Metal oxide nanomaterials based electrochemical and optical biosensors for biomedical applications: Recent advances and future prospectives.

The amalgamation of nanostructures with modern electrochemical and optical techniques gave rise to interesting devices, so-called biosensors. A biosensor is an analytical tool that incorporates various biomolecules with an appropriate physicochemical transducer. Over the past few years, metal oxide nanomaterials (MONMs) have significantly stimulated biosensing research due to their desired functionalities, versatile chemical stability, and low cost along with their unique optical, catalytic, electrical, and adsorption properties that provide an attractive platform for linking the biomolecules, for example, antibodies, nucleic acids, enzymes, and receptor proteins as sensing elements with the transducer for the detection of signals or signal amplifications. The signals to be measured are in direct proportionate to the concentration of the bioanalyte. Because of their simplicity, cost-effectiveness, portability, quick analysis, higher sensitivity, and selectivity against a broad range of biosamples, MONMs-based electrochemical and optical biosensing platforms are exhaustively explored as powerful early-diagnosis tools for point of care applications. Herein, we made a bibliometric analysis of past twenty years (2004-2023) on the application of MONMs as electrochemical and optical biosensing units using Web of Science database and the results of which clearly reveal the increasing number of publications since 2004. Geographical area distribution analysis of these publications shows that China tops the list followed by the United States of America and India. In this review, we first describe the electrochemical and optical properties of MONMs that are crucial for the creation of extremely stable, specific, and sensitive sensors with desirable characteristics. Then, the biomedical applications of MONMs-based bare and hybrid electrochemical and optical biosensing frameworks are highlighted in the light of recent literature. Finally, current limitations and future challenges in the field of biosensing technology are addressed.

Single-Cell Secretion Analysis via Microfluidic Cell Membrane Immunosorbent Assay for Immune Profiling.

Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).

Time-resolved fluorescence microspheres-antibody-penicillin-binding protein assisted construction of immunochromatographic assay for sensitive detection of 22 ß-lactams in milk.

A sensitive immunochromatographic assay (ICA) using time-resolved fluorescence microspheres (TRFMs) coupled with an indirect-labeling mode was developed for simultaneously determining 22 kinds of ß-lactams in milk samples. The TRFMs labeled anti-receptor monoclonal antibodies (mAbs) conjugated to penicillin-binding proteins (PBPs) as ternary TRFMs-mAb-PBPs (TMP) nanoscaffolds provide excellent solubility, brightness, and stability. Thanks to the fact that they not only fully expose the binding sites of PBPs, thereby enhancing the biological affinity of PBPs towards the target, but also generated superb fluorescence signals, the versatile TMP manifested unique possibilities as efficient probes for ICA with remarkable enhancement in sensitivity in ß-lactams screening. The results showed that the standard curves of the 22 varying ß-lactams displayed linearity in their respective concentration ranges (R2 > 0.98), with the cutoff values of 1-100 ng/mL. The constructed TMP-ICA was successfully applied to the analysis of real milk, with consistent results compared with liquid chromatography-tandem mass spectrometry (LC-MS), providing an effective method for sensing ß-lactams in food matrices.

Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure.

ABSTRACT: Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines.

Generation and characterization of avian IgY antibodies for detecting beta-casein A1 in bovine milk.

Beta-casein is a primary milk protein that constitutes approximately 30% of the casein in bovine milk, with the two most common types in cattle being A1 and A2. The A2 protein differs from the A1 version due to a mutation in the codon at position 67, resulting in a histidine to proline substitution. However, the bioactive peptide, beta-casomorphine-7 (BCM7), which originates from partial proteolysis of the A1 variant, has been linked to several gastrointestinal disorders in humans. Production of A1 beta casein-free products is increasing demand in the milk market, worldwide. This study generated and characterized a polyclonal IgY antibody that specifically recognizes the A1 beta-casein protein present in cow's milk. A commercially available IgY anti-A1 antibody was used as a positive control, and the sensitivity and specificity of both the commercial and produced anti-A1 antibodies were evaluated. The results showed 100% sensitivity and specificity of 100% of the commercial IgY anti-A1. The in-house produced anti-A1 antibody demonstrated a sensitivity of 95.2% and a specificity of 100%, indicating its potential as a reliable and cost effective tool for detecting A1 beta-casein protein in milk samples.

