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An electrochemical biosensor has been developed for detection of Escherichia coli O157 by integrating lateral flow with screen-printed electrodes. The screen-printed electrodes were attached under the lateral flow detection line, and organic-inorganic nanoflowers prepared from E. coli O157-specific antibodies as an organic component were attached to the lateral flow detection line. In the presence of E. coli O157, an organic-inorganic nanoflower-E. coli O157-antimicrobial peptide-labelled ferrocene sandwich structure is formed on the lateral flow detection line. Differential pulse voltammetry is applied using a smartphone-based device to monitor ferrocene on the detection line. The resulting electrochemical biosensor could specifically detect E. coli O157 with a limit of detection of 25 colony-forming units mL -1 . Through substitution of antibodies of organic components in organic-inorganic nanoflowers, biosensors have great potential for the detection of other pathogens in biomedical research and clinical diagnosis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

In this study, the covalently fixed "end-on" orientation of a monoclonal Listeria monocytogenes antibody (mAb- Lis ) to amino terminated oligo (ethylene glycol)-capped gold nanoparticles (NH 2 -TEG-AuNPs) was used to fabricate an in-house lateral flow strip (LFS), namely, the fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS. The aim was to evaluate the performance of the fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS in detecting L. monocytogenes . The proposed LFS enabled the sensitive detection of L. monocytogenes in 15 min with a visual limit of detection of 10 2 CFU/mL. Quantitative analysis indicated an LOD at 10 CFU/mL. The fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS showed no cross-reactivity with other pathogenic bacteria and practical performance across different food matrices, including human blood, milk, and mushroom samples. Furthermore, the clinical performance of the fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS for detecting L. monocytogenes was evaluated by using 12 clinical samples validated by the hemoculture method. It demonstrated excellent concordance with the reference methods, with no false-positive or false-negative results observed. Therefore, the fixed "end-on" Lis -mAb-NH-TEG-AuNPs LFS serves as a promising candidate for a point-of-care test (POCT), enabling the rapid, precise, and highly sensitive detection of L. monocytogenes in clinical samples and contaminated food.

Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development., 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.)

319-44 is a human monoclonal antibody capable of passively protecting mice against tick-mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319-44 has complement-dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.2 Å-resolution crystal structure of 319-44 Fab fragments in complex with Outer surface protein A (OspA), the ~30 kDa lipoprotein that was the basis of the first-generation Lyme disease vaccine approved in the United States. The 319-44 epitope is focused on OspA β-strands 19, 20, and 21, and the loops between β-strands 16-17, 18-19, and 20-21. Contact with loop 20-21 explains competition with LA-2, the murine monoclonal antibody used to estimate serum borreliacidal activities in the first-generation Lyme disease vaccine clinical trials. A high-resolution B-cell epitope map of OspA will accelerate structure-based design of second generation OspA-based vaccines., (© 2023 Wiley Periodicals LLC.)

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO 4 ) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO 4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

This study developed a novel method for the rapid detection of Escherichia coli ( E. coli ) O157:H7 on a microfluidic platform. First, the concentration of bacteria in a sample was determined with the adenosine triphosphate (ATP) method. Then, the specific detection of E. coli was achieved in a microfluidic chip by the immune-microsphere technique. The influences of the culture time, flow rate and capture time on the detection of the target bacteria were investigated systematically. Generally, with increasing capture time, more bacteria could be captured by the microspheres, which had a positive effect on bacterial detection. Furthermore, the sensitivity and specificity of the method were also tested. The results showed that this method could specifically detect E. coli with a sensitivity as high as 49.1 cfu/μL; the consumption of bacteria was 1 μL, and the reagent was at the microliter level. The testing time can be controlled within one and a half hours, and the cost of testing was approximately RMB 10. The method described in this article is simple and accurate and has great application value in bacterial detection for medical diagnostics.

Staphylococcal enterotoxin B (SEB) simultaneously crosslinks MHC class II antigen and TCR, promoting proliferation of T cells and releasing a large number of toxic cytokines. In this report, we computationally examined the possibility of using a single-chain biparatopic bispecific antibody to target SEB and prevent TCR binding. The design was inspired by the observation that mixing two anti-SEB antibodies 14G8 and 6D3 can block SEB-TCR activation, and we used 14G8-6D3-SEB tertiary crystal structure as a template. Twelve simulation systems were constructed to systematically examine the effects of the designed bispecific scFV MB102a, including isolated SEB, MB102a with different linkers, MB102a-SEB complex, MB102a-SEB-TCRβ complex, MB102a-SEB-TCR-MHC II complex, and MB102a-SEB-MHC II. Our all atom molecular dynamics simulations (total 18,900 ns) confirmed that the designed single-chain bispecific antibody may allosterically prevent SEB-TCRβ chain binding and inhibit SEB-TCR-MHC II formation. Subsequent analysis indicated that the binding of scFV to SEB correlates with SEB-TCR binding site motion and weakens SEB-TCR interactions., Competing Interests: Author BM was employed by company Molcell Biodesign, Inc. The remaining 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 © 2021 Bai, Ge, Su, Chen, Zeng, Lu and Ma.)

Tuberculosis (TB), a contagious disease mainly caused by Mycobacterium tuberculosis (M. tb), Mycobacterium bovis (M. bovis), and Mycobacterium caprae (M. caprae), poses a major global threat to the health of humans and many species of animals. Developing an ante-mortem detection technique for different species would be of significance in improving the surveillance employing a One Health strategy. To achieve this goal, a universal indirect ELISA was established for serologically detecting Mycobacterium tuberculosis complex infection for multiple live hosts by using a fusion protein of MPB70, MPB83, ESAT6, and CFP10 common in M. tb, M. bovis, and M. caprae as the coating antigen (MMEC) and HRP-labeled fusion protein A and G as a secondary antibody. After testing the known positive and negative sera, the receiver operating characteristic curves were constructed to decide the cut-off values. Then, the diagnostic sensitivity and specificity of MMEC/AG-iELISA were determined as 100.00% (95% CI: 96.90%, 100.00%) and 100.00% (95% CI: 98.44%, 100.00%) for M. bovis infection of cattle, 100.00% (95% CI: 95.00%, 100.00%) and 100.0% (95% CI: 96.80%, 100.00%) for M. bovis infection of sheep, 90.74% (95% CI: 80.09%, 95.98%) and 98.63% (95% CI: 95.14%, 99.76%) for M. bovis infection of cervids, 100.00% (95% CI: 15.81%, 100.00%) and 98.81% (95% CI: 93.54%, 99.97%) for M. bovis infection of monkeys, 100.00% (95% CI: 86.82%, 100.00%) and 94.85% (95% CI: 91.22%, 97.03%) for M. tb infection of humans. Furthermore, this MMEC/AG-iELISA likely detects M. caprae infection in roe deer. Thus this method has a promising application in serological TB surveillance for multiple animal species thereby providing evidence for taking further action in TB control., Competing Interests: The authors have declared that no competing interests exist.

