Probing an Ixodes ricinus salivary gland yeast surface display with tick-exposed human sera to identify novel candidates for an anti-tick vaccine.

In Europe, Ixodes ricinus is the most important vector of human infectious diseases, most notably Lyme borreliosis and tick-borne encephalitis virus. Multiple non-natural hosts of I. ricinus have shown to develop immunity after repeated tick bites. Tick immunity has also been shown to impair B. burgdorferi transmission. Most interestingly, multiple tick bites reduced the likelihood of contracting Lyme borreliosis in humans. A vaccine that mimics tick immunity could therefore potentially prevent Lyme borreliosis in humans. A yeast surface display library (YSD) of nymphal I. ricinus salivary gland genes expressed at 24, 48 and 72 h into tick feeding was constructed and probed with antibodies from humans repeatedly bitten by ticks, identifying twelve immunoreactive tick salivary gland proteins (TSGPs). From these, three proteins were selected for vaccination studies. An exploratory vaccination study in cattle showed an anti-tick effect when all three antigens were combined. However, immunization of rabbits did not provide equivalent levels of protection. Our results show that YSD is a powerful tool to identify immunodominant antigens in humans exposed to tick bites, yet vaccination with the three selected TSGPs did not provide protection in the present form. Future efforts will focus on exploring the biological functions of these proteins, consider alternative systems for recombinant protein generation and vaccination platforms and assess the potential of the other identified immunogenic TSGPs.

Bioreactor-Based Antigen Production Process Using the Baculovirus Expression Vector System.

Several vaccines are already produced using the baculovirus expression vector system (BEVS). This chapter describes methods for generating recombinant baculoviral DNA (also called bacmid) for cultivating Spodoptera frugiperda Sf-9 cells and producing a baculovirus stock from the recombinant bacmid and for producing a protein-based vaccine with the BEVS in a stirred tank reactor.

Nanofluidic-Based Accumulation of Antigens for Miniaturized Immunoassay.

The continuous advances of Nanofluidics have been stimulating the development of novel nanostructures and strategies to accumulate very diluted analytes, for implementing a new class of high sensitivity miniaturized polymeric sensors. We take advantage of the electrokinetic properties of these structures, which allow accumulating analytes inside asymmetric microfluidic structures to implement miniaturized sensors able to detect diluted solutions down to nearly 1.2 pg/mL. In particular, exploiting polydimethylsiloxane devices, fabricated by using the junction gap breakdown technique, we concentrate antigens inside a thin microfunnel functionalized with specific antibodies to favor the interaction and, if it is the case, the recognition between antigens in solution and antibodies anchored to the surface. The transduction mechanism consists in detecting the fluorescence signal of labeled avidin when it binds to biotinylated antigens. Here, we demonstrate that exploiting these electrokinetic phenomena, typical of nanofluidic structures, we succeeded in concentrating biomolecules in correspondence of a 1 pL sensing region, a strategy that grants to the device performance comparable to standard immunoassays.

Implicaciones pronósticas del daño miocárdico en pacientes con y sin diagnóstico confirmado de COVID-19 atendidos en un hospital universitario / Prognostic implications of myocardial injury in patients with and without COVID-19 infection treated in a university hospital