Azathioprine as an adjuvant therapy in severe Graves' disease: a randomized controlled open-label clinical trial.

Introduction: Azathioprine (AZA) interferes with the activation of T and B lymphocytes, which are the main cells involved in the pathogenesis of Graves' disease (GD). The aim of this study was to investigate the effectiveness of AZA as an adjuvant therapy to antithyroid drugs (ATDs) for moderate and severe GD. In addition, we conducted an incremental cost-effectiveness analysis of AZA to determine its cost-effectiveness. Methods: We conducted a randomized, open-label, and parallel-group clinical trial. We randomized untreated hyperthyroid patients with severe GD into three groups. All patients received 45-mg carbimazole (CM) as the starting dose and propranolol 40-120 mg daily. The first group (AZA1) received an additional 1 mg/kg/day AZA, the second group (AZA2) received an additional 2 mg/kg/day AZA, and the third group (control group) received only CM and propranolol. We measured thyroid-stimulating hormone (TSH) and TSH-receptor antibody (TRAb) levels at baseline and every 3 months, while free triiodothyronine (FT3) and free thyroxine (FT4) levels were measured at the time of diagnosis, 1 month after initiation of therapy, and every 3 months thereafter until 2 years after remission. Thyroid volume (TV) was assessed by ultrasound at baseline and 1 year after remission. Results: A total of 270 patients were included in this trial. By the end of follow-up, there was higher remission rate in the AZA1 and AZA2 groups compared with controls (87.5% and 87.5% vs. 33.4%, p = 0.002). Throughout the course of follow-up, FT3, FT4, TSH, and TRAb were significantly different between the AZA groups and the control group, but there was no significant difference regarding TV. The decline in the concentrations of FT4, FT3, and TRAb was significantly faster in the AZA2 group than in the AZA1 group. The relapse rate during the 12-month follow-up was insignificantly higher in the control group than in either the AZA1 or AZA2 group (10, 4.4, and 4.4%, p = 0.05, respectively). The median relapse time was 18 months for the control group and 24 months for the AZA1 and AZA2 groups. The incremental cost-effectiveness ratio for the AZA group compared with the conventional group was 27,220.4 Egyptian pounds per remission reduction for patients using AZA as an adjuvant for ATDs. Conclusion: AZA could be a novel, affordable, cost-effective, and safe drug offering hope for patients with GD to achieve early and long-lasting medical remission. Trial registry: The trial is registered at the Pan African Clinical Trial Registry (Registration number: PACTR201912487382180).

Research Note: A novel method for preparation of egg yolk immunoglobulin Y against Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis, P. g) is the main pathogen of periodontal disease, which is treated with egg yolk immunoglobulin Y (IgY) against P. gingivalis. In order to quickly obtain IgY, 30 hens were immunized with inactivated P. gingivalis. The purification of IgY was carried out by the oleic acid (OA) method and the classical method (AS), respectively. The IgY antibody characteristics and antibacterial effects in HPDLF cells were detected by SDS-PAGE, indirect ELISA, Western blot and viability/toxicity assays. SDS-PAGE and Western blot analysis showed that IgY molecules which were rapidly purified by OA method were complete and specific to P. gingivalis. In addition, the results of crystal violet staining and bacterial staining indicated that IgY could agglutinate with P. gingivalis, inhibiting bacterial invasion of host cells. This study is the first to rapidly and efficiently purify IgY by OA method, and the purified IgY is expected to be used in the detection and treatment of P. gingivalis.