Nanobodies against cell surface antigens of toxic cyanobacteria Microcystis aeruginosa were recovered by whole-cell biopanning of a naïve phage display library of nanobodies. Six unique sequences were identified and three sub-cloned and purified as fusion immunoreagents together with either green fluorescent protein or AviTag to be used for diagnostics. The yields of nanobody constructs were in the range of 5-10 mg/l and their specificity and sensitivity was initially evaluated by immunofluorescence and by fluorescent enzyme-linked immunosorbent assay (ELISA) using fluorescent nanobodies. The ELISA data confirmed the nanobody specificity but showed that the saturation of the fluorescence signal already in the presence of few hundreds of cells limited the dynamic range of the method. As an alternative, Avi-tagged nanobodies were used in combination with streptavidin-linked horseradish peroxidase for developing a diagnostic colorimetric cell ELISA, the limit-of-detection of which was 3.2 and 4.5 cells/ml for the two tested cyanobacteria strains, whereas the linear range of the assay was expanded from 10 to 10,000 cells. The fluorescent nanobodies were finally exploited for quantifying cyanobacteria by thermal lens spectrometry (TLS) that enabled to reach a limit-of-detection of 1.2 cells/ml and provided a linear range of measurement between 0 and 10,000 cells. No cross-reactivity with unrelated microalgae was detected and both colorimetric ELISA and TLS provided a linear range of detection of few logs. The data indicate that nanobodies are suitable capture reagents and that both TLS and colorimetric ELISA are reliable to monitor variations of cyanobacteria populations., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviously, detecting reagents such as a specific antibody is essential for the development of immune-based detection methods for C. jejuni or C. coli. In this study, in silico techniques were used to design a chimeric recombinant antigen, named multiepitope antigen (MEA), for the production of specific polyclonal antibody. To design MEA polypeptide based on C. jejuni fibronectin-binding protein or CadF, four conserved and unique antigenic peptides were identified and fused together directly. The C. jejuni CadF-based MEA polypeptide fused with two single six-histidine tags at both C- and N-terminal ends was expressed under Escherichia coli expression system. The recombinant MEA was successfully produced and purified by Ni-NTA resin with a high satisfactory yield. Indirect ELISA results showed that anti-MEA polyclonal antibody derived from rabbit serum had a titer of 16,000, indicating high antigenicity of MEA polypeptide. Dot blot results also confirmed that the produced anti-MEA antibody could specifically recognize both C. jejuni and C. coli whole cells as expected while there was no cross-reactivity to non-Campylobacter spp. tested in this study., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The immunochromatographic strip test (ICST) is a powerful on-site detection technology due to its unique advantages of simplicity, rapidity, and readability by the naked eye. Here we illustrate the potential of α-Fe 2 O 3 polyhedrons as a novel visual label, which exhibit advantages of high stability and economy, for the detection of Listeria monocytogenes (L. monocytogenes) as a model foodborne pathogen. A low-cost and simple one-step solvothermal approach was developed for the synthesis of α-Fe 2 O 3 polyhedrons; the average diameter of the α-Fe 2 O 3 polyhedrons is about 200 nm. The crystal structure and morphology of α-Fe 2 O 3 polyhedrons were characterized by x-ray diffraction and transmission electron microscope. α-Fe 2 O 3 polyhedrons were immunized with anti-L. monocytogenes antibody to prepare an antibody-colloidal α-Fe 2 O 3 polyhedron ICST. Visual detection can be obtained directly by the naked eye within 10 min. The detection limit of L. monocytogenes by α-Fe 2 O 3 polyhedron ICST assay was 3.8 × 10 6 and 5.6 × 10 6 CFU/ml of pure culture and artificially spiked orange juice drink sample, respectively. Results indicated that the antibody-colloidal α-Fe 2 O 3 polyhedron ICST is a rapid, simple, and low-cost assay. This approach showed great potential in the application of foodborne pathogen detection concerning food safety.

Detection of bacterial pathogens is the need of the hour due to the increase in antibiotic resistance and the infusion of multi-drug-resistant parasites. The conventional strategies such as ELISA, PCR, and MNP based tests for the detection are efficient but they are cost, time, lab, and manpower intensive. Thus, warranting a simple and effective technique for rapid detection of bacterial pathogens. Magnetic nanoparticles (NPs) have proved to be better alternatives for separation of bacterial pathogens from a variety of sample sources. However, the use of magnetic NPs has not been successful in the detection of these parasites. The current work involves the coating of magnetic NPs (Fe 3 O 4 ) with a conducting polymer (polypyrrole; Ppy) to facilitate simultaneous separation and detection. Electrical (conductivity) measurement was the mode of choice due to the sensitivity, accuracy, and ease it offers. To enhance the conductivity, carboxylic groups were expressed on the Fe 3 O 4 @Ppy complex and to ensure specificity, E. coli specific antibodies were conjugated. The resulting complex at various process parameters was characterized using FTIR, VSM, and SEM. SEM images were recorded to ensure bacterial separation at optimal process parameters. The impedance analysis and conductivity measurements were carried out for the sample volume of 15 μl. The bacterial suspension from 10 1 -10 6 CFU ml -1 was successfully detected with a limit of detection of 10 CFU ml -1 within 10 min using a simplistic detection method.

Background: Brucellosis is a serious zoonosis disease that frequently causes significant economic loss in animal husbandry and threatens human health. Therefore, we established a rapid, accurate, simple and sensitive fluorescent immunochromatographic strip test (ICST) based on quantum dots (QDs) for detection the antibodies of Brucella infection animals serum., Results: The test strips were successfully prepared by quantum dot fluorescent microspheres (QDFM) as tracers, which were covalently coupled to an outer membrane protein of Brucella OMP22. The outer membrane protein OMP28 and monoclonal antibodies of OMP22 were separately dispensed onto a nitrocellulose membrane as test and quality control lines, respectively. The critical threshold for determining negative or positive through the ratio of the fluorescent signal of the test line and the control line (H T / H C ) is 0.0492. The repeatability was excellent with an overall average CV of 8.78%. Under optimum conditions, the limit of detection was 1.05 ng/mL (1:512 dilution). With regard to the detection of brucellosis in 150 clinical samples, the total coincidence rate of ICST and Rose Bengal plate test (RBPT) was 97.3%, the coincidence rate of positive samples was 98.8%, the coincidence rate of negative samples was 95.3%, the sensitivity of RBPT is 1:32, and no cross reaction with the sera of other related diseases was observed., Conclusion: In our present study, the QDFM has promising application for on-site screening of brucellosis owing to its high detection speed, high sensitivity, high specificity and low cost.

