy, Transmission/Scanning electron microscopy, Fourier-transform infrared spectrometer, ELISA, and PCR, whereas MBs conjugated with detection antibodies were validated by magneto-ELISA/UV-vis spectroscopy., Results: We utilized the MB-AuNP-I-PCR for MPT-64 detection in 99 clinical specimens which displayed 85.2% sensitivity and 97.8% specificity to diagnose pleural TB cases. Markedly, the sensitivity achieved by MB-AuNP-I-PCR was noticeably higher ( p  < 0.01) than magneto-ELISA and GeneXpert., Conclusion: This is a preliminary report to diagnose pleural TB cases by MB-AuNP-I-PCR with promising results that require further corroboration in a higher number of specimens.

Foot-and-mouth disease virus (FMDV) causes significant financial losses in animal farms worldwide. Specific antibody monitoring of FMDV post-vaccination is important for evaluating vaccine efficacy, as well as the prevention and control of foot-and-mouth disease (FMD). In this study, we developed a fully automatic tubular chemiluminescence immunoassay method based on magnetic particles (A-MPCLIA) to specifically detect antibodies against FMDV serotype A. The diagnostic sensitivity (Dsn) and diagnostic specificity (Dsp) of A-MPCLIA were determined to be 96.05 % and 99 %, respectively, with a cut-off value of 30 activity units (U) by detecting the known background swine, cattle, and sheep serum. The developed method presented good Dsn, Dsp, and repeatability (CV < 10 %). The activity units of A-MPCLIA, determined using 23 animals vaccinated with FMDV serotypes O, A, and Asia 1 univalent inactivated vaccine and nine serum samples from naïve animals, were significantly positively correlated with the titers of liquid-phase-blocking enzyme-linked immunosorbent assay (R 2  = 0.992) and the titer of the virus neutralization test (R 2  = 0.9577). In addition, A-MPCLIA requires less than 20 min, and it is fully automatic in detection. These results showed that A-MPCLIA is a suitable method for the detection of antibodies against FMDV serotype A in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Mi Lin reports financial support was provided by Central Government’s Guide to Local Science and Technology Development Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Histamine may lead to low blood pressure, skin flushing and edema when it accumulates in large amounts in the body. Therefore, establishing sensitive methods for the detection of histamine in foods is extremely important to ensure food safety and human health., Results: The MI-Cu-GMP NPs (2-methylimidazole-copper-guanosine monophosphate nanozymes) with high laccase-like activity were synthesized. Using the prepared molecular imprinted membrane as biomimetic antibody and MI-Cu-GMP NPs as marker, a sensitive direct competitive biomimetic enzyme-linked immunoassay (BELISA) method for rapid detection of the histamine in foods was developed. Under optimal conditions, the limit of detection (LOD, IC 15 ) and sensitivity (IC 50 ) of the BELISA method for histamine was 0.05 mg L -1 and 1.22 mg L -1 , respectively. The liquor samples spiked with histamine was detected by the BELISA method with satisfactory recoveries ranging from 90.00% to 116.00%. Further, the level of histamine in three samples (cooking wine, rice vinegar and soy sauce) was tested by the BELISA and high-performance liquid chromatography (HPLC), with no significant difference found between the two methods., Conclusion: Given the advantages, the established BELISA method is expected to provide practical guidance for the monitoring of histamine in food and provides a foundation for the detection of other food hazards. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Sheeppox and Goatpox are highly contagious transboundary viral diseases of sheep and goats caused by Capripoxvirus, respectively. This study describes the development of indirect ELISA and its serodiagnostic potential as an alternative to the gold standard serum neutralization test (SNT). The homologue of vaccinia virus, ORF 117 (A27L) gene of the Romanian Fenner (RF) strain of Sheeppox virus (SPPV) was used for producing the full-length recombinant A27L (rA27L) protein (∼22 kDa) in a prokaryotic expression system. The immunogenic potential of purified rA27L was confirmed by detecting its specific reactivity with SPPV hyperimmune sera in Western Blot analysis. An indirect ELISA based on rA27L was developed and optimized. In comparison to SNT, the rA27L-ELISA assay exhibited a diagnostic specificity of 96% and 98%, while the sensitivity was 98% and 97%, with sheep and goat sera, respectively. No cross-reactivity was noted with other transboundary diseases such as Orf, Foot-and-Mouth disease, Peste des Petits Ruminants, Haemorrhagic septicemia and Brucella. A total of 508 serum samples were tested in total for the preliminary analysis. Among these, seropositivity rates for sheep (n = 270) and goats (n = 238) were 31.85% and 47.47%, respectively. Further, to determine accuracy of developed test a total of 1731 serum samples were analyzed from the states with high small ruminant populations in India which included Andhra Pradesh, Telangana, Karnataka, West Bengal, and Gujarat. Overall seropositivity recorded were 24% in sheep and 19.12% in goats. Among the population of sheep and goats examined, the females and animals in the age group of 1-2 years showed high Capripoxvirus antibodies. This study highlighted the potential utility of rA27L protein as a safe and alternative serodiagnostic reagent, unlike the SNT, for serosurveillance and seromonitoring of Capripoxvirus infection in sheep and goats., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

The M13 phage carries approximately 5 copies of the pIII protein, each of which is capable of displaying a single-chain variable fragment (scFv) that targets a specific antigen. This feature enables the M13 phage to be widely employed in the construction of scFv libraries, thereby facilitating the identification of antibodies with high specificity and affinity for target antigens. In this study, mice were immunized three times with Salmonella enteritidis (strain C50041) to induce diverse antibodies. The variable region sequences were subsequently amplified by PCR using genome extracted from the mice's splenic cells and fused to the pIII protein to construct the scFv phage display library (C50041-M13-scFv). Through biopanning with the C50041-M13-scFv library, a phage clone (C50041-scFv-4) exhibiting high affinity for the target bacteria was successfully obtained. Moreover, the scFv antibody (scFv-4) derived from C50041-scFv-4 was expressed in a prokaryotic expression system and validated to possess high specificity and affinity for C50041 through in vitro adsorption assays. Additionally, a phage-ELISA method was established: initially, bacteria were immobilized on the bottom surface of a 96-well plate. Next, the positive clone C50041-scFv-4 was introduced to specifically bind to the host cells. Finally, horseradish peroxidase (HRP)-conjugated anti-pVIII antibodies were used to detect the pVIII proteins of the bound phage clones. Owing to the capacity of multiple C50041-scFv-4 probes to simultaneously bind to a single target Salmonella and each phage clone's ability to accommodate hundreds of HRP-labeled antibodies, the proposed phage-ELISA demonstrated remarkable sensitivity (10 4 CFU mL -1 ) for detecting Salmonella enteritidis samples. This sensitivity surpasses that of traditional ELISA by one order of magnitude in this study. Our phage-ELISA technology exhibits broad applicability across various biological species and provides an improved and robust platform for pathogen detection including bacteria and viruses.