Synthesis of structure-defined ß-1,4-GlcNAc-modified wall teichoic acids as potential vaccine against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a high priority pathogen due to its life-threating infections to human health. Development of prophylactic or therapeutic anti-MRSA vaccine is a potential approach to treat S. aureus infections and overcome the resistance crisis. ß-1,4-GlcNAc glycosylated wall teichoic acids (WTAs) derived from S. aureus are a new type of antigen that is closely associated with ß-lactam resistance. In this study, structure-defined ß-1,4-GlcNAc-modified WTAs varied in chain length and numbers of GlcNAc modification were synthesized by an ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy in high efficiency and chromatography-free approach. Then the obtained WTAs were conjugated with tetanus toxin (TT) as vaccine candidates and were further evaluated in a mouse model to determine the structure-immunogenicity relationship. In vivo immunological studies revealed that the WTAs-TT conjugates provoked robust T cell-dependent responses and elicited high levels of specific anti-WTAs IgG antibodies production associated with the WTAs structure including chain length as well as the ß-1,4-GlcNAc modification pattern. Heptamer WTAs conjugate T6, carrying three copy of ß-1,4-GlcNAc modified RboP, was identified to elicit the highest titers of specific antibody production. The T6 antisera exhibited the highest recognition and binding affinity and the most potent OP-killing activities to MSSA and MRSA cells. This study demonstrated that ß-1,4-GlcNAc glycosylated WTAs are promising antigens for further development against MRSA.

Proteomic investigation and understanding on IgY purification and product development.

An increasing demand for the development of immunoglobin Y (IgY) illustrates the necessity of the component analysis in the process of conduction and quality control. This study investigated the proteomic changes in crude IgY extracts and purified IgY products obtained by sequential polyethylene glycol precipitation (PEG) of egg yolks followed by human mycoplasma protein-based affinity chromatography compared with intact egg yolks. After confirming the extraction efficiency and purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, liquid chromatography tandem-mass spectrometry (LC-MS/MS) was performed with samples including fresh yolk, IgY extracted product and purified product. A total of 348 proteins were identified, with 36 proteins deleted and 209 newly detected proteins in the purified product compared to the intact egg yolk. The significantly decreased proteins mainly included phosvitin, albumin, and apolipoprotein B whereas the significantly increased proteins were mainly IgY-related proteins. GO analysis showed that the purified IgY product had ATPase activity and purine ribonucleoside triphosphate binding activity, and was mainly involved in purine and nucleic acid metabolism. This study will inevitably fasten the commercial application of IgY antibodies and is of greater significance for promotion, development and approval for new antibody derived drug products.

Evaluación de un inmunoensayo para la medida de anticuerpos anti-tiroglobulina y anti-tiroperoxidasa / Evaluation of an immunoassay for the antithyroglobulin and antithyroperoxidase measuring

Introducción: La medición de los anticuerpos frente a tiroglobulina (ATG) y peroxidasa tiroidea (ATPO) es de interés para identificar pacientes con tiroiditis autoinmune.Este estudio pretende evaluar un inmunoensayo comercial de electroquimioluminiscencia para ATG y ATPO, estudiando la imprecisión, la linealidad, sensibilidad analítica, evaluación del arrastre, e influencia de interferentes endógenos.Material y métodos: La imprecisión se evaluó usando tres soluciones con diferentes concentraciones de analitos, analizándose 20 veces en la misma serie analítica y durante 20 días consecutivos, calculando el coeficiente de variación. Para el estudio de linealidad se combinaron una muestra con elevada concentración de analitos y un diluyente, obteniéndose concentraciones intermedias que se analizaron por triplicado. El límite de detección se calculó haciendo 20 determinaciones de una muestra de diluyente. El arrastre se evaluó analizando una muestra con alta concentración de anticuerpos seguida por otra con concentraciones muy bajas. El estudio de interferencias se realizó añadiendo a mezclas de suero hemolizado, Intralipid 30% y bilirrubina.Resultados: Las imprecisiones totales obtenidas (%) fueron 26,63, 9,53, y 14,9 para ATG y 21,19, 14,82 y 5,77 para ATPO. La linealidad queda definida por las ecuaciones de regresión: Y=6.61+1.01X(ATG) y Y=16.37+0.97X(ATPO). El límite de detección fue 17,17 para ATG y 5 para ATPO. El arrastre no fue significativo. La hemólisis produjo interferencia significativa en ambos ensayos.Conclusiones: Las imprecisiones obtenidas son comparables a las declaradas por el fabricante. La sensibilidad analítica cumple las especificaciones del fabricante. El comportamiento de ambos ensayos es lineal y no se halla arrastre significativo. La hemólisis interfiere ambos ensayos. (AU)

