An intense cathodic electrochemiluminescence from carbon-nanosheets in situ grown on glassy carbon electrode and application in immunoanalysis via biometallization strategy.

An intense cathodic electrochemiluminescence (ECL) is reported from a polarized glassy carbon electrode (GCE) in peroxydisulfate solution. After the polarization in 1 M Na2SO4 at the potential of - 3.7 V for 3 s, carbon nanosheets (C-NSs) were in situ grown on the surface of the GCE. Measured in 100 mM K2S2O8 solution, the ECL intensity of the GCE/C-NSs is 112-fold that of a bare GCE. The ECL spectrum revealed that the true ECL luminophore in the GCE/C-NSs-peroxydisulfate system is O2/S2O82- which is promoted by C-NSs. When Cu2+ was electrochemically enriched and reduced to Cu(0) on the catalytic sites of C-NSs, the ECL from GCE/C-NSs/Cu in K2S2O8 solution was decreased with increasing logarithmic concentration of Cu2+ in the range from 10 pM to 1 µM, with a limit of detection (LOD) of 3 pM. An immunoanalysis method is proposed via a biometallization strategy using CuS nanoparticles as the tags and carcinoembryonic antigen (CEA) as the model analyte. After the immune recognition in the microplate, the CuS tags in the immunocomplex were dissolved and the resultant Cu2+ was electrochemically enriched and reduced on the catalytic sites of C-NSs, quenching the ECL intensity of GCE/C-NSs-O2/S2O82- system. The proposed ECL immunoanalysis method was used to quantify CEA in actual serum samples with an LOD of 1.0 fg mL-1, possessing the advantages of simple electrode modification, high sensitivity and good reproducibility.

Electrochemical biosensor for the evaluation of monoclonal antibodies targeting the N protein of SARS-CoV-2 virus.

The emergence of COVID-19 caused by the coronavirus SARS-CoV-2 has prompted a global pandemic that requires continuous research and monitoring. This study presents a design of an electrochemical biosensing platform suitable for the evaluation of monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein. Screen-printed carbon electrodes (SPCE) modified with gold nanostructures (AuNS) were applied to design a versatile and sensitive sensing platform. Electrochemical techniques, including electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), were used to investigate the interactions between immobilised recombinant N (rN) protein and several monoclonal antibodies (mAbs). The electrochemical characterisation of SPCE/AuNS/rN demonstrated a successful immobilisation of rN, enhancing the electron transfer kinetics. Affinity interactions between immobilised rN and four mAbs (mAb-4B3, mAb-4G6, mAb-12B2, and mAb-1G5) were explored. Although mAb-4B3 showed some non-linearity, the other monoclonal antibodies exhibited specific and well-defined interactions followed by the formation of an immune complex. The biosensing platform demonstrated high sensitivity in the linear range (LR) from 0.2 nM to 1 nM with limits of detection (LOD) ranging from 0.012 nM to 0.016 nM for mAb-4G6, mAb-12B2, and mAb-1G5 and limits of quantification (LOQ) values ranging from 0.035 nM to 0.139 nM, as determined by both EIS and SWV methods. These results highlight the system's potential for precise and selective detection of monoclonal antibodies specific to the rN. This electrochemical biosensing platform provides a promising route for the sensitive and accurate detection of monoclonal antibodies specific to the rN protein.

Rapid Detection of Lipopolysaccharide and Whole Cells of Francisella tularensis Based on Agglutination of Antibody-Coated Gold Nanoparticles and Colorimetric Registration.

The paper presents development and characterization of a new bioanalytical test system for rapid detection of lipopolysaccharide (LPS) and whole cells of Francisella tularensis, a causative agent of tularemia, in water samples. Gold nanoparticles (AuNPs) coated by the obtained anti-LPS monoclonal antibodies were used for the assay. Their contact with antigen in tested samples leads to aggregation with a shift of absorption spectra from red to blue. Photometric measurements at 530 nm indicated the analyte presence. Three preparations of AuNPs with different diameters were compared, and the AuNPs having average diameter of 34 nm were found to be optimal. The assay is implemented in 20 min and is characterized by detection limits equal to 40 ng/mL for LPS and 3 × 104 CFU/mL for whole cells of F. tularensis. Thus, the proposed simple one-step assay integrates sensitivity comparable with other immunoassay of microorganisms and rapidity. Selectivity of the assay for different strains of F. tularensis was tested and the possibility to choose its variants with the use of different antibodies to distinguish virulent and non-virulent strains or to detect both kinds of F. tularensis was found. The test system has been successfully implemented to reveal the analyte in natural and tap water samples without the loss of sensitivity.

Fast and Accurate Determination of Minute Ochratoxin A Levels in Cereal Flours and Wine with the Label-Free White Light Reflectance Spectroscopy Biosensing Platform.