Background: Tamdy virus (TAMV) was first isolated in Uzbekistan and Turkmenistan. In 2018, it was found in China, marking its entry into the molecular research era. TAMV is linked to febrile diseases, but its epidemiology and spillover risks are poorly understood, necessitating urgent molecular research and detection method development., Methods: The secondary structure of TAMV glycoprotein Gn was predicted, and the results showed that it had rich antigenic epitopes. According to the predicted results, glycoprotein Gn was divided into 46 truncated 16-peptides by modified synthetic peptide method, and the antigenicity of 46 truncated 16-peptides was verified by western blotting analysis., Results: The results showed that P8, P9, P24, P25, P28, P29, and P39 had antigenicity. Subsequently, the seven positive 16-peptide sequences with antigenicity were truncated to form 8-peptide sequences with an overlap of seven amino acids. After analysis with the same method, eight fine antigenic epitopes E1 ( 58 VINSTLDH 65 ), E2 ( 65 HVGSWGMP 72 ), E3 ( 68 SWGMPVTT 75 ), E4 ( 187 IRNQPFKS 194 ), E5 ( 195 FNVEVQ 200 ), E6 ( 226 AVVEHH 231 ), E7 ( 228 VEHHGNKA 235 ), and E8 ( 310 RGGRR 314 ) were identified, all of which were located on the three-dimensional surface of glycoprotein Gn and were highly conserved in different TAMV strains., Conclusions: Eight precise epitopes were identified, and an indirect ELISA method based on fusion multiepitope peptide (r-Gn-MEPX 2 ) was developed and implemented, featuring high sensitivity, accuracy, and specificity., Competing Interests: Declarations. Ethics approval and consent to participate: All procedures involving experimental animals were conducted following the Animal Husbandry Guidelines of the Wuhan Institute of Virology. The animal experiments were approved by the ethics committee of Wuhan Institute of Virology (permit no. WIVAF33202301). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Schistosoma haematobium is the causative pathogen for urogenital schistosomiasis. To achieve progress towards schistosomiasis elimination, there is a critical need for developing highly sensitive and specific tools to monitor transmission in near-elimination settings. Although antibody detection is a promising approach, it is usually unable to discriminate active infections from past ones. Moreover, crude antigens such as soluble egg antigen (SEA) show cross-reactivity with other parasitic infections, and it is difficult to formulate the standard preparations. To resolve these issues, the performances of recombinant antigens have been evaluated. The antibody responses against recombinant S. haematobium serine-protease inhibitor (ShSerpin) and RP26 were previously shown to reflect active schistosome infection in humans. Furthermore, antibody detection using multiple recombinant antigens has been reported to improve the accuracy of antibody-based assays compared to single-target assays. Therefore, we examined the performances of ShSerpin, RP26 and the mixture of these antigens for detecting S. haematobium low-intensity infection and assessed the potential for transmission monitoring., Methodology/principal Findings: We collected urine and plasma samples from school-aged children in Kwale, Kenya and evaluated S. haematobium prevalence by number of eggs in urine and worm-derived circulating anodic antigen (CAA) in plasma. Among 269 pupils, 50.2% were CAA-positive by the lateral flow test utilizing up-converting phosphor particles (UCP-LF CAA), while only 14.1% were egg-positive. IgG levels to S. haematobium SEA (ShSEA), ShSerpin, RP26, and the mixture of ShSerpin and RP26 were measured by ELISA. The mixture of ShSerpin and RP26 showed the highest sensitivity, 88.7%(125/141)among the four antigens in considering indecisive UCP-LF CAA results as negative., Conclusion/significance: IgG detection against the ShSerpin-RP26 mixture demonstrated better sensitivity for detection of active S. haematobium infection. This recombinant antigen mixture is simpler to produce with higher reproducibility and can potentially replace ShSEA in monitoring transmission under near-elimination settings., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Kokubo-Tanaka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: Ocular toxoplasmosis, caused by Toxoplasma gondii , is a significant cause of posterior uveitis and vision impairment globally. Accurate diagnosis is essential to prevent retinal damage and optimise treatment. This study aimed to compare three diagnostic methods funduscopy, serology (ELISA), and PCR in detecting ocular toxoplasmosis in patients at ECWA Eye Hospital, Kano, Nigeria., Methods and Analysis: A total of 264 patients suspected of having ocular toxoplasmosis were enrolled. Funduscopy was performed to identify retinal lesions typical of T. gondii . Serological analysis was conducted using ELISA to detect anti- Toxoplasma IgG antibodies. Additionally, PCR was used to confirm the presence of T. gondii DNA in blood samples. The results from these methods were compared with assess their sensitivity, specificity and diagnostic value., Results: Funduscopy detected T. gondii in 25.4% (67/264) of the patients, while ELISA showed a higher detection rate of 36.7% (97/264). PCR, the most specific method, confirmed infection in only 3.8% (10/264) of cases, with 60% (6/10) of these being male and 40% (4/10) female. T. gondii detected had 196 base pairs. The study found that ELISA had the highest sensitivity (90.0%) but lower specificity (64.9%), whereas funduscopy showed high specificity (74.4%) but low sensitivity (20.0%). PCR had the highest specificity but a low detection rate, emphasising its role in confirming diagnosis., Conclusion: Although ELISA and funduscopy detected more positive cases, PCR remains the gold standard for confirming T. gondii infection due to its high specificity. In resource-limited settings like ECWA Eye Hospital, combining serology and PCR offers a practical approach for improving diagnostic accuracy and ensuring timely treatment of ocular