Introduction: The measuring of antibodies against thyroglobulin (ATG) and thyroperoxydase (ATPO) is useful for identifying patients with autoimmunethyroiditis. This study tries to assess an electrochemiluminescent immunoassay for ATG and ATPO, evaluating imprecision, linearity, analytic sensitivity, carry-over and the influence of endogenous interferents.Material and methods: Imprecision was assessed using three pools with different analytes concentrations, performing within run and between run 20 times. Fort the linearity study a sample containing high analyte concentration and a solvent devoid of analyte were combined, obtaining intermediates concentrations, which were analyzed by triplicate. The limit of detection was calculated analyzing 20 times a sample devoid of analyte. Carry-over was evaluated analyzing a sample with a high antibody concentration followed by other one containing low antibody concentration. The interference study was carried-out adding hemolyzed, Intralipid 30% and bilirubin into sera pool.Results: Total imprecision obtained (%) were 26.63, 9.53, and 14.9 for ATG and 21.19, 14.82, and 5.77 for ATPO. Linearity was defined for the following regression equations: Y=6.61+1.01X (ATG) and, Y=16.37+0.97X (ATPO). The limit of detection was 17.17 for ATG and 5 for ATPO. Carry-over was not significant. Hemolysis caused significant interference in both assays.Conclusions: Imprecision obtained were similar to the manufacturer declared ones. Analytic sensibility complies the manufacturer’s specifications. The behavior of both assays was linear and significant carry-over was not found. Hemolysis interferes in both assays. (AU)

Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody.

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.

Gradient method for accurate affinity determinations.

The value of the affinity constants (kd, ka, and KD) that are determined by label free interaction analysis methods are strongly affected by the ligand density at the sensor surface [1]. This paper outlines a new SPR-imaging method that applies a ligand density gradient enabling the analyte response to be extrapolated to Rmax = 0 µRIU. The mass transport limited region is used to determine the analyte concentration. Cumbersome optimization procedures for tuning the ligand density is prevented and surface dependent effects as rebinding, strong biphasic behavior etcetera are minimized. The method can be fully automated for e.g. accurate determination of the quality of antibodies from commercial sources.

Uma análise dos principais diagnósticos de Sars-cov-2 disponíveis no Brasil: uma revisão de literatura / Diagnosis and main types of coronavirus in humans: a narrative review / Diagnóstico y principales tipos de coronavirus en humanos: una revisión narrativa

Introdução: Com a emergência do SARS-CoV-2 foi disponibilizado uma grande quantidade de ferramentas de diagnóstico. Neste contexto, a falta de vacina, de tratamento e o grande número de casos graves e morte, possibilitou a aprovação emergencial de diversos testes, que ainda necessitam de estudos populacionais para seu registro definitivo. Objetivo: Realizar uma revisão de literatura para avaliar as metodologias de diagnóstico disponíveis no Brasil, de acordo com a realidade local de saúde, explorando o momento epidemiológico a complexidade do teste e a finalidade da sua aplicação. Metodologia: Trata-se de um estudo bibliográfico, descritivo do tipo revisão de literatura. Foram utilizadas as seguintes bases de dados científicos para buscas: PUBMED, MEDLINE, LILACS E COCHRANE LIBRARY, através de descritores selecionados na plataforma DECS. Resultados: O cenário de diversos ensaios, baseados em diferentes metodologias, como os testes baseados em RNA viral, em detecção de antígenos virais ou de anticorpos, associados ao conhecimento da história natural do vírus, possibilita uma análise crítica do melhor diagnóstico de acordo com a clínica do paciente, os epidemiológicos, o objetivo do diagnóstico e a acurácia do ensaio. Atualmente, há mudança no padrão imunológico da população e a descrição de tipos e subtipos de SARS-CoV-2 com mudanças gênicas, que podem levar a mudanças na acurácia diagnóstica ou a re-emergência em surtos de doença grave. Conclusão: Ainda é incerto o caminho evolutivo da história natural da Covid-19 e os ensaios diagnósticos estão em diferentes estágios de desenvolvimento, validação e produção e cada tipo de teste tem suas próprias vantagens e desvantagens distintas inerentes a plataforma tecnológica de origem e uma combinação de tipos de testes usados em momentos diferentes pode ser útil para a condução clínica dos pacientes e no controle da pandemia por SARS-CoV-2.