Ochratoxin A (OTA) is one of the most toxic naturally encountered contaminants and is found in a variety of foods and beverages, including cereals and wine. Driven by the strict regulations regarding the maximum allowable OTA concentration in foodstuff and the necessity for on-site determination, the development of fast and sensitive methods for the OTA determination in cereal flours and wine samples, based on white light reflectance spectroscopy, is presented. The method relied on appropriately engineered silicon chips, on top of which an OTA-protein conjugate was immobilized. A polyclonal antibody against OTA was then employed to detect the analyte in the framework of a competitive immunoassay; followed by the subsequent addition of a biotinylated secondary antibody and streptavidin for signal enhancement. A small size instrument performed all assay steps automatically and the bioreactions were monitored in real time as the software converted the spectral shifts into effective biomolecular adlayer thickness increase. The assay developed had a detection limit of 0.03 ng/mL and a working range up to 200 ng/mL. The assay lasted 25 min (less than 1h, including calibrators/antibody pre-incubation) and was accomplished following a simple sample preparation protocol. The method was applied to corn and wheat flour samples and white and red wines with recovery values ranging from 87.2 to 111%. The simplicity of the overall assay protocol and convenient instrumentation demonstrates the potential of the immunosensor developed for OTA detection at the point of need.

Comparative study of human growth hormone measurements: impact on clinical interpretation.

OBJECTIVES: Human growth hormone (hGH) provocation test is an essential tool to assess growth hormone deficiency (GHD) in children and young adults. It is important to have a robust method to determine the hGH peak of stimulation. This work aimed to compare three common automated immunoassays for hGH quantification and to ascertain whether there are still result-related differences which can impact clinical decision. METHODS: We analyzed the GH provocation test for 39 young subjects from pediatric department of Montpellier hospital, admitted for suspicion of growth hormone deficiency. The full range of measurements as well as the peak level of serum GH were compared using three automated immunoassays on three different immunoanalyzers: IDS-hGH on iSYS, LIAISON-hGH on Liaison XL and Elecsys ROCHE-hGH, on COBAS 8000. RESULTS: A good correlation was obtained between methods for all measurements (r2>0.99) by using Passing-Bablok regression analysis. Bland-Altman analysis showed the best agreement between IDS-hGH and LIAISON-hGH systems (bias=-14.5%) compared to Elecsys ROCHE-hGH (bias=28.3%). When considering stratification of the study population and a unique cutoff, there were some discrepancies in interpretation of the results especially concerning the more recent Elecsys ROCHE-hGH assay. Nevertheless, when the adequate cutoff for each method was taken into account results were well correlated for all systems. CONCLUSIONS: A cutoff for Elecsys Roche-hGH method was established to better explain the results. Clinician must be aware of the use of assay-specific cutoff to correctly integrate the results of GH tests in the GHD diagnosis.

A Review on the Role of Ethylenediaminetetraacetic Acid (EDTA) in the Treatment and Understanding of Psoriasis.

Psoriasis is a long-term, autoimmune inflammatory condition characterized by red, scaly plaques that can range from a few patches to total skin coverage. Over the past 60 years, and more recently, the metal-chelating agent ethylenediaminetetraacetic acid (EDTA) has proven increasingly useful in the treatment and understanding of psoriasis and related conditions. This review will analyze the current role and effectiveness of EDTA in clinical and non-clinical studies designed to improve the diagnosis and treatment of psoriasis in patients. Currently, EDTA demonstrates great medical benefit in the treatment of psoriasis as an antioxidant and as an inhibitor of beta-lipoprotein production. EDTA additionally functions well in research applications due to its ability to maintain red blood cell structural integrity. The authors find that the perceived impact of EDTA in the understanding and combating of psoriasis to be greatly underestimated and is therefore in need of increased awareness and attention by healthcare professionals, dermatologists, and clinical researchers.

Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2.

BACKGROUND: The detection of serum antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a new tool for the coronavirus disease 2019 (COVID-19) diagnosis. Since many coronaviruses are sensitive to heat, heating inactivation of samples at 56°C prior to testing is considered a possible method to reduce the risk of transmission, but the effect of heating on the measurement of SARS-CoV-2 antibodies is still unclear. METHODS: By comparing the levels of SARS-CoV-2 antibodies before and after heat inactivation of serum at 56°C for 30 minutes using a quantitative fluorescence immunochromatographic assay RESULTS: We showed that heat inactivation significantly interferes with the levels of antibodies to SARS-CoV-2. The IgM levels of all the 34 serum samples (100%) from COVID-19 patients decreased by an average level of 53.56%. The IgG levels were decreased in 22 of 34 samples (64.71%) by an average level of 49.54%. Similar changes can also be observed in the non-COVID-19 disease group (n = 9). Of note, 44.12% of the detected IgM levels were dropped below the cutoff value after heating, suggesting heat inactivation can lead to false-negative results of these samples. CONCLUSION: Our results indicate that heat inactivation of serum at 56°C for 30 minutes interferes with the immunoanalysis of antibodies to SARS-CoV-2. Heat inactivation prior to immunoanalysis is not recommended, and the possibility of false-negative results should be considered if the sample was pre-inactivated by heating.

Is insulin intoxication still the perfect crime? Analysis and interpretation of postmortem insulin: review and perspectives in forensic toxicology.