INTRODUCCIÓN Y OBJETIVOS: La elevación de la troponina cardiaca como marcador de daño miocárdico es un predictor pronóstico en pacientes con COVID-19. Sin embargo, se desconoce su rendimiento en pacientes coetáneos con sospecha de COVID-19 pero con prueba de reacción en cadena de la polimerasa negativa. MÉTODOS: Estudio de cohortes retrospectivo que incluyó a todos los pacientes consecutivos atendidos en un hospital universitario con sospecha de COVID-19, confirmada o descartada mediante prueba de reacción en cadena de la polimerasa, todos ellos con determinaciones de troponina cardiaca I. Se analizó el impacto de la positividad de la troponina cardiaca I en la mortalidad a 30 días. RESULTADOS: Un total de 433 pacientes quedaron distribuidos en los siguientes grupos: COVID-19 confirmada (n=186), el 22% de ellos con daño miocárdico (n=41), y COVID-19 descartada (n=247), el 21,5% de ellos con daño miocárdico (n=52). Los grupos de COVID-19 confirmada y descartada tuvieron similares edad, sexo y antecedentes cardiovasculares. La mortalidad en el grupo de COVID-19 confirmada frente al de descartada fue significativamente superior (el 19,9 frente al 5,3%; p <0,001). En ambos grupos, el daño miocárdico fue predictor de mortalidad en el análisis multivariado de regresión de Cox (grupo de COVID-19 confirmada, HR=3,54; IC95%, 1,70-7,34; p = 0,001; grupo de COVID-19 descartada, HR=5,57; IC95%, 1,70-18,20; p = 0,004). El modelo predictivo analizado por curvas ROC fue similar en ambos grupos: COVID-19 confirmada, AUC=0,808 (0,750-0,865); COVID-19 descartada, AUC=0,812 (0,760-0,864) (p = 0,701). CONCLUSIONES: Se detecta daño miocárdico en 1 de cada 5 pacientes con infección por COVID-19 confirmada o descartada. En ambas circunstancias, el daño miocárdico es predictor de mortalidad a 30 días en similar grado

INTRODUCTION AND OBJECTIVES: Cardiac troponin, a marker of myocardial injury, is frequently observed in patients with COVID-19 infection. Our objective was to analyze myocardial injury and its prognostic implications in patients with and without COVID-19 infection treated in the same period of time. METHODS: The present study included patients treated in a university hospital with cardiac troponin I measurements and with suspected COVID-19 infection, confirmed or ruled out by polymerase chain reaction analysis. The impact was analyzed of cardiac troponin I positivity on 30-day mortality. RESULTS: In total, 433 patients were distributed among the following groups: confirmed COVID-19 (n=186), 22% with myocardial injury (n=41); and ruled out COVID-19 (n=247), 21.5% with myocardial injury (n=52). The confirmed and ruled out COVID-19 groups had a similar age, sex, and cardiovascular history. Mortality was significantly higher in the confirmed COVID-19 group than in the ruled out group (19.9% vs 5.3%, P <.001). In Cox multivariate regression analysis, cardiac troponin I was a predictor of mortality in both groups (confirmed COVID-19 group: HR, 3.54; 95%CI, 1.70-7.34; P=.001; ruled out COVID-19 group: HR, 5.57; 95%CI, 1.70-18.20; P=.004). The predictive model analyzed by ROC curves was similar in the 2 groups (P=.701), with AUCs of 0.808 in the confirmed COVID-19 group (0.750-0.865) and 0.812 in the ruled out COVID-19 group (0.760-0.864). CONCLUSIONS: Myocardial injury is detected in 1 in every 5 patients with confirmed or ruled out COVID-19 and predicts 30-day mortality to a similar extent in both circumstances

Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay / Identificación de antígenos definidos molecularmente para un ensayo de liberación de interferón-gamma específico para Histoplasma capsulatum

Background: In a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides. Aims: To identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test. Methods: We surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides. Results: Seven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a "brute force" approach for peptide design. Conclusions: The H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate

Antecedentes: En un trabajo anterior mostramos la viabilidad de un ensayo de liberación de interferón-gamma (IGRA) para detectar la infección latente por Histoplasma capsulatum. En esa prueba de concepto utilizamos extractos crudos del hongo como antígenos; sin embargo, los IGRA de última generación desarrollados para otras infecciones se basan en antígenos definidos molecularmente, principalmente en mezclas de péptidos inmunogénicos. Objetivos Identificar proteínas de H. capsulatum que podrían servir como antígenos definidos molecularmente en una prueba IGRA. Métodos: Examinamos la literatura en busca de proteínas inmunogénicas de H. capsulatum ya conocidas, y ensayamos dos de ellas como antígenos en una prueba IGRA, en un estudio donde participaron 80 voluntarios. Además, utilizamos varias herramientas bioinformáticas para identificar proteínas específicas de H. capsulatum y analizar posibles estrategias para el diseño de péptidos inmunogénicos específicos. Resultados: Encontramos siete proteínas de H. capsulatum caracterizadas como inmunogénicas en la literatura. Las pruebas IGRA donde utilizamos la proteína de choque térmico 60 o el antígeno M, mostraron una alta sensibilidad, pero baja especificidad, debido probablemente a la alta similitud de secuencia con los ortólogos correspondientes en otros microorganismos patógenos. Identificamos unas 2000 proteínas específicas de H. capsulatum, la mayoría de las cuales permanecen sin anotar. Las predicciones de epítopos de células T de clase II realizadas para un pequeño número de estas proteínas mostraron una gran variabilidad entre los diferentes alelos, sustentando la aplicación de un enfoque de «fuerza bruta» en el diseño de estos péptidos. Conclusiones: El genoma de H. capsulatum codifica una gran cantidad de proteínas específicas que representan una fuente valiosa de posibles antígenos para una prueba IGRA. Entre ellos, la proteína Cfp4 resulta un candidato muy atractivo

Simultaneous in situ visualization and quantitation of dual antigens adsorbed on adjuvants using high content analysis.

Aim: Traditional antigenicity assay requires antigen recovery from the particulate adjuvants prior to analysis. An in situ method was developed for interrogating vaccine antigens with monoclonal antibodies while being adsorbed on adjuvants. Materials & methods: The fluorescence imaging-based high content analysis was used to visualize the antigen distribution on adjuvant agglomerates and to analyze the antigenicity for adsorbed antigens. Results: Simultaneous visualization and quantitation were achieved for dual antigens in a bivalent human papillomavirus vaccine with uniquely labeled antibodies. Good agreement was observed between the in situ multiplexed assays with well-established sandwich enzyme-linked immunosorbent assays. Conclusion: The streamlined procedures and the amenability for multiplexing make the in situ antigenicity analysis a favorable choice for in vitro functional assessment of bionanoparticles as vaccine antigens.

A disposable fiber optic SPR probe for immunoassay.

Immunoassay can be divided into two aspects, one is immobilization of antibodies for the efficient detection of corresponding antigens, and the other is immobilization of antigens to search for antibodies which work against them. In this paper, we demonstrated these two aspects of immunoassay by using the disposable fiber optic biosensors based on surface plasmon resonance (SPR) through surface decoration with half-antibody fragments, which has been scarcely ever reported to the best of our knowledge. We first fabricated the fiber optic SPR biosensor which consists of one gold film coated single-mode fiber sandwiched by two multimode fibers. Then, we decorated the fiber optic SPR biosensors with antibody fragments and antigen fragments, respectively, and compared the specific detection performances of these two kinds of sensors. After surface decoration with half-antibody fragments, the antigen-decorated fiber probe has a demonstrated sensitivity and limit of detection of 0.9771nm/(µg/mL) and 0.1 µg/mL, respectively, which improves by 10 times compared with the performance of the antibody-decorated fiber probe. Additionally, the selective detection results indicate that our proposed biosensor can be employed as a reliable antigen detector or an effective antibody filter. Our proposed sensor has the advantages of miniaturization, low cost, simple usage, label-free detection, high efficiency and sensitivity, and can effectively avoid cross-contamination caused by reuse. Given the reliable and clean detection method for immunoassay, our work should open a new window for the utilization of miniaturized fiber optic sensors in biochemical sensing.

[Clinical Pharmaceutical Research Based on New Proteome Analysis Based on Chromatographic Separation].