Introduction: With the emergence of SARS-CoV-2, a large number of diagnostic tools were made available. In this context, the lack of vaccine, treatment and the large number of severe cases and death, allowed the emergency approval of several tests, which still require population studies for their definitive registration. Objective: To carry out a literature review to evaluate the diagnostic methodologies available in Brazil, according to the local health reality, exploring the epidemiological moment, the complexity of the test and the purpose of its application. Methodology: This is a bibliographic, descriptive study of the literature review type. The following scientific databases were used for searches: PUBMED, MEDLINE, LILACS AND COCHRANE LIBRARY, through selected descriptors on the DECS platform. Results: The scenario of several tests, based on different methodologies, such as tests based on viral RNA, on detection of viral antigens or antibodies, associated with knowledge of the natural history of the virus, allows a critical analysis of the best diagnosis according to the patient's clinical, epidemiological, diagnostic objective and assay accuracy. Currently, there is a change in the immune pattern of the population and the description of types and subtypes of SARS-CoV-2 with genetic changes, which can lead to changes in diagnostic accuracy or the re-emergence in outbreaks of severe disease. Conclusion: The evolutionary path of the natural history of Covid-19 is still uncertain and diagnostic assays are at different stages of development, validation and production and each type of test has its own distinct advantages and disadvantages inherent in the technology platform of origin and a combination of types of tests used at different times can be useful for the clinical management of patients and in the control of the SARS-CoV-2 pandemic.

Introducción: Con la aparición del SARS-CoV-2, se dispuso de un gran número de herramientas diagnósticas. En este contexto, la falta de vacuna, tratamiento y el gran número de casos graves y muerte, permitieron la aprobación de urgencia de varias pruebas, que aún requieren estudios poblacionales para su registro definitivo. Objetivo: Realizar una revisión bibliográfica para evaluar las metodologías diagnósticas disponibles en Brasil, de acuerdo con la realidad sanitaria local, explorando el momento epidemiológico, la complejidad de la prueba y la finalidad de su aplicación. Metodología: Se trata de un estudio bibliográfico, descriptivo, del tipo revisión de literatura. Para las búsquedas se utilizaron las siguientes bases de datos científicas PUBMED, MEDLINE, LILACS Y COCHRANE LIBRARY, a través de descriptores seleccionados en la plataforma DECS. Resultados: El escenario de varias pruebas, basadas en diferentes metodologías, como pruebas basadas en el ARN viral, en la detección de antígenos virales o anticuerpos, asociado al conocimiento de la historia natural del virus, permite un análisis crítico del mejor diagnóstico de acuerdo con la clínica del paciente, epidemiológica, objetivo diagnóstico y precisión de la prueba. Actualmente, hay un cambio en el patrón inmunológico de la población y la descripción de tipos y subtipos de SARS-CoV-2 con cambios genéticos, que pueden conducir a cambios en la precisión diagnóstica o la reaparición en brotes de enfermedad grave. Conclusiones: El camino evolutivo de la historia natural del Covid-19 es aún incierto y los ensayos de diagnóstico se encuentran en diferentes etapas de desarrollo, validación y producción y cada tipo de prueba tiene sus propias ventajas y desventajas distintas inherentes a la plataforma tecnológica de origen y una combinación de tipos de pruebas utilizadas en diferentes momentos puede ser útil para el manejo clínico de los pacientes y en el control de la pandemia de SARS- CoV-2.

Epidemiological aspects of the occurrence of anti-Sarcocystis spp. andanti-Toxoplasma gondii antibodies in dogs in the Northern region of Brazil / Aspectos epidemiológicos da ocorrência de anticorpos anti-Sarcocystisspp. e anti-Toxoplasma gondii em cães da região norte do Brasil

We conducted aseroepidemiological study on the occurrence of anti-Sarcocystisspp. and anti-Toxoplasma gondii antibodies in dogs from family farming properties in the municipality of Ji-Paraná, Rondônia.Blood samples were collected from apparently healthy dogs between September 2012 and November 2013. In total, 181 blood serum samples were analyzed using an indirect immunofluorescence assay, among which 57 (31.49%) and 20 (11.04%) were positive for anti-T. gondii and anti-Sarcocystis spp., respectively. Statistical analyses showed that the type of food fed to the dogs was associated with the occurrence of anti-Sarcocystisspp. antibodies. In contrast, age and access to bovine carcasses were the risk factors for anti-T. gondii.The high occurrence of seropositive dogs for Sarcocystis spp. and T. gondii evidences the wide distribution of these agents in the studied area, possibly due to human and animal exposure to these protozoan species. In addition, anti-T. gondii antibodies were directly proportional to dog age. The increase in the number of positive animals with age was statistically significant. Furthermore, high antibody titers (up to 800) against Sarcocystis spp. in dogs suggest the possibility of recent exposure, in addition to environmental contamination by oocysts/sporocysts eliminated by the feces of these animals.