toxoplasmosis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Despite its wide use to treat various inflammatory diseases, infliximab becomes ineffective in some patients due to inadequate drug levels and production of anti-drug antibodies (ADA). The aim of this study was to compare the prevalence and ADA levels in a large cohort of patients. ADA and infliximab (IFX) through levels measured by enzyme-linked immunosorbent assay were collected from 505 patients within a period of 4 years. The results indicate that (i) 13.5% of patients produce ADA, (ii) male patients were more likely to produce ADA at levels above 10 000 ng/ml than female patients, (iii) ADA levels were lower when associated with immunosuppressant drugs, (iv) there was an inverse relationship between ADA presence and IFX detection, and (v) no correlation was observed between ADA levels and number of injections or brand of IFX administered. This study improves our understanding of the factors promoting IFX immunogenicity and highlights the need to develop personalized treatment strategies., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Dirofilaria immitis and D. repens are mosquito-borne filaroids that primarily infect dogs but also cats. Diagnosing feline dirofilariosis is challenging because of the low parasitic burdens and transient or absent microfilaremia. To improve detection of antibodies against Dirofilaria spp. in cats, an indirect enzyme-linked immunosorbent assay (ELISA) using somatic antigens of D. immitis was standardized., Methods: Serum samples from cats positive for D. immitis, D. repens, bronchopulmonary metastrongylids and gastrointestinal helminths as well as negative sera were tested to evaluate the sensitivity (Se) and specificity (Sp). Three different antigen concentrations (2, 3 and 4 μg/ml) and two concentrations of horseradish peroxidase (HRP) IgG anti-cat conjugate (1:10,000 and 1:20,000) were used to screen the optimal titration of the test. Once the best conditions were established, Se and Sp were assessed by testing 151 serum samples positive for D. immitis, D. repens and other parasites (i.e. feline lungworms, gastrointestinal helminths) or negative., Results: The optimized ELISA showed 89% Se and 98% Sp in detecting D. immitis infections, presenting a cross-reactivity with D. repens. A cut-off point of 1.150 was established to classify positive and negative samples., Conclusions: The developed ELISA may improve the detection of antibodies against Dirofilaria spp. in cats, also in epidemiological contexts characterized by multiple helminth infections. Future efforts will focus on reducing the cross-reactivity with D. repens, therefore increasing ELISA Sp., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

We report here the use of antibody-DNA conjugates (Ab-DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab-DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab-DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.

Infection with H. pylori (Helicobacter pylori) is the most prevalent human infection worldwide and is strongly associated with many gastrointestinal disorders, including gastric cancer. Endoscopy is mainly used to diagnose H. pylori infection in gastric biopsies. However, this approach is invasive, time-consuming and expensive. On the other hand, serology-based methods can be considered as a non-invasive approach to detecting H. pylori infection. The LFA (lateral flow assay) serves as a rapid point-of-care diagnostic tool. This paper-based platform facilitates the detection and quantification of analytes within human fluids such as blood, serum and urine. Due to ease of production, rapid results, and low costs, LFAs have a wide application in clinical laboratories and hospitals. In this comprehensive review, we examined LFA-based approaches for detection of H. pylori infection from human fluids and compare them with other high-sensitivity methods like ELISA (Enzyme-linked immunosorbent assay). Furthermore, we reviewed methods to elevate LFA sensitivity during H. pylori infection including, CRISPR/Cas system and isothermal amplification approaches. The development and optimization of novel labeling agents such as nanozyme to enhance the performance of LFA devices in detecting H. pylori were reviewed. These innovations aim to improve signal amplification and stability, thereby increasing the diagnostic accuracy of LFA devices. A combination of advances in LFA technology and molecular insight could significantly improve diagnostic accuracy, resulting in a significant improvement in clinical and remote diagnostic accuracy., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Brucellosis, a significant zoonotic infectious disease, poses a global health threat. Accurate and efficient diagnosis is crucial for prevention, control, and treatment of brucellosis. VirB proteins, components of the Type IV secretion system (T4SS) in Brucella , play a pivotal role in bacterial virulence and pathogenesis but have been understudied for their diagnostic potential., Methods: Tandem Mass Tag (TMT) proteomics technology was utilized to identify highly expressed VirB proteins from wild-type Brucella strains. Recombinant T4SS proteins were prepared, and an indirect ELISA method was established for serological diagnosis of human brucellosis., Results: Seven T4SS proteins (rVirB3, rVirB4, rVirB9, rBMEII0036, rVirB8, rVirB11, and rVirB10) were expressed used to construct the indirect ELISA method which showed high diagnostic accuracy. Sensitivity and specificity of the proteins exceeded 0.9100 and 0.9167, respectively, demonstrating good performance comparable to traditional LPS and Rose Bengal Ag antigens. Cross-reactivity was observed in a limited number of serum samples from febrile patients without brucellosis., Conclusions: The study highlights the potential of VirB proteins as novel diagnostic antigens for human brucellosis. Future research can further optimize the use of VirB proteins in diagnostic assays and explore their applications in vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Wu, Sun, Guo, Xie, Zhang and Yin.)