Comprehensive identification of antigens in immune complexes (IC-antigens) is beneficial to provide insights into pathophysiology and could form the basis for novel diagnostic and treatment strategies for many immune-related diseases. Immune complexome analysis is a method for comprehensively identifying and profiling IC-antigens in biological fluids (such as serum and cerebrospinal fluid). We applied this strategy to the analysis of circulating ICs in autoimmune diseases (rheumatoid arthritis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus), infectious diseases, and cancers. Fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) consists of fluorogenic derivatization of proteins, followed by HPLC of the derivatized proteins, isolation of the proteins differentially expressed in a certain group, enzymatic digestion of the isolated proteins followed by LC-tandem MS using a database-searching algorithm for protein identification. We have applied this method to understand the cardioprotective effect of pre-administration of docetaxel in adriamycin/docetaxel combination anti-cancer therapy, and the cellular processes that are affected by non-steroidal anti-inflammatory drugs (NSAIDs) in mouse stomach tissue during ulcer formation.

Efecto de la alimentación del lactante en la excreción de inmunopéptidos del gluten en heces / Influence of infant feeding on the excretion of gluten immunopeptides in feces

Introducción: la secreción de antígenos de la dieta en la leche materna ha sido ampliamente documentada. La transferencia de gliadina podría ser crítica para el desarrollo de una respuesta inmune. Objetivos: investigar la presencia de péptidos inmunogénicos de gluten en heces de lactantes alimentados con diversos regímenes. Material y métodos: estudio ciego, prospectivo, controlado, colaborativo entre tres centros hospitalarios y desarrollado entre septiembre de 2016 y enero de 2017. El protocolo del estudio fue aprobado previamente por el Comité de Ética de los hospitales de Sevilla. Resultados: se reclutaron 90 niños divididos en tres grupos de 30 niños. Un grupo experimental (edad media de 9,2 ± 2,8 semanas) con lactancia materna exclusiva; un grupo control1 (edad media 10,3 ± 3,3 semanas), alimentados con fórmula de inicio de forma exclusiva y en el que en ninguno de sus integrantes se detectó un test positivo del péptido 33-mer de gliadina en heces; y un grupo control2 con lactantes que consumían gluten en forma habitual (edades promedio 56 semanas ± 3,7 semanas) y que en un 23% (siete casos) tampoco excretaron en sus heces el PIG 33-mer. No se apreció diferencia en la cantidad de gluten ingerida por estos niños y por los que sí excretaron el péptido 33-mer en sus heces. Conclusiones: el hecho de no detectar gluten en heces de lactantes alimentados con leche materna exclusiva indica que probablemente este se encuentre por debajo de los límites para su detección. Hay niños sanos de un año de edad que, aunque consumen gluten, no lo excretan en heces

Introduction: the secretion of antigens from the diet into breast milk has been extensively documented. The transfer of gliadin could be critical for the development of an immune response. Objectives: to investigate the presence of immunogenic gluten peptides in the feces of infants fed with different diets. Material and methods: a blind, prospective, controlled, collaborative study was performed in three hospitals, between September 2016 and January 2017. The study protocol was approved by the Ethics Committee of the hospitals in Seville prior to starting the study. Results: the cohort was divided into three groups of 30 infants: an experimental group (average age 9.2 ± 2.8 weeks) with exclusive breastfeeding, a control group 1 (average age 10.3 ± 3.3 weeks) exclusively fed with onset formula and a control group 2 (average age 56 ± 3.7 weeks) with infants that consumed gluten on a regular basis. The peptide 33-mer of gliadin was negative in all feces samples from both the experimental and control group 1. With regard to control group 2, the peptide 33-mer of gliadin was negative in 23% of cases (seven children). There was no difference in the amount of gluten ingested by these children compared to those who excreted the 33-mer peptide. Conclusions: the failure to detect gluten in the feces of infants that were exclusively breastfed indicates that it is probably below the limits of detection. Healthy children who consume gluten may not excrete it in feces

Label-free immunosensors based on a novel multi-amplification signal strategy of TiO2-NGO/Au@Pd hetero-nanostructures.