Conduzimos um estudo soroepidemiológico sobre a ocorrência de anticorpos anti- Sarcocystis spp. e anti-Toxoplasma gondiiem cães de propriedades de agricultura familiar no município de Ji-Paraná, Rondônia. Amostras de sangue foram coletadas de cães aparentemente saudáveis, entre setembro de 2012 e novembro de 2013. Ao todo, foram analisados 181 soros sanguíneos por meio do ensaio de imunofluorescência indireta, sendo positivas 57 (31,49%) e 20 (11,04%) amostras para anticorpos anti-T. gondii e anti-Sarcocystis spp., respectivamente. As análises estatísticas demonstraram que o tipo de alimentação fornecida aos cães esteve associado à ocorrência de anticorpos anti-Sarcocystis spp. Em contraste a idade e o acesso à carcaça bovina foram fatores de risco para a presença de anticorpos anti-T. gondii. A alta ocorrência de cães soropositivos para Sarcocystis spp. e T. gondii evidencia a ampla distribuição desses agentes na área estudada, possivelmente devido à exposição humana e animal a essas espécies de protozoários. Além disso, o resultado dos anticorpos anti-T. gondii relacionados a idade do cão mostraram diferença estatística, com aumento significativo no número de animais positivos com a idade. Além disso, altos títulos de anticorpos (até 800) contra Sarcocystis spp. em cães sugerem a possibilidade de exposição recente, além da contaminação ambiental por oocistos/esporocistos eliminados pelas fezes desses animais.

Colloidal Au sphere and nanoflower-based immunochromatographic strips for sensitive detection of zearalenone in cereals.

Zearalenone (ZEN), also known as an F-2 toxin, is a secondary metabolite in the toxic Fusarium species with estrogen properties. ZEN and its derivatives can cause developmental and reproductive disorders in humans and other mammals. In this study, colloidal Au spheres (AuSPs) and Au nanoflowers (AuNFs) were used as signal labels to detect ZEN in cereals, and the critical factors affecting the sensitivity of the immunochromatographic strip (ICS), namely the volume of antigen, antibody, and probe quantities were optimized and compared in detail. Since the large specific surface area of AuNFs reduces the steric hindrance of proteins, it is more conducive to improving the fixation rate of antibodies and proteins. Compared with the traditional colloidal AuSP immunochromatographic strip (AuSP-ICS), the volume of the antibody used in the AuNF immunochromatographic strip (AuNF-ICS) was 0.6 times that in the AuSPs-ICS. At the same antigen volume, a lower amount of probe can achieve the desired visual detection effect and higher sensitivity. For the AuNF-ICS, the limit of detection (LOD) was as low as 0.08 ng mL-1. ZEN could be detected quickly and accurately from 0.08-10.2 ng mL-1. And the AuNF-ICS had a high degree of specificity and sensitivity to ZEN. In summary, the AuNF-ICS serves as a valuable tool in large-scale on-site detection of ZEN.

Antibody-Coated Wearable Organic Electrochemical Transistors for Cortisol Detection in Human Sweat.

The dysregulation of the hormone cortisol is related to several pathological states, and its monitoring could help prevent severe stress, fatigue, and mental diseases. While wearable antibody-based biosensors could allow real-time and simple monitoring of antigens, an accurate and low-cost antibody-based cortisol detection through electrochemical methods is considerably challenging due to its low concentration and the high ionic strength of real biofluids. Here, a label-free and fast sensor for cortisol detection is proposed based on antibody-coated organic electrochemical transistors. The developed devices show unprecedented high sensitivities of 50 µA/dec for cortisol sensing in high-ionic-strength solutions with effective cortisol detection demonstrated with real human sweat. The sensing mechanism is analyzed through impedance spectroscopy and confirmed with electrical models. Compared to existing methods requiring bulky and expensive laboratory equipment, these wearable devices enable point-of-care cortisol detection in 5 min with direct sweat collection for personalized well-being monitoring.