Ademetionine 1,4-Butanedisulfonate (SAMe) is widely used as a prescription drug to treat cholestasis associated with liver disease, and is also used to treat depression, Alzheimer's disease and other diseases. Currently, the main way to produce SAMe is by fermentation of S-adenosylmethionine synthetase（SAMS）. However, during the fermentation process, host cells produce contaminants unrelated to the treatment. Among them, host cell protein (HCP), which is co-purified with the drug, is the main impurity in the production process. HCP-induced antigenic reactions can lead to allergies or other adverse reactions and affect drug quality, efficacy, and safety. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) are currently used as the main analytical methods for measuring HCP. The ELISA method may ignore some HCPs with low immunogenicity, while mass spectrometry is primarily employed for the characterization of proteomes. Orthogonal methods can more effectively evaluate impurities in drugs. In this study, ELISA was initially utilized to quantify the content of Saccharomyces cerevisiae host protein in 11 batches of SAMe active pharmaceutical ingredient (API) from two suppliers, then the above APIs were qualitatively analyzed by MS method. Following the identification of surrogate peptide of SAMS, an Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry（UPLC-MS/MS）method was established and validated, the SAMS protein residues in 11 batches of samples were subsequently determined. The results demonstrated that the levels of Saccharomyces cerevisiae host protein and SAMS in the samples were both low, with values below 20 ppm and 1.19 ppm, respectively. The qualitative results also indicate that the types of peptide residues in the samples are more diverse than those of protein residues. Furthermore, residues of proteins and peptides can be detected in all samples, which underscores the importance of evaluating the HCP in API. This study employs a range of complementary detection methods to conduct a comprehensive and sensitive evaluation of total HCP and specific proteins in drugs, thereby guiding more informed assessments of the risks posed by HCP impurities in raw materials during pharmaceutical processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aflatoxin B 1 (AFB 1 ) is a potent carcinogen, and is among the most hazardous mycotoxins in agricultural products. Therefore, the development of sensitive and convenient detection methods for AFB 1 is significant for food safety against mycotoxins. Herein, a bioluminescent enzyme immunoassay (BLEIA) was developed for ultrasensitive detection of AFB 1 , based on the novel Fc-specific antibody-nanoluciferase (Ab-Nluc) conjugates which were fabricated using an IgG-binding protein-assisted photo-conjugation strategy. In indirect competitive immunoassay format, the proposed BLEIA exhibited the detection limit of 0.0232 ng mL -1 , which was 37.4-fold lower than that obtained using the classical enzyme-linked immunosorbent assay (ELISA) based on Ab-horseradish peroxidase (Ab-HRP) chemical conjugates (0.868 ng mL -1 ). Meanwhile, the BLEIA exhibited high accuracy and precision. Thus, the proposed Fc-specific Ab-Nluc conjugates-based BLEIA provides an ultrasensitive and reliable method for detecting toxins and has potential for use in food safety monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The development of traditional enzyme-linked immunosorbent assay (ELISA) systems for the detection of small-molecule residues in foods is limited because of the poor stability of biological enzymes and the lower sensitivity of absorption-based signals. Herein, two ELISAs based on Prussian blue nanoparticles (PBNPs) were developed to establish ultrasensitive and stable methods for detecting norfloxacin (NOR) in milk. The results show that the detection limit (IC 15 ) of NOR was 0.77 μg L -1 and the sensitivity (IC 50 ) was 18.28 μg L -1 in the standard solution using the PBNPs-based nano-ELISA. When the fluorescence quenching ELISA based on PBNPs was used, the detection limit was 0.06 μg L -1 and the sensitivity was 4.21 μg L -1 in the standard solution. The recoveries and precision were good, as confirmed by analysis of real milk samples. The results were consistent with those of commercial ELISA kits, indicating the high accuracy of these two methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

This study presents the first successful generation of polyclonal antibodies (pAbs) and oligonucleotide aptamers specifically targeting fusaric acid (FA). Utilizing these pAbs and aptamers, three highly sensitive and specific assays were developed for the detection of FA in cereals with limits of detection (LOD) ranging from 5 to 50 ng/g: an antibody-based enzyme-linked immunosorbent assay (ELISA), an aptamer-based enzyme-linked aptamer-sorbent assay (ELASA), and a hybrid enzyme-linked aptamer-antibody sandwich assay (ELAAA). The recovery rates of FA in spiked cereal samples ranged from 87 % to 112 % across all assays. Analysis of 15 cereal feed samples revealed FA contamination levels of 459 to 1743 ng/g (ELISA), 427 to 1960 ng/g (ELASA), and 381 to 1987 ng/g (ELAAA). These results were further validated by HPLC analysis, confirming high consistency within developed assays. Overall, the ELISA, ELASA, and ELAAA are promising tools for the rapid detection of FA, significantly contributing to food safety monitoring., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

H. pylori (Hp) is highly causative agent of chronic gastritis, gastric cancer and human death worldwide. To address the challenge of H. pylori infection, numerous immunological assays have been developed for its diagnosis and management. However, the limited availability of these assays in certain laboratories, coupled with their high cost, inconsistent specificity, and sensitivity, has hampered their widespread adoption, particularly in developing countries where H. pylori infection is prevalent. Therefore, this study aimed to develop and validate a competitive enzyme-linked immunosorbent assay (cELISA) assay for detecting H. pylori infections by targeting the Protective Surface Antigen (PSA) and Cytotoxic-Associated Gene Pathogenesis Island (Cag11) proteins in H. pylori stool antigen sample. In the current study, the optimal conditions including the dilution of anti-rPSA D15 and anti-rCag11 antibodies at 1:1000, coating antigens (rPSA D15 and rCag11) at a concentration of 1 μg/well, the dilution of HRP-labelled antibody at 1:5000 and H. pylori stool antigen dilution at 1:1000 with a 1hour incubation and color development time of 30 minutes for cELISA were determined using an ELISA checkerboard titration assay. Based on the optimized conditions, novel rPSA D15-cELISA and rCag11-cELISA assays with a respective optimum cut-off value of 20.80% PI and 24.16% PI were developed. According to the receiver operating characteristic (ROC) curve analysis on the diagnostic performance of the newly developed rPSA D15-cELISA and rCag11-cELISA assays using 60 clinical H. pylori stool samples, the rPSA D15-cELISA test assay established an optimum cut-off point of 20.80% with sensitivity and specificity of 90% (95% confidence of interval (CI) 74.38-96.54), Area under the curve (AUC) of 0.9556 (95% CI = 0.896-1.000) and P value <0.0001. Similarly, the rCag11-cELISA assay revealed optimum cut-off value of 24.16% with