An electrochemical immunosensor based on self-assembled gold nanorods on glassy carbon electrode was developed for label-free and sensitive detection of Staphylococcus aureus (S. aureus). The gold nanorods were firstly assembled on the electrode surface by using poly-(diallyldimethylammonium chloride) (PDDA) and poly-(styrenesulfonate) (PSS) as the linkers, followed by the functionlization of anti-S. aureus antibodies. The immobilized antibodies on self-assembled gold nanorods could efficiently capture S. aureus to the modified electrode by the specific immune reaction, which clearly blocked the electron transfer of electrochemical probes on the electrode surface due to the resistance of S. aureus. Atomic force microscopy and electrochemical impedance spectroscopy were used to verify the stepwise assembly of the immunosensor fabrication. The immunosensor could detect S. aureus in a linear range from 1.8 × 10 3 to 1.8 × 10 7  CFU mL -1 with a low detection limit of 2.4 × 10 2  CFU mL -1 . Furthermore, the designed electrochemical immunosensor was successfully used to determine S. aureus in milk samples with acceptable results. The proposed immunosensor could be further expanded to sensitive detect other pathogens with the addition of specific antibodies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The identification of rice bacterial leaf blight disease requires a simple, rapid, highly sensitive, and quantitative approach that can be applied as an early detection monitoring tool in rice health. This paper highlights the development of a turn-off fluorescence-based immunoassay for the early detection of Xanthomonas oryzae pv. oryzae (Xoo), a gram-negative bacterium that causes rice bacterial leaf blight disease. Antibodies against Xoo bacterial cells were produced as specific bio-recognition molecules and the conjugation of these antibodies with graphene quantum dots and gold nanoparticles was performed and characterized, respectively. The combination of both these bio-probes as a fluorescent donor and metal quencher led to changes in the fluorescence signal. The immunoreaction between AntiXoo-GQDs, Xoo cells, and AntiXoo-AuNPs in the immuno-aggregation complex led to the energy transfer in the turn-off fluorescence-based quenching system. The change in fluorescence intensity was proportional to the logarithm of Xoo cells in the range of 10 0 -10 5  CFU mL -1 . The limit of detection was achieved at 22 CFU mL -1 and the specificity test against other plant disease pathogens showed high specificity towards Xoo. The detection of Xoo in real plant samples was also performed in this study and demonstrated satisfactory results., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Vibrio cholerae , cause of the life-threatening diarrheal disease cholera, can be divided into different serogroups based on the structure of its lipopolysaccharide (LPS), which consists of lipid-A, corepolysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2- O -methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11, and F7) that specifically react with Ogawa LPS. The mAbs bound to Vibrio cholerae vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to demonstrate the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent V. cholerae vaccines.

A point-of-care (POC) immunoassay was established for the sensitive and rapid detection of pathogenic Escherichia coli O157:H7, using magnetic Fe 3 O 4 organic-inorganic composites (Ab@Fe 3 O 4 ) for immunomagnetic separation, nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp) for signal amplification, and thermometer readings. Antibodies and Fe 3 O 4 were incubated in Cu 2+ phosphate buffer to synthesize the magnetic composite Ab@Fe 3 O 4 with antibodies, to specifically capture E. coli O157:H7. Antimicrobial peptides and PtNp were incubated in Cu 2+ phosphate buffer to synthesize the signal composites Ap@PtNp with antimicrobial peptides (magainin I), recognizing and labeling E. coli O157:H7. In the presence of E. coli O157:H7, magnetic microcomposites targeted bacteria and signal microcomposites to form the sandwich structure: Ab@Fe 3 O 4 -bacteria-Ap@PtNp for magnetic separation. Ap@PtNp of signal composites catalyzed H 2 O 2 to generate thermo-signals (temperature rise), which were determined by a thermometer. This point-of-care bioassay detected E. coli O157:H7 in the linear range of 10 1 -10 7  CFU mL -1 and with a detection limit of 14 CFU mL -1 . One-pot process magnetic Fe 3 O 4 organic-inorganic composites (Ab@Fe 3 O 4 , magnetic microcomposites, MMC) for immunomagnetic separation and nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp, signal microcomposites, SMC) were used as signal amplification and thermometer readings for E. coli O157:H7 detection.

Meningococcal meningitis remains a substantial cause of mortality and morbidity worldwide. Until recently, countries in the African meningitis belt were susceptible to devastating outbreaks, largely attributed to serogroup A Neisseria meningitidis (MenA). Vaccination with glycoconjugates of MenA capsular polysaccharide led to an almost complete elimination of MenA clinical cases. To understand the molecular basis of vaccine-induced protection, we generated a panel of oligosaccharide fragments of different lengths and tested them with polyclonal and monoclonal antibodies by inhibition enzyme-linked immunosorbent assay, surface plasmon resonance, and competitive human serum bactericidal assay, which is a surrogate for protection. The epitope was shown to optimize between three and six repeating units and to be O -acetylated. The molecular interactions between a protective monoclonal antibody and a MenA capsular polysaccharide fragment were further elucidated at the atomic level by saturation transfer difference NMR spectroscopy and X-ray crystallography. The epitope consists of a trisaccharide anchored to the antibody via the O - and N -acetyl moieties through either H-bonding or CH-π interactions. In silico docking showed that 3- O -acetylation of the upstream residue is essential for antibody binding, while O -acetate could be equally accommodated at three and four positions of the other two residues. These results shed light on the mechanism of action of current MenA vaccines and provide a foundation for the rational design of improved therapies., Competing Interests: Competing interest statement: P.H. is a student at the Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa and participated in a postgraduate studentship program at GlaxoSmithKline (GSK). L.D.I., A.B., M.R.R., F.B., R.R., and R.A. are employees of the GSK group of companies. F.B., R.R., and R.A. are shareholders of the GSK group of companies. R.A. and F.B. are listed as inventors on a patent filed by GSK. A.G., A.A., G.J.L.B., and J.J.-B. have nothing to disclose., (Copyright © 2020 the Author(s). Published by PNAS.)

To overcome the drawbacks of antibody labeling dependence and single-readout system in the conventional lateral flow immunoassays (LFIAs) as well as the non-targeted combination of new capture agents reported recently for pathogen detection, in this work, a multi-readout and label-free LFIA was proposed for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) based on a nanozyme-bacteria-antibody sandwich pattern. A type of functional nanozyme-mannose modified Prussian blue (man-PB), was introduced as the recognition agent as well as signal indicator. Apart from original signal intensity on the T-line, the peroxidase-like catalytic activity-driven generation of colorimetric signal could be used as another format of quantitation. Importantly, such LFIA could exhibit excellent performance for target pathogens detection separately with a quantitative range of 10 2 -10 8 cfu·mL -1 and a low detection limit of 10 2 cfu·mL -1 based on different readout formats, indicating the application potential of the proposed LFIA in real samples., 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 © 2020 Elsevier Ltd. All rights reserved.)

