High purity and recovery of native filamentous hemagglutinin (FHA) from Bordetella pertussis using affinity chromatography.

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.

Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design.

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 (NaIO4) 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 NaIO4 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.

Computational Construction of a Single-Chain Bi-Paratopic Antibody Allosterically Inhibiting TCR-Staphylococcal Enterotoxin B Binding.

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-TCRß 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-TCRß 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.

Serological detection of Mycobacterium Tuberculosis complex infection in multiple hosts by One Universal ELISA.

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.

Nanobody-Dependent Detection of Microcystis aeruginosa by ELISA and Thermal Lens Spectrometry.

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.

A quantum dot fluorescent microsphere based immunochromatographic strip for detection of brucellosis.

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 (HT / HC) 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.

Microfluidics for the rapid detection of Escherichia coli O157:H7 using antibody-coated microspheres.

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.

Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

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.

Development of nano-immunosensor with magnetic separation and electrical detection of Escherichia coli using antibody conjugated Fe3O4@Ppy.

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 (Fe3O4) 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 Fe3O4@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 101-106 CFU ml-1 was successfully detected with a limit of detection of 10 CFU ml-1 within 10 min using a simplistic detection method.

Solvothermal synthesis of α-Fe2O3 polyhedrons and its application in an immunochromatographic strip test for the detection of foodborne pathogen Listeria monocytogenes.

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-Fe2O3 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-Fe2O3 polyhedrons; the average diameter of the α-Fe2O3 polyhedrons is about 200 nm. The crystal structure and morphology of α-Fe2O3 polyhedrons were characterized by x-ray diffraction and transmission electron microscope. α-Fe2O3 polyhedrons were immunized with anti-L. monocytogenes antibody to prepare an antibody-colloidal α-Fe2O3 polyhedron ICST. Visual detection can be obtained directly by the naked eye within 10 min. The detection limit of L. monocytogenes by α-Fe2O3 polyhedron ICST assay was 3.8 × 106 and 5.6 × 106 CFU/ml of pure culture and artificially spiked orange juice drink sample, respectively. Results indicated that the antibody-colloidal α-Fe2O3 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.

Structure of a protective epitope reveals the importance of acetylation of Neisseria meningitidis serogroup A capsular polysaccharide.

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.

Immunoassay for foodborne pathogenic bacteria using magnetic composites Ab@Fe3O4, signal composites Ap@PtNp, and thermometer readings.

A point-of-care (POC) immunoassay was established for the sensitive and rapid detection of pathogenic Escherichia coli O157:H7, using magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4) for immunomagnetic separation, nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp) for signal amplification, and thermometer readings. Antibodies and Fe3O4 were incubated in Cu2+ phosphate buffer to synthesize the magnetic composite Ab@Fe3O4 with antibodies, to specifically capture E. coli O157:H7. Antimicrobial peptides and PtNp were incubated in Cu2+ 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@Fe3O4-bacteria-Ap@PtNp for magnetic separation. Ap@PtNp of signal composites catalyzed H2O2 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 101-107 CFU mL-1 and with a detection limit of 14 CFU mL-1. One-pot process magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4, 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.

Electrochemical immunosensor based on self-assembled gold nanorods for label-free and sensitive determination of Staphylococcus aureus.

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 × 103 to 1.8 × 107 CFU mL-1 with a low detection limit of 2.4 × 102 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.

Toward a nanopaper-based and solid phase immunoassay using FRET for the rapid detection of bacteria.

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.

Rapid detection of brucellosis using a quantum dot-based immunochromatographic test strip.

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.

Generation and Characterization of Monoclonal Antibodies to the Ogawa Lipopolysaccharide of Vibrio cholerae O1 from Phage-Displayed Human Synthetic Fab Library.

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.

Fluorescence-based immunoassay for the detection of Xanthomonas oryzae pv. oryzae in rice leaf.

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 100-105 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.

Functional nanozyme mediated multi-readout and label-free lateral flow immunoassay for rapid detection of Escherichia coli O157:H7.

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 102-108 cfu·mL-1 and a low detection limit of 102 cfu·mL-1 based on different readout formats, indicating the application potential of the proposed LFIA in real samples.

Selective Killing of Shiga Toxin-Producing Escherichia coli with Antibody-Conjugated Chitosan Nanoparticles in the Gastrointestinal Tract.

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.

A molecular rack and pinion actuates a cell-surface adhesin and enables bacterial gliding motility.

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.