sensitivity of 93.33% (95% CI 78.68-98.82), specificity of 90% (95% CI 74.38-96.54), AUC of 0.986 (95% CI = 0.967-1.000) and P <0.0001. Furthermore, the reproducibility assay coefficients of variation (CV) of the newly developed rPSA D15-cELISA and rCag11-cELISA assay were less than 10%, indicating that the two cELISA assays exhibits excellent reproducibility and reliability. To validate their clinical diagnostic application, the comparative study results of rPSA D15-cELISA and rCag11-cELISA showed a high agreement (k = 0.766 and 0.799) with the commercially available H. pylori antigen test immunochromatographic kit and more accurate than the reference kit by detecting stool antigen of H. pylori strain, indicating it is promising for clinical testing. In conclusion, these results indicated that the newly developed rPSA D15-cELISA and rCag11-cELISA H. pylori stool antigen test assays were a potential reliable and a clinically useful assay for rapid, specifically, sensitively and accurately diagnosis and large-scale epidemiological investigation of H. pylori infection., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2025 Eskeziyaw et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aflatoxin B1 (AFB1) contamination of food crops pose severe public health risks, particularly in decentralized agricultural systems common in low-resource settings. Effective monitoring tools are critical for mitigating exposure, but their adoption is limited by barriers such as cost, infrastructure, and technical expertise. The objectives of this study were: (1) to evaluate common AFB1 detection methods, including enzyme-linked immunosorbent assays (ELISA) and lateral-flow assays (LFA), validated via high-performance liquid chromatography (HPLC), focusing on their suitability for possible applications in decentralized, low-resource settings; and (2) to conduct a barriers-to-use assessment for commonly available AFB1 detection methods and their applicability in low-resource settings. Among four ELISA kits, the AgraQuant Aflatoxin B1 2/50 ELISA Kit demonstrated the highest accuracy and precision, reliably quantifying AFB1 in maize and tortillas across 5-150 ppb with minimal cross-reactivity. For LFA, a smartphone-based algorithm achieved a high presence/absence accuracy rate of 84% but struggled with concentration prediction. The barriers-to-use analysis highlighted the practicality of low-cost tools like moisture readers for field screening but underscored their qualitative limitations. Advanced methods like HPLC and LC-MS offer greater precision but remain impractical due to their high costs and infrastructure requirements, suggesting a potential role for adapted ELISA or LFA methods as confirmatory approaches. These findings support the development of multi-tiered frameworks integrating affordable field tools with regional or centralized confirmatory testing. Addressing systemic barriers through capacity building, partnerships, and improved logistics will enhance AFB1 monitoring in decentralized systems, protecting public health in vulnerable communities.

Plant signaling peptides, also known as phytocytokines, play a crucial role in cell-to-cell communication during plant development and immunity. The detection of small peptides in plant tissues is challenging and often relies on time-consuming and cost-intensive approaches. Here, we present an ELISA-based assay as a rapid and cost-effective method for the detection of naturally released peptides in plant tissues. Our ELISA-based method was developed to detect Zip1, a 17-amino-acid phytocytokine derived from Zea mays that elicits salicylic acid signaling in maize leaves. Using a custom peptide-antibody, we designed an experimental pipeline to achieve peptide specificity, selectivity, and sensitivity allowing the detection of the Zip1 peptide in complex biological samples. As a proof of concept, we first overexpressed the precursor molecule PROZIP1 in Nicotiana benthamiana and in transfected maize protoplasts and monitored the release of Zip1-containing peptides. In a second approach we treated maize leaves with salicylic acid to induce native PROZIP1 expression and processing. Using ELISA, we were able to quantify native Zip1 signals with a detection limit in the nanogram range, which allowed us to detect different Zip1-containing peptides in plant material. This method can be adapted for the detection and quantification of a variety of plant signaling peptides., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

An obstacle for many microfluidic developments is the fabrication of its structures, which is often complex, time-consuming, and expensive. Additive manufacturing can help to reduce these barriers. This study investigated whether the results of a microfluidic assay for the detection of the promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion protein (PML::RARA), and thus for the differential diagnosis of acute promyelocytic leukemia (APL), could be transferred from borosilicate glass microfluidic structures to additively manufactured fluidics. Digital light processing (DLP) and stereolithography (SLA) printers as well as different photopolymerizable methacrylate-based resins were tested for fabrication of the fluidics. To assess suitability, both print resolution and various physical properties, serializability, biocompatibility, and functionalization with biological molecules were analyzed. The results show that additively manufactured microfluidics are suitable for application in leukemia diagnostics. This was demonstrated by transferring the microfluidic sandwich enzyme-linked immunosorbent assay (ELISA) for PML::RARA onto the surface of magnetic microparticles from a glass structure to three-dimensional (3D)-printed parts. A comparison with conventional glass microstructures suggests lower sensitivity but highlights the potential of additive manufacturing for prototyping microfluidics. This may contribute to the wider use of microfluidics in biotechnological or medical applications.

The enzyme-linked immunosorbent assay (ELISA) stands as one of the most frequently employed rapid detection techniques for both chemical and biological contaminants. Horseradish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ), serving as signal generators, play indispensable roles throughout the entire ELISA process. However, HRP and H 2 O 2 exhibit high sensitivity to elevated temperatures, hindering the broader utilization and transportation of ELISA. Hereby, being inspired by the cascade catalysis of glucose by glucose oxidase (GOx) and HRP as well as the advantageous properties of metal-organic frameworks (MOFs), we developed a cascade catalysis-based competitive fluorescent ELISA (CCF-ELISA) for mycotoxin detection in maize using zearalenone (ZEN) as a model. The results revealed the remarkable protective effects of MOFs on enzymatic activities. Based on upon characteristics, the built CCF-ELISA enabled the detection of ZEN with a half-maximal inhibitory concentration of 0.54 ng/mL and allowed a detection range from 0.19 to 1.51 ng/mL. When applied to naturally contaminated maize samples, the outcomes obtained from the CCF-ELISA showed a high level of comparability with high-performance liquid chromatography-tandem mass spectrometry, demonstrating the promising potential of the developed CCF-ELISA in the rapid detection for food security.