Novel diagnostic tools are a major challenge for brucellosis research, especially in developing countries. Herein, we established a handheld quantum dot (QD) immunochromatographic device for the fast detection of brucellosis antibodies in the field. Total bacterial protein extracted from Brucella 104M served as labelling and coating antigen. QD labelling and immunochromatography methods were used to optimise reaction conditions, labelling conditions, reaction temperature and storage temperature. QD test strips were employed to test brucellosis serum to determine their sensitivity, specificity and stability. Test strips were compared with Rose Bengal test, standard agglutination test and colloidal gold immunochromatographic assay. Labelled Brucella total protein displayed good specificity and no cross-reactivity. The concentration of labelled total bacterial protein was 3.9 mg/ml, the coating concentration was 2.0 mg/ ml, and the serum titre with the lowest detection sensitivity was 1:25. The optimal reaction temperature for the test strip was 25-30°C. The test strip was stable after storage at room temperature and the repeatability was high, with a coefficient of variation of 4.0%. After testing 199 serum samples, the sensitivity of the QD test strip was 98.53%, the specificity was 93.57%, and the coincidence rate with the standard agglutination test was 96.98%. The developed QD immunochromatographic method can be used for rapid detection and preliminary screening of brucellosis in the field., Competing Interests: The authors have declared that no competing interests exist.

In this study, we propose a novel sensitive solid-based immunosensor developed on a plasmonic nanopaper platform for the detection of Escherichia coli (E. coli) bacteria. This plasmonic nanopaper that comprises of carboxylated bacterial cellulose (CBC) impregnated with gold nanoparticles (AuNP-CBC), employed as a quencher and a sustainable functionalized platform to be conjugated with protein A. Thus, the conjugated protein A allows the aligned linkage of EAb-QD (anti-E. coli conjugated quantum dot) and EAb-AF (anti-E. coli conjugated Alexa Fluor 488). Interestingly, once E. coli was captured by the AuNP-CBC/EAb-QD or AuNP-CBC/EAb-AF, the energy transfer from the QD or Alexa Fluor fluorophores is triggered due to the conformational change in the antibody structure and this, in turn, causes a decrease in the distance between fluorophores and the quencher nanopaper and, therefore diminishing their photoluminescence. The immunosensors performed successfully to recognize E. coli at concentrations as low as 50 CFU mL -1 in the standard buffer. The examined functionality of the immunosensors in a real matrix such as chicken extract and lettuce juice demonstrated a highly efficient response while QD is the main fluorophore with a limit of detection around 100 CFU mL -1 .

Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis is the cause of one of the diseases with the highest mortality and morbidity rate in the Americas and in the world. In developing countries, the diagnosis of tuberculosis (TB) is based on baciloscopy and bacteriological cultures. The first method has a low sensitivity, and the second can take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control this disease and favors the transmission of tuberculosis to the susceptible population. In this work, we present the synthesis, amine-silanization, characterization and bio-functionalization of magnetic nanoparticles (MNPs) to develop a sandwich ELISA to detect and concentrate antigens from M. tuberculosis. For this purpose, a recombinant mycobacterial heat shock protein Hsp16.3, which contributes to the persistence of TB, was cloned and expressed in the E. coli system. Polyclonal antibodies anti-Hsp16.3 were produced in a rabbit and in mice. Magnetic nanoparticles were synthesized by co-precipitation, amine-functionalized and characterized by several physical-chemical methods. The XRD, Mossbauer spectroscopy, zeta potential, TEM, and FTIR all proved the successful preparation of the MNPs showing a diffraction crystal diameter of 10.48 ± 2.56 nm, superficial net charge of [Formula: see text]: +23.57 ± 2.87 mV, characteristic patterns of magnetite and a structure similar to a sphere. Additionally, it showed a magnetization saturation of 37.06 emu.g -1 . For the functionalization of nanoparticle surfaces with anti-Hsp16.3, the active ester method was used for bond formation, and parameters such as time of incubation, coupling agents ratio (EDC/NHS) and concentration as well as surface saturation level of amine-silanized MNPs (MNP@Si@NH 2 ) were standardized. Finally, bio-functionalized MNPs were used to detect, fix and concentrate the recombinant antigen Hsp16.3 from M. tuberculosis in a sandwich ELISA-MNP assay.

Shiga toxin-producing Escherichia coli (STEC) are critical foodborne pathogens, which cause serious human health issues, including hemolytic uremic syndrome. Illnesses caused by STEC lack effective treatments that target the elimination of these bacteria from the gastrointestinal tract without causing an adverse effect. Reducing this pathogen from a reservoir of STEC is an effective strategy, but the challenges remain due to the lack of efficient, selective antimicrobial agents. We developed specific antibody-conjugated chitosan nanoparticles (CNs) to selectively target and treat STEC in the gastrointestinal tract. Given the great broad-spectrum antimicrobial activity of CN, we conjugated antibodies to CN. Antibodies were raised and purified from egg yolks after immunization of hens with seven different O-side-chain antigens isolated from STEC (O26, O45, O103, O111, O121, O145, and O157). We prepared CN-immunoglobulin Y (IgY) conjugates by forming amide bonds at different ratios of CN:IgY (10:1, 10:2, and 10:4). The CN-IgY conjugated at a 10:2 ratio demonstrated significantly enhanced antimicrobial activity against E. coli O157:H7. Conjugates of CN and anti-STEC IgY antibodies killed corresponding STEC serotypes specifically and selectively, while showing no significant impact on nontargeted bacteria, including Salmonella enterica and Lactobacillus plantarum . The enhanced antimicrobial activity of CN-IgY against STEC was also confirmed in synthetic intestinal fluid, as well as an in vivo animal model of Caenorhabditis elegans . These results suggest that the CN-IgY conjugates have strong and specific antimicrobial activity and that they are also great candidates to eliminate pathogens selectively in the gastrointestinal tract without inhibiting beneficial bacteria.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752-862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752-862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752-862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752-862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

In this work a new fluorescence immunosensor with use of graphene oxide and graphene quantum dot for detection Campylobacter jejuni whole cell in food samples was designed. This biosensor was designed based on interaction of poly clonal antibody conjugated with graphene quantum dot with surface protein in Campylobacter jejuni cell membrane. Specific binding of graphene quantum dot with Campylobacter jejuni membrane leads to generate a distance among graphene dot and graphene oxide and fluorescence is ON. In lack of Campylobacter jejuni or in existence of other bacterial cells, distance between of graphene dot and graphene oxide is very low and graphene quantum dot fluorescence emission was OFF. Experiment revealed that step by step increase in bacterial target cells caused to gradually increased fluorescence emission and this process was linear. Limit of detection for this bacterial sensor was 10 CFU/ml and ability of this FRET immunosensor for Campylobacter jejuni sensing in comparison with other bacterial cells was significant. Also, this method for monitoring Campylobacter jejuni in poultry liver was applied and results revealed that this immunosensor could be used for analysis bacterial cell in food samples., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