Evaluación de los niveles de anticuerpos para COVID-19 en pacientes que concurrieron a un laboratorio de la Ciudad Autónoma de Buenos Aires / Evaluation of antibody levels for COVID-19 patients who attended a laboratory in Buenos Aires city

Resumen A fines de 2019 se describieron en China los primeros casos de neumonía asociada a SARS-CoV-2. La OMS la llamó COVID-19 y declaró emergencia sanitaria internacional en enero de 2020, ante la rápida diseminación de la infección a nivel mundial. En la Argentina los primeros casos se detectaron en marzo de 2020 y casi inmediatamente comenzaron a utilizarse métodos directos para detección de SARS-CoV-2 (RT PCR, LAMP, entre otros). Los métodos para detección de anticuerpos fueron aprobados posteriormente y no son de elección para realizar el diagnóstico de la enfermedad. En este laboratorio estos últimos comenzaron a utilizarse durante la primera ola de COVID-19 y con estos datos se realizó un estudio observacional retrospectivo de una serie de pacientes con resultados de anticuerpos IgG positivos. Se calculó la tasa de notificación al Sistema Integrado de Información Sanitaria Argentino (SISA) y se evaluaron los niveles de anticuerpos, agrupándolos de acuerdo a: si estaban notificados y si tenían resultado de RT PCR/LAMP, los síntomas presentados y el tiempo transcurrido post RT PCR/LAMP. No fue posible demostrar diferencias entre los pacientes con RT PCR/LAMP detectable y no detectable, tampoco con el tipo de síntomas declarados ni con respecto a los días transcurridos posinfección. Sin embargo, se observó que existía una diferencia significativa entre el grupo de pacientes notificados y no notificados y una alta tasa de pacientes con anticuerpos positivos que no fueron declarados en SISA, por lo que su detección podría considerarse como marcador subrogante de contacto cuando no fuera posible arribar al diagnóstico por métodos moleculares.

Abstract At the end of 2019 the first cases of SARS-CoV-2-associated pneumonia were reported in China. Consequently, the World Health Organization (WHO) named it COVID-19 and in January 2020, it declared the international health emergency due to the worldwide rapid spread of the infection. The first cases in Argentina were detected in early March 2020. Molecular tests like RT PCR and LAMP were immediately used. Serological tests for antibody detection were approved a few months later; however, these are still not the preferred evidiagnostic method for the disease. In our laboratory, the latter began to be used during the first wave of COVID-19. With the results obtained in that moment, an observational retrospective study in a cohort of patients who came voluntarily to test for SARS-CoV-2 IgG antibodies and whose results were positive was performed. The notification rate to the Argentine Integrated System for Health Information (SISA for its acronym in Spanish) was calculated and antibody levels were evaluated, clustering them according to the following facts: if the event had been notified to the SISA and if they had a previous RT PCR/LAMP result, the symptoms experienced by these patients and the time elapsed between RT PCR/LAMP and antibody test results. It was not possible to demonstrate differences between patients with detectable and undetectable RT PCR/LAMP, neither with the type of declared symptoms nor with respect to the days elapsed post-infection. However, it was found that there was a significant difference between notified and non-notified patients, and a high rate of non-notified patients with positive antibodies. Therefore, antibodies level might be considered as a surrogate marker of SARS-CoV-2 contact when a diagnosis through molecular methods is not available.