Objective: Gastric adenocarcinoma (GAC) is the 17th most common cancer in the UK with a 5-year survival rate of 22%. GastroPanel (Biohit Oyj; Helsinki, Finland) is an ELISA kit that measures pepsinogen I (PGI); pepsinogen II (PGII); gastrin-17 (G-17); and Helicobacter pylori IgG antibodies (Hp IgG). PGI and the PGI/PGII ratio correlate inversely with the severity of chronic atrophic gastritis (AG). The aim of this study was to assess GastroPanel performance in the identification of moderate to severe AG in dyspepsia., Methods: In this UK, single-centre, prospective diagnostic accuracy study, 324 patients [56.8% (n=184) female; median age 57 years (range 39-92 years)] were recruited for gastroscopy with biopsy and histology according to the updated Sydney System (USS). Blood (plasma) samples were collected for GastroPanel analysis. Paired samples were obtained from 268 patients [56.3% (n=151) female; median age=57 (range 39-92 years)]. GastroPanel results were interpreted using the GastroSoft app (Biohit)., Results: Overall agreement between GastroPanel and the USS classification was 90% (95% CI=86.7 to 93.8%), with a weighted kappa (κw) of 0.828 (95% CI=0.781 to 0.865). In receiver operating characteristics (ROC) curve analysis, using moderate/severe atrophic gastritis of the corpus (AGC2+) as the endpoint, AUC=0.840 (95% CI 0.630 to 1.000) and 0.960 (95% CI 0.907 to 1.000) for PGI and the PGI/PGII ratio, respectively., Conclusion: GastroPanel is a reliable dyspepsia triage test distinguishing patients who can be safely treated conservatively from those with moderate to severe corpus atrophic gastritis at high risk of developing GAC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Aflatoxin M1 (AFM1) is known to be carcinogenic, mutagenic, and teratogenic and poses a serious threat to food safety and human health, which makes its surveillance critical. In this study, an indirect competitive ELISA (icELISA) based on a nanobody (Nb M4) was developed for the sensitive and rapid detection of AFM1 in dairy products. In our previous work, Nb M4 was screened from a Bactrian-camel-immunized phage-displayed library. It exhibits VH-like features, possesses higher thermal stability than monoclonal antibody (mAb 1E6) and tightly binds to AFM1-BSA with a K D value of 2.5 nM. Under the optimal conditions, its half-maximal inhibitory concentration was 0.338 ng/mL, the limit of detection was 0.051 ng/mL, and linearity was noted in the range of 0.168-0.679 ng/mL. Nb M4 displayed almost no cross-reactivity with other mycotoxins. No matrix effect was observed in milk and milk powder samples, and the matrix effect in yogurt samples could be weakened by 2-fold dilution. Furthermore, validation studies in spiked samples (milk, yogurt, and milk powder) resulted in good recoveries of 95.40-111.33%, with a low coefficient of variation (2.89-6.78%). High-performance liquid chromatography was used to evaluate the accuracy and reliability of the developed icELISA, which indicated a satisfactory consistent correlation (R 2  = 0.9722). This study highlights the potential of Nb M4 as a promising component for detecting AFM1 in dairy products., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Host cell proteins (HCPs) coexpressed during the production of biotherapeutics can affect the safety, efficacy, and stability of the final product. As such, monitoring HCP populations and amounts throughout the production and purification process is an essential part of the overall quality control framework. Mass spectrometry (MS) is used as an orthogonal method to enzyme-linked immunosorbent assays (ELISA) for the simultaneous identification and quantification of HCPs, particularly for the analysis of downstream processes. In this study, we present an MS-based analytical protocol with improvements in both speed and identification performance that can be implemented for routine analysis to support upstream process development. The protocol adopts a streamlined sample preparation strategy, combined with a high-throughput MS analysis pipeline. The developed method identifies and quantifies over 1000 HCPs, including 20 proteins listed as high risk in the literature, in a clarified cell culture sample with repeatability and precision shown for digest replicates. In addition, we explore the effects of varying standard spike-ins and changes to the data processing pipeline on absolute quantification estimates of the HCPs, which highlight the importance of standardization for wider use in the industry. Data are available via ProteomeXchange with the identifier PXD053035.

This study is the first to conduct a sero-surveillance of Bovine Tropical Theileriosis (BTT) caused by the protozoan parasite Theileria annulata (T. annulata) using a recombinant Tams1 protein-based dot-ELISA in cattle, and to compare its efficacy with plate-ELISA, single PCR, nested PCR, and blood microscopy. The goal was to identify the most effective method for the early and accurate detection of theileriosis, which significantly impacts livestock through reduced milk yield and increased mortality. A total of 101 field blood samples were examined using blood smear analysis, single PCR, nested PCR, and dot-ELISA. The recombinant Tams1 protein was successfully cloned and expressed using a pET-30b (+) expression vector in a prokaryotic system. The protein was purified with Ni-NTA chromatography, confirmed for immunoreactivity with T. annulata positive serum via Western blot analysis, and used to optimize both dot-ELISA and plate-ELISA. Both dot-ELISA and plate-ELISA using recombinant Tams1 protein exhibited comparable diagnostic performance, with a kappa value of 0.826 and similar analytical productivity (P = 0.6165). Dot-ELISA revealed a BTT seroprevalence of 58.4% in the cattle population, demonstrating good sensitivity (93.33%) and specificity (90%). The diagnostic performance of dot-ELISA was found to be superior to other molecular techniques, including microscopy, single PCR, and nested PCR. Dot-ELISA is also a sustainable solution in comparison to other laboratory diagnostic techniques with benefits of early diagnosis, reduced waste generation, resource efficiency, cost-effective point of care disease surveillance. With its minimal antigen requirement, Tams1 molecule based dot-ELISA is recommended as an effective tool for epidemiological studies and field surveys of BTT., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Rose Bengal antigen and smooth lipopolysaccharide (s-LPS) were produced from a field strain of Brucella ceti ("homologous" antigens) and from the reference strain B. abortus S99 ("heterologous" antigens); they are currently used for the diagnosis of brucellosis in cattle, water buffaloes, sheep, goats, and pigs, as recommended in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (WOAH). "Homologous" and "heterologous" antigens were used in a rapid serum agglutination test (Rose Bengal test, RBT) and a competitive ELISA assay (c-ELISA) to test a panel of sera, blood, and other body fluids (cerebrospinal fluid, pericardial fluid, tracheal fluid, and aqueous humor) collected from 71 individuals belonging to five cetacean species ( Stenella coeruleoalba ; Tursiops truncatus ; Grampus griseus ; Globicephala melas ; and Ziphius cavirostris ), which were found stranded on the Italian coastline. Six animals were positive for Brucella spp. for bacterial isolation and/or PCR, and 55 animals were negative; for the remaining 10 animals, no PCR/isolation data were available. A total of 90 samples were tested. Results obtained from the two tests were compared in order to identify the most suitable antigen for the serological diagnosis of Brucella infection in cetaceans. The RBT performed with the "homologous" antigen showed the best results in comparison with the "heterologous" antigen: diagnostic sensitivity, specificity, and accuracy were 80.0%, 44.1%, and 46.9% for the "homologous" antigen and 80.0%, 17.0%, and 21.9% for the "heterologous" antigen. For the c-ELISA, "homologous" and "heterologous" s-LPS showed similar results (diagnostic sensitivity 66.7%, diagnostic specificity 97.3%, and diagnostic accuracy 95.0%). Therefore, the RBT using the "homologous" antigen and c-ELISA with "homologous" or "heterologous" s-LPS could be used in parallel for the detection of antibodies against Brucella spp. in cetaceans.