An Immuno-Nano-Biosensor with high sensitivity was designed based on iron and silica nanoparticles to detect B. abortus. Briefly explain, primary polyclonal antibody (IgG 1 ) was conjugated on surface magnetic nanoparticles (MNPs) to form MNP-IgG 1 . Secondary polyclonal antibody (IgG 2 ) and Horseradish Peroxidase enzyme were conjugated on silica nanoparticles (SNPs) to form HRP-SNP-IgG 2 . HRP-SNP-IgG 2 . MNP-IgG 1 and HRP-SNP-IgG 2 were added to B. abortus. The MNP-IgG 1 -B.abortus-IgG 2 -SNP-HRP complex was isolated from the reaction mixture using a magnet. After that, tetramethylbenzidine was added to the complex. The reaction was stopped with HCl and investigated using UV-Vis spectrophotometry. The nanoparticles' structure and size were investigated using SEM and DLS. Immuno-Nano-Biosensor sensitivity and specificity were determined. The SEM and DLS results indicated that the SNPs, MNPs, HRP-SNP-IgG 2 and MNP-IgG 1 size and structure were 35, 44, 60 and 56 nm, respectively. In addition, a good linear correlation was observed at 10 2 -10 7  CFU mL - 1 concentrations, which their linear equation and regression were Y = 0.3× + 0.18 and R 2 0.982, respectively. The limitation of detecting B. abortus was 160 CFU mL - 1 . Finally, the results demonstrated that those designed Immuno-Nano-Biosensor could be specifically detected B. abortus and B. melitensis in real samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The gliding bacterium Flavobacterium johnsoniae is known to have an adhesin, SprB, that moves along the cell surface on a spiral track. Following viscous shear, cells can be tethered by the addition of an anti-SprB antibody, causing spinning at 3 Hz. Labeling the type 9 secretion system (T9SS) with a YFP fusion of GldL showed a yellow fluorescent spot near the rotation axis, indicating that the motor driving the motion is associated with the T9SS. The distance between the rotation axis and the track (90 nm) was determined after adding a Cy3 label for SprB. A rotary motor spinning a pinion of radius 90 nm at 3 Hz would cause a spot on its periphery to move at 1.5 μm/s, the gliding speed. We suggest the pinion drives a flexible tread that carries SprB along a track fixed to the cell surface. Cells glide when this adhesin adheres to the solid substratum., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

This study explores the adoption of laser-induced breakdown spectroscopy (LIBS) for the analysis of lateral-flow immunoassays (LFIAs). Gold (Au) nanoparticles are standard biomolecular labels among LFIAs, typically detected via colorimetric means. A wide diversity of lanthanide-complexed polymers (LCPs) are also used as immunoassay labels but are inapt for LFIAs due to lab-bound detection instrumentation. This is the first study to show the capability of LIBS to transition LCPs into the realm of LFIAs, and one of the few to apply LIBS to biomolecular label detection in complete immunoassays. Initially, an in-house LIBS system was optimized to detect an Au standard through a process of line selection across acquisition delay times, followed by determining limit of detection (LOD). The optimized LIBS system was applied to Au-labeled Escherichia coli detection on a commercial LFIA; comparison with colorimetric detection yielded similar LODs (1.03E4 and 8.890E3 CFU/mL respectively). Optimization was repeated with lanthanide standards to determine if they were viable alternatives to Au labels. It was found that europium (Eu) and ytterbium (Yb) may be more favorable biomolecular labels than Au. To test whether Eu-complexed polymers conjugated to antibodies could be used as labels in LFIAs, the conjugates were successfully applied to E. coli detection in a modified commercial LFIA. The results suggest interesting opportunities for creating highly multiplexed LFIAs. Multiplexed, sensitive, portable, and rapid LIBS detection of biomolecules concentrated and labeled on LFIAs is highly relevant for applications like food safety, where in-field food contaminant detection is critical. Graphical abstract.

Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14 TM helix transporter, NorC, from Staphylococcus aureus We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from Escherichia coli after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn 2+ -binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn 2+ -binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn 2+ also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing E. coli spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant S. aureus strains that express efflux transporters such as NorC in hospital and community settings., (© 2020 Kumar et al.)

Lateral flow immunoassay (LFA) strips are extensively used for rapid tests of various biochemical molecules, but these strips still have some limitations in bacterial detection due to their low sensitivity and poor stability in complex samples. In this study, we reported a highly sensitive and quantitative fluorescent LFA strip for bacterial detection by using novel magnetic-core@dual quantum dot (QD)-shell nanoparticles (Fe3O4@DQDs) as multifunctional fluorescent labels. The Fe3O4@DQDs were prepared through a polyethyleneimine (PEI)-mediated layer-by-layer (LBL) assembly method, and they possess monodispersity, high magnetic responsiveness, good stability, and superior fluorescence properties. Based on these merits, the Fe3O4@DQDs were used to capture and enrich bacteria from complex samples and then used as advanced fluorescent labels of LFA strips for the quantitative detection of bacteria. Under optimal conditions, the assay ultra-sensitively detected Streptococcus pneumoniae with a low limit of detection of 8 cells per mL and a wide dynamic linear range of 10 cells per mL to 107 cells per mL. Systematic comparison revealed that the fluorescence detection limit of the Fe3O4@DQD-based strip was 55 and 1000 times higher than those of Fe3O4-core@QD-shell nanocomposite (Fe3O4-QD)-based and conventional QD microsphere-based strips, respectively. The proposed method also exhibited high specificity and selectivity for biological samples (human whole blood and sputum) and is thus a promising tool for real bacterial sample testing.

Aim: This work reports a new method for the use of lasers for the selective killing of bacteria targeted using light-absorbing Silver nanoparticles (Ag-NPs) conjugated with a specific antibody against the Gram-positive bacterium Staphylococcus aureus (S. aureus)., Methods and Results: Ag-NPs were synthesized using a chemical reduction method and characterized with respect to their surface plasmon resonance, surface morphology via transmission electron microscopy (TEM) and dynamic light scattering (DLS). The bacterial surface was targeted using 20 nm Ag-NPs conjugated with an anti-protein A antibody. Labelled bacteria were irradiated with blue visible laser at 2·04 W/cm 2 . The antibacterial activity of functionalized Ag-NPs was investigated by fluorescence microscopy after irradiation, and morphological changes in S. aureus after laser treatment were assessed using scanning electron microscopy (SEM). The laser-irradiated, functionalized Ag-NPs exhibited significant bactericidal activity, and laser-induced bacterial damage was observed after 10 min of laser irradiation against S. aureus. The fluorescence microscopic analysis results supported that bacterial cell death occurred in the presence of the functionalized Ag-NPs., Conclusions: The results of this study suggest that a novel method for the preparation of functionalized nanoparticles has potential as a potent antibacterial agent for the selective killing of resistant disease-causing bacteria., Significance and Impact of the Study: This study shows that Ag-NPs functionalized with a specific antibody, could be used in combination with laser radiation as a novel treatment to target resistant bacterial and fungal pathogens with minimal impact on normal microflora., (© 2019 The Society for Applied Microbiology.)