A label-free electrochemical immunosensor for quantitative detection of human epididymis specific protein 4 antigen (HE4 Ag) was developed by a novel multi-amplification signal system. The multi-amplification signal system was formed by loading bimetallic Au@Pd holothurian-shaped nanoparticles (Au@Pd HSs) on titanium oxide nanoclusters functionalized nitrogen-doped reduced graphene oxide (TiO2-NGO). The Au@Pd HSs were obtained via seed-mediated approach with in-situ grown palladium nanoarms on gold nanorods (Au NRs) surfaces, which possessed good electrocatalysis for hydrogen peroxide (H2O2) reduction and excellent biocompatibility. The TiO2-NGO with the high catalytic activity and large specific surface area was synthesized by hydrothermal method. Using H2O2 as an electrochemically active substrate, the prepared label-free electrochemical immunosensor based on the TiO2-NGO/Au@Pd HSs hetero-nanostructures as the signal amplification platform exhibited excellent selectivity, reproducibility and stability for the detection of HE4 Ag. Meanwhile, the linear range from 40 fM to 60 nM with the detection limit of 13.33 fM (S/N = 3) was obtained, indicating the immunosensor offers a promising method for clinical detection of HE4 Ag.

Mechanical Compression of Coverslipped Tissue Sections During Heat-induced Antigen Retrieval Prevents Section Detachment and Preserves Tissue Morphology.

Heat-induced antigen retrieval (HIAR) is routinely employed on aldehyde-fixed tissue sections to enhance the reactivity of antibodies that exhibit weak or no specific interactions with tissue antigens when applied in conventional immunohistochemical protocols. A major drawback of HIAR protocols is, however, the heat-induced detachment of sections from the microscope slide with resultant impaired tissue morphology or loss of the section. We developed a method in which tissue sections mounted on glass slides are temporally coverslipped, and a clamp is used to compress the sections on the microscope slide during HIAR treatment. This "pressurized coverslipping" during HIAR was tested on various formalin-fixed tissues (murine kidneys and temporal bones, human tonsils and temporal bones) that were embedded in paraffin or celloidin. The method reliably kept the sections adherent to the slide, preserved the tissue morphology, and effectively retrieved tissue antigens for improved results in immunohistochemical labeling, even for exceptionally delicate, large, and poorly adhering sections, that is, decalcified human temporal bone sections. In summary, we present a simple method for improved slide adherence and morphological preservation of tissue sections during HIAR treatment that can be combined with all HIAR protocols and that requires only basic lab equipment.

Isolation of Proteins from Polyacrylamide Gels.

Minute amounts of proteins are required for immunization of mice for the development of antibodies including monoclonal antibodies. Here we describe a rapid procedure for the isolation of proteins from polyacrylamide gels after sodium dodecyl sulfate polyacrylamide gel electrophoresis in sufficient amounts for immunization of animals.

Disposable Voltammetric Immunosensors Integrated with Microfluidic Platforms for Biomedical, Agricultural and Food Analyses: A Review.

Disposable immunosensors are analytical devices used for the quantification of a broad variety of analytes in different areas such as clinical, environmental, agricultural and food quality management. They detect the analytes by means of the strong interactions between antibodies and antigens, which provide concentration-dependent signals. For the herein highlighted voltammetric immunosensors, the analytical measurements are due to changes in the electrical signals on the surface of the transducers. The possibility of using disposable and miniaturized immunoassays is a very interesting alternative for voltammetric analyses, mainly, when associated with screen-printing technologies (screen-printed electrodes, SPEs), and microfluidic platforms. The aim of this paper is to discuss a carefully selected literature about different examples of SPEs-based immunosensors associated with microfluidic technologies for diseases, food, agricultural and environmental analysis. Technological aspects of the development of the voltammetric immunoassays such as the signal amplification, construction of paper-based microfluidic platforms and the utilization of microfluidic devices for point-of-care testing will be presented as well.

How to Improve Sensitivity of Sandwich Lateral Flow Immunoassay for Corpuscular Antigens on the Example of Potato Virus Y?