The California serogroup (CSG) viruses are orthobunyaviruses endemic in North America and responsible for the second most common cause of mosquito-borne viral encephalitis in the United States. As the CSG viruses have been neglected and are poorly studied, there are no commercial diagnostic serological assays or reagents available for detection. Therefore, diagnostic laboratories have had to rely on the development of their own in-house serological assays. To develop serological assays, antigenic materials such as recombinant protein, virus-like particles (VLP), or inactivated virus can be used in the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) to detect CSG virus-specific IgM antibodies. All positive MAC-ELISA results should be confirmed by a well-defined gold standard test method such as the plaque-reduction neutralization test (PRNT)., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: In the diagnosis of linear IgA bullous dermatosis (LABD), detection of IgA at the epidermal basement membrane zone and circulating IgA autoantibodies are essential. The disease has two subtypes, lamina lucida-type and sublamina densa-type, with 120 kDa LAD-1 and 97 kDa LABD97 as major autoantigens for lamina lucida-type. Normal human epidermal keratinocytes (NHEK) and HaCaT cells are widely used for immunoblotting (IB) in the diagnosis process, but they do not provide high sensitivity and semiquantitative analysis., Objective: To develop a more sensitive and convenient method for detecting IgA antibodies in lamina lucida-type LABD patients., Methods: The expressions of LAD-1 and LABD97 in lysates and culture supernatants from Ker-CT, HaCaT, DJM-1, and NHEK were compared. The sensitivity of IBs using concentrated culture supernatants of HaCaT and Ker-CT and ELISAs using several recombinant proteins (RPs) corresponding to BP180 ectodomain were compared using 55 sera from LABD patients., Results: In culture supernatant, Ker-CT expressed higher amounts of LAD-1 and LABD97. IBs using concentrated culture supernatant of HaCaT and Ker-CT showed 43 % and 46 % positivity to sera from LABD patients, respectively. In ELISAs, the RP of amino acids 490-1421 of BP180 showed the highest positivity (80.0 %) among several proteins. Additionally, this ELISA showed reduced OD values in LABD and related diseases patients' sera at remission., Conclusion: The ELISA using the RP coding amino acids 490-1421 of BP180 is useful for identifying IgA antibodies and monitoring disease activity in lamina lucida-type LABD patients., (Copyright © 2024 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Schistosoma mansoni poses a considerable global public health challenge. In Egypt, approximately 60% of the inhabitants in the Northern and Eastern areas of the Nile Delta are affected by this parasite, whereas the Southern region experiences a significantly lower infection rate of 6%., Aim: Construction of an immune phage display Nbs library based on the VHH framework for selecting S. mansoni -specific Nbs for seeking cost-effective, sensitive, and specific diagnostic tools for rapidly detecting Schistosoma mansoni., Methods: Camel was immunized using soluble adult worm antigens (SAWP) for the production of Variable domains of heavy chains of camelid heavy-chain only antibodies (VHHs), which are known as nanobodies (Nb). The PBMCs repertoires VHH sequences library have been constructed with a high percentage of insertion and right orientation using pADL-23c phagmid and M13 phage followed by three rounds of bio-panning against SAWP using phage display technique. Evaluations using polyclonal phage ELISA and other techniques have been carried out to reveal the successful enrichment of anti-SAWP Nbs (VHH) clones. Evaluation of the diagnostic potentiality of these Nbs was carried out using ELISA on human serum samples confirmed for S. mansoni infection. Receiver Operator of Characteristics (ROC) curve analysis was used for discrimination between S. mansoni infection and both negative controls and the Fasciola hepatica group., Results: Using monoclonal ELISA, Nbs of 22 clones out of 24 selected clones showed binding affinity to SAWP. The cutoff values of the produced anti- S. mansoni Nbs was > 0.19, leading to 80% sensitivity, 95% specificity, and 90% accuracy. Sequence analysis of three of these Nbs with high binding affinities showed diversity in their targets, considering their CDR3 aa sequences., Conclusion: This study successfully generated a diverse phage library enriched with anti-S. mansoni VHHs. The nanobodies produced exhibit high diagnostic potential for detecting S. mansoni infection in human patients, offering a promising avenue for the development of efficient diagnostic tools. The innovative approach described herein may have potential applications for patent considerations in the field of the field of diagnostic technology., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This study assessed the diagnostic potential of nonpolar lipid extracts in enzyme-linked immunosorbent assays (ELISAs) for tuberculosis (TB) serodiagnosis. Nonpolar lipid extracts were harvested from Mycobacterium tuberculosis (Mtb) knockout in mce1 operon (∆mce1) and its parental wild type (WT) strains. IgM and IgG anti-nonpolar lipid serum levels were measured in TB patients (n=45), healthy individuals with positive (n=22) and negative (n=44) interferon-gamma release assay (IGRA) results, and symptomatic respiratory (SR) patients with negative TB tests (n=9). IgG anti-WT lipid distinguished TB patients from IGRA-positive individuals with 60% sensitivity and 77.3% specificity. Conversely, IgG anti-∆mce lipid levels didn't vary significantly across groups. Interestingly, most SR patients exhibited significantly higher IgM and IgG anti-WT lipid titers than the IGRA-positive and -nega groups. While the overall diagnostic potential