We have developed a method for rapid detection of pathogenic bacteria from water using a virtual net comprising magnetic nanoparticle clusters (MNC). When an external magnetic field was applied to the antibody-functionalized MNC (Ab-MNC) solution in a glass tube (GT), the Ab-MNCs were aligned along the direction of the applied magnetic field to form a wall of MNCs. The injection of a liquid into the GT pushed the MNCs to flow when the drag force exceeded the magnetic force that held the MNCs. In contrast, injection of a liquid into the GT wrapped with a copper tape (Cu-GT) created a magnetic field in the opposite direction of the liquid flow according to Lenz's law, which retained the MNCs inside Cu-GT even at a flow rate 2.5 times higher than the maximum flow rate at which the MNCs were retained inside the GT. As proof of concept, E. coli O157:H7-spiked aqueous solutions were injected into Cu-GT containing Ab-MNCs. The structural flexibility of the Ab-MNC wall allowed the liquid to pass through but induced binding of the bacteria to the Ab-MNC wall, just as the wall acted like a virtual net. The detection limit was 10 2 CFU/mL of E. coli as measured by an ATP luminometer, and the total assay time was 15 min including 10 min for the isolation and separation steps.

Multidrug-resistant gram-negative bacteria infection is particularly severe within the designated ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which underscores the urgent need to explore alternative therapeutic strategies. The type III secretion system (T3SS) is considered to be a key virulence factor in many gram-negative bacteria, and T3SS is in turn regulated by SpuE in P. aeruginosa, which is a spermidine binding protein from an ATP-binding cassette transporter family and highly conserved within ESKAPE pathogens. Here, we identified a potent anti-SpuE antagonistic antibody that allosterically inhibits the expression of T3SS and attenuates virulence of P. aeruginosa. X-ray crystallography and molecular dynamics simulations revealed that binding of antibody to SpuE induces a change in the dynamics of SpuE, which in turn may reduce spermidine uptake by P. aeruginosa. The antibody could serve as a template for developing novel biologics to target a broad spectrum of gram-negative bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus (S. aureus) infections are a leading cause of death by an infectious agent. Survival within host phagocytic cells is one mechanism by which S. aureus evades antibiotic treatment. A novel THIOMAB™ antibody-antibiotic conjugate (TAC) strategy was developed to kill S. aureus intracellularly and mitigate the spread of infection. In this report, we used a longitudinal whole-body bioluminescence imaging method to study the antibacterial dynamics of TAC alone or in combination with vancomycin in a mouse infection model. Injections of stably luminescent S. aureus bacteria into mice resulted in exponential increases in whole body bioluminescence with a reduction in body weight and survival rate. Vancomycin, a standard-of-care antibiotic, suppressed bacterial growth in mice. However, bacterial growth rebounded in these animals once treatment was discontinued. In contrast, single dose of TAC showed rapid reduction of bioluminescence intensity, which persisted for up to 19 days. The combination of TAC and vancomycin achieved a more sustained and significantly greater reduction of bioluminescence compared with vancomycin alone. In summary, the present study showed an imaging method to longitudinally assess antibacterial drug dynamics in mice and demonstrated that TAC monotherapy or in combination with vancomycin had superior and sustained activity compared to vancomycin alone., Competing Interests: CZ, HC, RD, WH, NLK, and AK are current or former employees of Genentech Inc. at the time of these studies. MY, FG, and DX are current employees of WuXi AppTec (Shanghai) Co., Ltd. WuXi AppTec is a contract research organization that was sponsored by Genentech to perform the in vivo experiments in this report. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhea in children, travelers, and deployed military personnel. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with antigen/adjuvant toxoids as an ETEC vaccine. Here, we investigated the intradermal (i.d.) or sublingual (s.l.) delivery of CFA/I fimbrial antigens, including CfaEB and a CfaE-heat-labile toxin B subunit (LTB) chimera admixed with double mutant heat-labile toxin (LT) LT-R192G/L211A (dmLT). In addition, we compared dmLT with other LT proteins to better understand the generation of adjuvanted fimbrial and toxoid immunity as well as the influence on any local skin reactogenicity. We demonstrate that immunization with dmLT admixed with CfaEB induces robust serum and fecal antibody responses to CFA/I fimbriae and LT but that i.d. formulations are not optimal for s.l. delivery. Improved s.l. vaccination outcomes were observed when higher doses of dmLT (1 to 5 μg) were admixed with CfaEB or, even better, when a CfaE-LTB chimera antigen was used instead. Serum anti-CFA/I total antibodies, detected by enzyme-linked immunosorbent assay, were the best predictor of functional antibodies, based on the inhibition of red blood cell agglutination by ETEC. Immunization with other LT proteins or formulations with altered B-subunit binding during i.d. immunization (e.g., by addition of 5% lactose, LTA1, or LT-G33D) minimally altered the development of antibody responses and cytokine recall responses but reduced skin reactogenicity at the injection site. These results reveal how formulations and delivery parameters shape the adaptive immune responses to a toxoid and fimbria-derived subunit vaccine against ETEC., (Copyright © 2019 American Society for Microbiology.)

Objective: Research suggests human norovirus binding to histo-blood group antigen (HBGA)-like molecules on enteric bacteria may enhance viral pathogenesis; however, the properties of these bacterial ligands are not well known. Previous work identified, but did not characterize, seven norovirus-binding bacteria. To further examine this bacteria-virus binding interaction, enteric bacteria were analyzed via Western blot with anti-HBGA antibodies and lectins targeting HBGA-associated sugar components. Virus overlay assays using capsids from six different human norovirus strains further identified responsible ligands and strain dependent binding properties., Results: Each bacterial species possessed varying degrees of HBGA-like activity, and lectin binding further elucidated potential sugar residues involved (N-acetyl-galactosamine, α-D-galactose or α-L-fucose). Both GI and GII norovirus capsids bound specific bacterial ligand sizes, and generally corresponded to anti-HBGA Western blot patterns. A 35-kDa band reacted with all HBGA antibodies, bound all six of the noroviruses tested, and had a high affinity for the lectins. Collectively, this work characterizes the varying carbohydrate residues potentially responsible for norovirus-bacteria interactions and provides a basis for future ligand identification.