A simple approach was proposed to decrease the detection limit of sandwich lateral flow immunoassay (LFIA) by changing the conditions for binding between a polyvalent antigen and a conjugate of gold nanoparticles (GNPs) with antibodies. In this study, the potato virus Y (PVY) was used as the polyvalent antigen, which affects economically important plants in the Solanaceae family. The obtained polyclonal antibodies that are specific to PVY were characterized using a sandwich enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). For LFIA, the antibodies were conjugated with GNPs with a diameter of 17.4 ± 1.0 nm. We conducted LFIAs using GNP conjugates in a dried state on the test strip and after pre-incubation with a sample. Pre-incubating the GNP conjugates and sample for 30 s was found to decrease the detection limit by 60-fold from 330 ng∙mL-1 to 5.4 ng∙mL-1 in comparison with conventional LFIA. The developed method was successfully tested for its ability to detect PVY in infected and uninfected potato leaves. The quantitative results of the proposed LFIA with pre-incubation were confirmed by ELISA, and resulted in a correlation coefficient of 0.891. The proposed approach is rapid, simple, and preserves the main advantages of LFIA as a non-laboratory diagnostic method.

Phage Display and Selections on Purified Antigens.

The isolation of antibody fragments targeting proteins implicated in cancers and other diseases remains a crucial issue on targeted therapy or diagnostic tool development. In many case, the protein of interest, or a relevant portion of this protein such as its extracellular domain, is available as purified protein. In such cases, phage display on purified antigen is an easy and fast way to select antibody fragment able to efficiently bind this antigen. However the output of phage selection can vary significantly depending on the way to immobilize the purified antigen during selection. The following protocols describe the selection of phage antibody on purified antigen adsorbed on plastic, i.e., panning, or a selection in solution, using a biotinylated antigen as well as the corresponding screening produces, and give hints on the advantage and drawbacks of each approach.

Novel monoclonal antibody-based immunochromatographic strip for detecting citrinin in fruit from Zhejiang province, China.

Citrinin (CIT) is a hepato-nephrotoxic fungal metabolite produced by the genera Penicillium, Aspergillus and Monascu. There is an increasing demand for rapid and economical methods for detection CIT residues in fruit. In this study, we developed an immunochromatographic strip (ICS) for detection of citrinin (CIT) residues in fruit for the first time. Anti-CIT monoclonal antibody (McAb) 2B9 was prepared, with a binding affinity of 9.39 × 108 L/moL. Conjugates CIT-BSA and McAb 2B9 were used to develop the ICS which could be completed in 5 min, with the detection limit of 50 ng/mL and no cross reactivity with other mycotoxins. Analysis of CIT in 64 fruit samples revealed that data obtained from the ICS test were in good agreement with indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs) and high performance liquid chromatography (HPLC). This result demonstrated that the ICS test could be used as a rapid, reliable, cost-effective and user-friendly qualitative tool for detection of CIT residues on-site.

Evaluation of the protective efficacy of Ornithodoros moubata midgut membrane antigens selected using omics and in silico prediction algorithms.