Resumo No final de 2019 foram reportados na China os primeiros casos de pneumonia associados a SARS-CoV-2. A Organização Mundial da Saúde (OMS) chamou-a de COVID-19 e declarou emergência sanitária internacional em janeiro de 2020, frente à rápida disseminação da infecção em nível mundial. Na Argentina os primeiros casos foram detectados no início de março de 2020 e de forma quase imediata, começaram a ser utilizados métodos diretos para detectar SARS-CoV-2 (RT PCR, LAMP, entre outros). Os métodos para detectar anticorpos foram posteriormente aprovados e não são de eleição para realizar o diagnóstico da doença. Em nosso laboratório, a utilização destes últimos começou durante a primeira onda de COVID-19 e com os resultados obtidos nesse momento foi realizado um estudo observacional retrospectivo de uma série de pacientes com resultados de anticorpos IgG positivos. Foi calculada a taxa de notificação ao Sistema Integrado de Informação em Saúde da Argentina (SISA) e foram avaliados os níveis de anticorpos agrupando- os de acordo a: se estavam notificados e se eles tinham resultado de RT PCR/LAMP, os sintomas apresentados e o tempo decorrido pós RT PCR/LAMP. Não foi possível demonstrar diferenças entre pacientes com RT PCR/LAMP detectável e não detectável, nem com o tipo de sintomas declarados nem com relação aos dias decorridos após a infecção. No entanto, verificou-se que existia uma diferença significativa entre o grupo de pacientes notificados e não notificados, e uma alta taxa de pacientes com anticorpos positivos que não foram declarados no SISA, portanto, sua detecção poderia ser considerada como um marcador substituto de contato quando não fosse possível chegar ao diagnóstico por métodos moleculares.

Correlación entre dos metodologías para la medición de anticuerpos contra la proteína spike del coronavirus del Síndrome Respiratorio Agudo Grave 2 / Correlation between two methodologies for the measurement of antibodies against the spike protein of coronavirus of the Severe Acute Respiratory Syndrome 2

Resumen El coronavirus del Síndrome Respiratorio Agudo Grave 2 (SARS-CoV-2) posee diversas proteínas estructurales que incluyen la proteína spike (S), principal blanco de las vacunas actuales. Existen diversas metodologías para la medición de anticuerpos contra ésta que brindan información acerca de la respuesta inmune frente a la vacunación. El objetivo de este trabajo fue determinar la correlación entre quimioluminiscencia (CLIA) y enzimoinmunoanálisis de adsorción (ELISA) para la medición de anticuerpos IgG anti-proteína S (IgG anti-S). Se recolectaron resultados serológicos de 169 individuos y se determinaron los niveles de anticuerpos por ambas metodologías. Del total de muestras, 106 arrojaron un resultado positivo por ambas metodologías y 15 resultaron discordantes (CLIA+, ELISA-), con índice Kappa de 0,80. La correlación entre ambas metodologías fue buena. Este estudio podría aportar al manejo y seguimiento de la población vacunada, con la finalidad de obtener un valor de corte para evaluar la aplicación de una dosis adicional.

Abstract Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has several structural proteins including the spike (S) protein, which is the main target of current vaccines. There are various methodologies for the measurement of antibodies against it that provide information about the immune response to vaccination. The objective of this study was to determine the correlation between chemiluminescence (CLIA) and enzyme-linked immunoassay (ELISA) for the measurement of IgG anti-S protein (IgG anti-S) antibodies. Serological results were collected from 169 individuals and antibody levels were determined by both methodologies. Out of the total samples, 106 were positive by both methodologies and 15 were discordant (CLIA+, ELISA-), with a Kappa index of 0.80. The correlation between both methodologies was good. This study could contribute to the management and follow-up of the vaccinated population, in order to obtain a cut-off value to evaluate the application of an additional dose.

Resumo O coronavírus da Síndrome Respiratória Aguda Grave 2 (SARS-CoV-2) possui várias proteínas estruturais, incluindo a proteína spike (S), principal alvo das vacinas atuais. Existem várias metodologias para medir anticorpos contra ela que fornecem informações sobre a resposta imune diante da vacinação. O objetivo deste trabalho foi determinar a correlação entre quimioluminescência (CLIA) e enzimoimunoanálise de absorção (ELISA) para a medição de anticorpos IgG anti-proteína S (IgG anti-S). Foram coletados resultados sorológicos de 169 indivíduos e os níveis de anticorpos foram determinados por ambas as metodologias. Do total de amostras, 106 deram resultados positivos nas duas metodologias e 15 foram discordantes (CLIA+, ELISA-), com índice Kappa de 0,80. A correlação entre as duas metodologias foi boa. Este estudo poderia contribuir para a gestão e seguimento da população vacinada, visando a obter um valor de corte para avaliar a aplicação de uma dose adicional.

Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies.

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.