Salmonella , a common foodborne pathogen, causes many cases of foodborne illness and poses a threat to public health worldwide. Immunological detection systems can be combined with nanoparticles to develop sensitive and portable detection technologies for timely screening of Salmonella infections. Here, we developed an antibody-probe-based immuno-N-hydroxysuccinimide (NHS) bead (AIB) system to detect Salmonella. After adding the antibody probe, Salmonella accumulated in the samples on the surfaces of the immuno-NHS beads (INBs), forming a sandwich structure (INB- Salmonella -probes). We demonstrated the utility of our AIB diagnostic system for detecting Salmonella in water, milk, and eggs, with a sensitivity of 9 CFU mL -1 in less than 50 min. The AIB diagnostic system exhibits highly specific detection and no cross-reaction with other similar microbial strains. With no specialized equipment or technical requirements, the AIB diagnostic method can be used for visual, rapid, and point-of-care detection of Salmonella .

Introduction. Timely detection of invasive aspergillosis (IA) caused by fungal pathogens, i.e. Aspergillus fumigatus and Aspergillus flavus , in immunocompromised patients is crucial in preventing high mortality. Aim. To develop a simple immunoassay for the detection of galactomannan (GM), an IA biomarker. Methodology . GM from A. fumigatus and A. flavus clinical strains was purified and characterized by X-ray diffraction, IR spectroscopy and 13 C/ 1 H nuclear magnetic resonance (NMR) for polyclonal antibody (pAb) production in rabbits. An enzyme-linked immunosorbent assay (ELISA) was standardized using concanavalin A to capture Aspergillus GM and pAbs to detect it. Gold nanoparticles (AuNPs) were synthesized and conjugated to pAbs for the development of a dot-blot immunoassay. The developed dot-blot was evaluated with 109 clinical serum and bronchoalveolar lavage samples. Results . Spectroscopy studies characterized the d-galactofuranosyl groups of GM responsible for the immune response and generation of pAbs. The ELISA employing pAbs showed a sensitivity of 1 ng ml -1 for Aspergillus GM. Furthermore, a sensitive, visual, rapid dot-blot assay developed by the conjugation of pAbs to AuNPs (~24±5 nm size, -36±2 mV zeta potential) had a detection limit of 1 pg ml -1 in serum. The pAbs interacted with Aspergillus spp. but did not cross-react with other fungal pathogen genera such as Penicillium and Candida . Evaluation of the dot-blot with 109 clinical samples showed high sensitivity (80 %) and specificity (93.2 %), with an overall assay accuracy of 89%. Conclusion . The developed nano-gold immunodiagnostic assay has immense potential for practical use in rapid, specific and sensitive on-site diagnosis of IA, even under resource-limited settings.

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. The pathological effects of CDI are primarily attributed to toxins A (TcdA) and B (TcdB). Adequate toxin-specific antibody responses are associated with asymptomatic carriage, whereas insufficient humoral responses are associated with recurrent CDI. While the data supporting the importance of anti-toxin antibodies are substantial, clarity about the toxin domain specificity of these antibodies is more limited. To investigate this matter, combinations of human mAbs targeting multiple domains of TcdB were assessed using toxin neutralization assays. These data revealed that a combination of mAbs specific to all major toxin domains had improved neutralizing potency when compared to equivalent concentrations of a single mAb or a combination of mAbs against one or two domains. The function and toxin domain binding specificity of serum antibodies elicited by immunization of hamsters with a toxoid vaccine candidate was also assessed. Immunization with a toxoid vaccine candidate provoked toxin neutralizing antibodies specific to multiple domains of both TcdA and TcdB. When assessed in a toxin neutralization assay, polyclonal sera displayed greater activity against elevated concentrations of toxins than equivalent concentrations of individual mAbs. These data suggest a potential benefit of any antibody based therapeutic or prophylactic treatment that targets multiple toxin domains., (Copyright © 2019. Published by Elsevier Ltd.)

A dual sites affinity protocol was developed for fluorescent analysis of Staphylococcus aureus (S. aureus) by employing daptomycin and immunoglobulin G (IgG) as the recognition elements. Pig IgG immobilized on microplate was employed as the first recognition element to capture S. aureus owing to the fact that the Fc segment of mammal IgG can selectively bind with protein A on the surface of the target bacteria. Meanwhile, fluorescein isothiocyanate-conjugated daptomycin was employed as the second recognition element as well as the signal tracer for the target bacteria utilizing the binding capability of daptomycin to Gram-positive bacteria. S. aureus can be analyzed within a concentration range of 5.0 × 10 3 -5.0 × 10 8  CFU mL -1 with a detection limit of 3.6 × 10 3  CFU mL -1 . The analytical process can be accomplished within 1.5 h by using a pre-coated microplate. The dual sites affinity protocol can exclude the interference led by Gram-negative bacteria and other common Gram-positive bacteria. We have successfully applied it to analyze S. aureus in spiked lake water and physiological saline injection samples, and the recovery values ranged from 88.0% to 120.0%. The results demonstrate its application potential for environmental sanitation and drug safety control., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Weligama coconut leaf wilt disease (WCLWD) causes heavy losses in the coconut cultivations of southern Sri Lanka. The in-house developed and validated indirect ELISA was based on specific polyclonal antibodies raised in female New Zealand White rabbits, against partially purified WCLWD associated phytoplasma. This ELISA has the potential to distinguish secA PCR confirmed, WCLWD associated phytoplasma positive palms from phytoplasma free palms at high accuracy (93%) and sensitivity (92.7%), but with marginal specificity (79%). The calculated ELISA cross reactivity index (CRI) values were low for sugarcane white leaf (7%) and sugarcane grassy shoot (8%) infected leaves, but with marked highCRIfor both Bermuda grass white leaf (69%) and areca nut yellow leaf (70%) infected leaves. SecA gene based phylogenetic relationships of the WCLWD associated phytoplasma with these other locally prevalent phytoplasma strains elucidated this immunological cross reactivity, which was further reiterated by virtual restriction fragment length polymorphism analysis. Based on scanning electron microscopy, this study provides additional visual evidence, for the presence of phytoplasmas in WCLWD infected tissues., Competing Interests: The authors have declared that no competing interests exist.