of Mtb nonpolar lipids was limited, the impaired immunogenecity of Δmce1 lipid extract suggests that some missing lipid classes in this extract can potentially induce antibody production in TB patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sandwich enzyme-linked immunosorbent assays (ELISAs), which quantify antigens captured between two layers of antibodies, are sensitive and fundamental tools used to diagnose diseases, evaluate the effects of countermeasures, and quantify target proteins. Many filoviruses use transcriptional editing to express three different glycoproteins (GPs) from the GP gene. The main product transcribed from the GP gene, the soluble glycoprotein (sGP), is detected at high levels in the blood during the acute phase of infection and correlates with viremia, making it an attractive target for molecular diagnostics. Here, we describe a sandwich ELISA-based method for quantifying filovirus sGP., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Myxoma (MYXV) and rabbit hemorrhagic disease (RHDV) viruses are pathogens of economic relevance for cuniculture and conservation concern for wild European rabbits (Oryctolagus cuniculus), recently classified as 'Endangered' in its native range. Large-scale serological surveys, facilitated by sample collection using dried blood spots (DBS), allow monitoring seroprevalence in the wild reservoir but require evaluating the technique for the host and pathogen of interest. This study aimed to evaluate Protein Saver 903 DBS for MYXV and RHDV (genotype GI.2) serological surveys in European rabbits. Paired serum and DBS collected from 172 rabbits harvested or found dead in the Iberian Peninsula were tested for IgG antibodies specific against MYXV and RHDV GI.2 using indirect ELISA. We found an almost perfect agreement between serum and DBS for MYXV (Cohen's κ=0.914, CI 95 0.847 - 0.981) and a strong agreement for RHDV GI.2 (Cohen's κ=0.808, CI 95 =0.722 - 0.893). The diagnostic sensitivity of DBS was 95.4 % (CI 95 90.3 - 97.9 %) for MYXV and 82.1 % (CI 95 73.2 - 88.5 %) for RHDV GI.2. The diagnostic specificity and positive predictive value were 100 % for both pathogens. This study supports DBS as a suitable sampling strategy for serological surveys of antibodies specific to MYXV and RHDV GI.2 in European rabbits, which generally agrees with results from other hosts and pathogens where this technique was evaluated., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Human trichinosis is a serious foodborne parasitic zoonosis. Diagnosing human trichinosis is usually difficult due to the nonspecific clinical picture and the limited effectiveness of serological tests in acute infections. While ELISA can detect circulating Trichinella antigens, aiding in early diagnosis, its sensitivity may be low. The application of nanoparticles can improve the sensitivity of ELISA and allow a specific early diagnosis of the disease. This work compares the nano chitosan beads-based ELISA (NCSB-ELISA) and traditional sandwich ELISA for the detection of circulating Trichinella spiralis (T. spiralis) crude extract-antigen (CEA) in serum samples of experimentally infected mice. Fifty-seven mice included in this study were classified into 3 groups: T. spiralis infected group (Group I) (36 mice), which was equally subdivided into six subgroups according to the time of sacrifice (6, 8, 10, 12, 14 and 16) days post-infection (dpi), cross-reactivity group (Group II) (9 mice) and negative control group (Group III) (12 mice). T. spiralis AW-CEA prepared from the adult worms were used to produce anti- T. spiralis IgG-polyclonal antibodies in rabbits; these antibodies were utilized to detect AW-CEA in serum samples by traditional sandwich ELISA and NCSB-ELISA. Using NCSB-ELISA, T. spiralis AW-CEA was detected in sera collected at 8 dpi, with a sensitivity of 50% and a specificity of 100%. Meanwhile, traditional sandwich ELISA could not detect the antigen at the same time interval. Both ELISA were able to detect the antigen in samples collected at 10, 12, 14 and 16 dpi with a sensitivity of 16.67%, 50%, 67.67% and 83.67%, respectively, for traditional sandwich-ELISA and a specificity of 100% at 10, 12 and 14 dpi while at 16 dpi specificity was decreased to 90.91%. In contrast, the sensitivity of NCSB-sandwich ELISA on the same days was 66.67%, 83.34%, 100% and 100%, respectively, with a specificity of 100% at all days. False positive detection of T. spiralis AW-CEA in the serum of mice in GII was recorded on day 16 pi by only traditional sandwich ELISA. This study concluded that NCSB-ELISA is a promising and sensitive technique for the early and specific diagnosis of acute trichinosis in an animal model., Competing Interests: Declaration of competing interest Authors declare that no conflict of interest exists. The manuscript has not been published and is not under consideration elsewhere., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Identifying and quantifying host cell proteins (HCPs) are crucial in developing and manufacturing biopharmaceutical products. ELISA has been used widely for identifying and quantifying HCPs in biopharmaceutical products. Liquid chromatography-mass spectrometry (LC-MS) has recently emerged as an orthogonal tool for HCP analysis, providing comprehensive and quantitative information on individual HCPs. This chapter explores the advancements in LC-MS/MS methodologies for HCP analysis, including sample preparation, data acquisition modes, and data analysis strategies. It also discusses the challenges of HCP identification and quantitation, and recent innovations to overcome these limitations. The application of LC-MS/MS methods in HCP profiling holds significant promise for improving process control and ensuring product safety in biopharmaceutical manufacturing., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)