The African argasid tick Ornithodoros moubata transmits two important pathogens, the African swine fever virus and the spirochete Borrelia duttoni, the cause of human relapsing fever. To date, only conventional control measures such as widespread application of acaricides, strict control measures, and animal movement restrictions have been implemented to confine these diseases. Vaccines against tick infestations have the potential to be among the most efficacious interventions for the management of these diseases. Plasma membrane-associated proteins upregulated in tick midgut cells in response to blood feeding and digestion are thought to play vital functions in tick physiology and in the transmission of tick-borne pathogens. In addition, their antigenic extracellular regions are easily accessible to antibodies synthesised by immunised hosts, which makes them interesting targets for tick vaccine design. The mialomes (midgut transcriptomes and proteomes) of unfed O. moubata females and of engorged females at 48 h post-feeding have recently been obtained, providing a wealth of predicted midgut protein sequences. In the current study, these mialomes were screened using in silico tools to select predicted antigenic transmembrane proteins that were upregulated after feeding (516 proteins). The functionally annotatable proteins from this list (396 proteins) were then manually inspected following additional criteria in order to select a finite and easy-manageable number of candidate antigens for tick vaccine design. The extracellular antigenic regions of five of these candidates were obtained either as truncated recombinant proteins or as KLH-conjugated synthetic peptides, formulated in Freund's adjuvant, and individually administered to rabbits to assess their immunogenicity and protective potential against infestations by O. moubata and the Iberian species Ornithodoros erraticus. All candidates were highly immunogenic, but provided low protection against the O. moubata infestations (ranging from 7% to 39%). Interestingly, all candidates except one also protected against infestations by O. erraticus, achieving higher efficacies against this species (from 20% to 66%). According to their protective potential, three of the five antigens tested (Om17, Om86 and OM99) were considered little suitable for use in tick vaccines, while the other two (OM85 and OM03) were considered useful antigens for tick vaccine development, deserving further studies.

Fabrication and assessment of an electrospun polymeric microfiber-based platform under bulk flow conditions with rapid and efficient antigen capture.

This study investigated the fabrication and proof of concept design demonstrating rapid and highly sensitive antigen capture utilizing electrospun polystyrene (PS) microfiber mat substrates paired with vacuum pump pressurization to induce bulk flow. In comparison with conventional flat PS surfaces used for immunoassay purposes, this system optimizes the increased surface area of the electrospun polystyrene (ESPS) fiber mat substrates and the accelerated propagation of the antigen through the detection platform by using a vacuum pump to enable efficient and rapid antigen capture. The novelty of this work was demonstrated through a parametric study detailing how a fiber substrate can capture antigen sensitively and at high speeds. In terms of sensitivity, the current system is comparable to the conventionally used flat PS substrates. Additionally, the amount of antigen captured on a flat PS substrate in 60 minutes was surpassed in under 5 seconds when utilizing the ESPS-vacuum system. Three-dimensional ESPS fiber mats were then noted as a comparison between Damkohler numbers and between flat PS and ESPS-vacuum systems. The bulk flow of the ESPS-vacuum system allows for a Damkohler number of 0.37 indicating a balance between the flow rate and the reaction rate as opposed to a PS flat platform of 5.80 × 104 which illustrates a diffusion rate limited system. Finally, the overall ESPS-vacuum system was tested for its immunoassay capability. A sandwich fluorescence-based immunoassay was performed on both PS flat-diffusion and ESPS-vacuum systems. The ESPS-vacuum system indicated a wider detection range capability from 5 to 1000 ng mL-1 in comparison with the PS flat-diffusion system at 5 to 100 ng mL-1.

Paper-based diagnostics in the antigen-depletion regime: High-density immobilization of rcSso7d-cellulose-binding domain fusion proteins for efficient target capture.

In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760µM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system.

Electrochemical immunosensors - A powerful tool for analytical applications.

Immunosensors are biosensors based on interactions between an antibody and antigen on a transducer surface. Either antibody or antigen can be the species immobilized on the transducer to detect antigen or antibody, respectively. Because of the strong binding forces between these biomolecules, immunosensors present high selectivity and very high sensitivity, making them very attractive for many applications in different science fields. Electrochemical immunosensors explore measurements of an electrical signal produced on an electrochemical transductor. This signal can be voltammetric, potentiometric, conductometric or impedimetric. Immunosensors utilizing electrochemical detection have been explored in several analyses since they are specific, simple, portable, and generally disposable and can carry out in situ or automated detection. This review addresses the potential of immunosensors destined for application in food and environmental analysis, and cancer biomarker diagnosis. Emphasis is given to the approaches that have been used for construction of electrochemical immunosensors. Additionally, the fundamentals of immunosensors, technology of transducers and nanomaterials and a general overview of the possible applications of electrochemical immunosensors to the food, environmental and diseases analysis fields are described.