Pathogenic bacterial infection has become a growing threat to public health; therefore, exploration of a sensitive and specific method for the identification of bacteria is very important. In this report, we fabricated a new magnetic core-shell nanocomposite with a homogenous morphology. The Fe 3 O 4 nanoparticle core with a diameter of 15 nm was coated with a thin silica layer, and the thickness of the layer was finely adjusted to about 10 nm. Pt nanoclusters with a diameter of 2-3 nm were anchored uniformly on the surface to form Fe 3 O 4 @SiO 2 -Pt nanocomposites. These nanocomposites exhibited excellent peroxide enzyme activity and acted as a signal-output probe for the identification of pathogenic bacteria. This strategy was also based on using vancomycin (Van) as the capture agent in an ELISA procedure. The detection limit for S. aureus was around 1 × 10 1 cfu mL -1 . Furthermore, Fe 3 O 4 @Si-Pt nanocomposites also show ideal bacteria separation and inhibition effects, and can act as a multifunctional platform for bacterial detection and antibacterial applications.

A MEMS-based impedance biosensor was designed, fabricated, and tested to effectively detect the presence of bacterial cells including E. coli O157:H7 and Salmonella typhimurium in raw chicken products using detection region made of multiple interdigitated electrode arrays. A positive dielectrophoresis based focusing electrode was used in order to focus and concentrate the bacterial cells at the centerline of the fluidic microchannel and direct them toward the detection microchannel. The biosensor was fabricated using surface micromachining technology on a glass substrate. The results demonstrate that the device can detect Salmonella with concentrations as low as 10 cells/mL in less than 1 h. The device sensitivity was improved by the addition of the focusing electrodes, which increased the signal response by a factor between 6 and 18 times higher than without the use of the focusing electrodes. The biosensor is selective and can detect other types of pathogen by changing the type of the antibody immobilized on the detection electrodes. The device was able to differentiate live from dead bacteria., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Existing clinical methods for bacteria detection lack speed, sensitivity, and, importantly, point-of-care (PoC) applicability. Thus, finding ways to push the sensitivity of clinical PoC biosensing technologies is crucial. Here we report a portable PoC device based on lens-free interferometric microscopy (LIM). The device employs high performance nanoplasmonics and custom bioprinted microarrays and is capable of direct label-free bacteria ( E. coli) quantification. With only one-step sample handling we offer a sample-to-data turnaround time of 40 min. Our technology features detection sensitivity of a single bacterial cell both in buffer and in diluted blood plasma and is intrinsically limited by the number of cells present in the detection volume. When employed in a hospital setting, the device has enabled accurate categorization of sepsis patients (infectious SIRS) from control groups (healthy individuals and noninfectious SIRS patients) without false positives/negatives. User-friendly on-site bacterial clinical diagnosis can thus become a reality.

Campylobacter jejuni (C. jejuni) is a foodborne intestinal pathogen and major cause of gastroenteritis worldwide. C. jejuni proteins that are immunogenic have been sought for their potential use in the development of biomarkers, diagnostic assays, or subunit vaccines for humans or livestock. To identify new immunogenic C. jejuni proteins, we used a native protein microarray approach. A protein chip, with over 1400 individually purified GST-tagged C. jejuni proteins, representing over 86% of the proteome, was constructed to screen for antibody titers present in test sera raised against whole C. jejuni cells. Dual detection of GST signals was incorporated as a way of normalizing the variation of protein concentrations contributing to the antibody staining intensities. We detected strong signals to 102 C. jejuni antigens. In addition to antigens recognized by antiserum raised against C. jejuni, parallel experiments were conducted to identify antigens cross-reactive to antiserum raised against various serotypes of E. coli or Salmonella or to healthy human sera. This led to the identification of 34 antigens specifically recognized by the C. jejuni antiserum, only four of which were previously known. The chip approach also allowed identification of conformational antigens. We demonstrate in the case of Cj1621 that antigen signals are lost to denaturing conditions commonly used in other approaches to identify immunogens. Antigens identified in this study include those possessing sequence features indicative of cell surface localization, as well as those that do not. Together, our results indicate that the unbiased chip-based screen can help reveal the full repertoire of host antibodies against microbial proteomes., Competing Interests: The authors have declared that no competing interests exist. JL is currently employed by Idexx Laboratories, which played no role in this study.

Background: Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published., Objective: Here, the protealysin cellular localization was studied for the first time using immunochemical methods., Methods and Results: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm., Conclusion: The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Our previous results showed higher level of anti-Hib IgG (2.41 μg/ml) average geometric mean concentration (GMC) regardless of age and gender, followed by levels of IgM (0.91 μg/ml) and IgA (0.34 μg/ml), reflecting the prevalence of immunity against Hib. Current study was evaluated the avidity profile for these high anti-Hib concentration. The IgM avidity analysis did not revealed any significant results between male and female values. While, the average of IgA avidity in male samples were significantly (P< 0.05) higher than the average of female. Also, The result showed that high percentage of population has moderate to high IgG avidity. The study showed lower avidity percentages of both IgM and IgA comparing to IgG. More studies are suggested to be done on individuals with low avidity considering the secondary immunodeficiency in participant's history.

Monoclonal antibody (MAb)-based ELISA was developed to detect leptospiral antigens from the plasma of febrile patients. The MAb reacts specifically with pathogenic leptospires and the assay possesses excellent diagnostic test parameters compared to PCR, indicating that this new ELISA is useful for the early diagnosis of leptospirosis with low operational cost., (Copyright © 2018 Elsevier B.V. All rights reserved.)

DSTA4637A, a THIOMAB™ antibody-antibiotic conjugate targeting Staphylococcus aureus, has shown promising bactericidal activity in a mouse model. DSTA4637A consists of a monoclonal anti-S. aureus antibody with an average of two rifalogue antibiotic molecules, dmDNA31, linked to its light chains. The goal of this study was to develop a minimal physiologically-based pharmacokinetic (mPBPK) model to characterize the pharmacokinetic (PK) properties of three analytes of DSTA4637A (i.e., total antibody, antibody-conjugated dmDNA31, and unconjugated dmDNA31) in mice, and to predict pharmacokinetics of DSTA4637A analytes in humans, as well as to provide an initial assessment for potential PK drug-drug interactions (DDI) in clinical trials via cross-species scaling of the mPBPK model. In the proposed model, selected organs, including heart, liver, and kidney, were connected anatomically with plasma and lymph flows. Mouse plasma and tissue concentrations of the three analytes of DSTA4637A were fitted simultaneously to estimate the PK parameters. Cross-species scaling of the model was performed by integrating allometric scaling and human physiological parameters. The final mPBPK model was able to successfully capture PK profiles of three DSTA4637A analytes in mouse plasma and in investigated organs. The model predicted a steady-state peak unbound dmDNA31 concentration lower than 5% of the IC 50 of dmDNA31 towards cytochrome P450 following 100 mg/kg weekly intravenous dose, which suggests a low risk of PK DDI in humans for DSTA4637A with co-administered cytochrome P450 substrates. The proposed mPBPK modeling and cross-species scaling approaches provide valuable tools that facilitate the understanding and translation of DSTA4637A disposition from preclinical species to humans.