Insulin is an anabolic hormone essential to glucose homeostasis. Insulin therapy, comprising human insulin (HI) or biosynthetic analogs, is critical for the management of type-1 diabetes and many of type-2 diabetes. However, medication error including non-adapted dose and confusion of insulin type, and misuse, such as massive self-administration or with criminal intent, can have lethal consequences. The aim of this paper is to review the state of knowledge of insulin analysis in biological samples and of the interpretation of insulin concentrations in the situation of insulin-related death investigations. Analytic aspects are considered, as quantification can be strongly impacted by methodology. Immunoanalysis, the historical technique, has a prominent role due to its sensitivity and ease of implementation. Recently, liquid chromatography coupled to mass spectrometry has provided indispensable selectivity in forensic contexts, distinguishing HI, analogs, and degradation products. We review the numerous antemortem (dose, associated pathology, injection-to-death interval, etc.) and postmortem parameters (in corpore degradation, in vitro degradation related to hemolysis, etc.) involved in the interpretation of insulin concentration. The interest and limitations of various alternative matrices providing a valuable complement to blood analysis are discussed. Vitreous humor is one of the most interesting, but the low diffusion of insulin in this matrix entails very low concentrations. Injection site analysis is relevant for identifying which type of insulin was administered. Muscle and renal cortex are matrices of particular interest, although additional studies are required. A table containing most case reports of fatal insulin poisoning published, with analytical data, completes this review. A logic diagram is proposed to highlight analytical issues and the main parameters to be considered for the interpretation of blood concentrations. Finally, it remains a challenge to provide reliable biological data and solid interpretation in the context of death related to insulin overdose. However, the progress of analytical tools is making the "perfect crime" ever more difficult to commit.

Determination of Mycotoxin Production of Fusarium Species in Genetically Modified Maize Varieties by Quantitative Flow Immunocytometry.

Levels of mycotoxins produced by Fusarium species in genetically modified (GM) and near-isogenic maize, were determined using multi-analyte, microbead-based flow immunocytometry with fluorescence detection, for the parallel quantitative determination of fumonisin B1, deoxynivalenol, zearalenone, T-2, ochratoxin A, and aflatoxin B1. Maize varieties included the genetic events MON 810 and DAS-59122-7, and their isogenic counterparts. Cobs were artificially infested by F. verticillioides and F. proliferatum conidia, and contained F. graminearum and F. sporotrichoides natural infestation. The production of fumonisin B1 and deoxynivalenol was substantially affected in GM maize lines: F. verticillioides, with the addition of F. graminearum and F. sporotrichoides, produced significantly lower levels of fumonisin B1 (~300 mg·kg-1) in DAS-59122-7 than in its isogenic line (~580 mg·kg-1), while F. proliferatum, in addition to F. graminearum and F. sporotrichoides, produced significantly higher levels of deoxynivalenol (~18 mg·kg-1) in MON 810 than in its isogenic line (~5 mg·kg-1). Fusarium verticillioides, with F. graminearum and F. sporotrichoides, produced lower amounts of deoxynivalenol and zearalenone than F. proliferatum, with F. graminearum and F. sporotrichoides. T-2 toxin production remained unchanged when considering the maize variety. The results demonstrate the utility of the Fungi-Plex™ quantitative flow immunocytometry method, applied for the high throughput parallel determination of the target mycotoxins.

Seroprevalence of Toxoplasma gondii and associated risk factors in operators of a slaughter plant in Bío-Bío (Chile) / Seroprevalencia de Toxoplasma gondii y factores de riesgo asociados en operarios de una planta de beneficio animal del Bío-Bío, Chile / Soro prevalência de Toxoplasma gondii e fatores de associados em operários de uma planta de beneficiamento animal de Bío Bío, no Chile

Abstract: Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii, a protozoan whose definitive hosts are cats, among them domestic cat, which can transmit the infection to humans. In Chile, there are no published studies on seroprevalence in people with occupational risk. Thus, this study aimed to determine the seroprevalence of Toxoplasma gondii and associated risk factors in operators of a slaughter plant in Bío Bío (Chile). Serum samples from 39 operators were collected and studied by chemiluminescence analysis in order to detect IgG and IgM antibodies, with a sensitivity and specificity of 93 and 96%, respectively. An epidemiological survey was conducted and odds ratio was calculated for the analysis of the variables of hygiene, food, and exposure. Evidence showed that 24 individuals had IgG antibodies for an apparent seroprevalence of 61.5%, while this was 0% for IgM. In addition, the highest seropositivity was observed in operators who did not use masks (64%) and did not disinfect the working material (100%), as well as in those who consumed undercooked meat (62.5%). Regarding exposure time, 72.7% was obtained for the group of more than 10 years, and 62.2% of seropositivity was found in those exposed between four and seven days a week. There were no significant differences for any of the analyzed variables (p > 0.05). The study concludes that there is a high seroprevalence of Toxoplasma gondii in workers with occupational risk at the Bío-Bío slaughter plant.

Resumen: La toxoplasmosis es una zoonosis de distribución mundial causada por Toxoplasma gondii, protozoo que tiene como hospederos definitivos a los felinos, entre estos el gato doméstico, el cual puede transmitir la infección al ser humano. En Chile no existen estudios publicados de seroprevalencia en personas con riesgo ocupacional. Por eso el presente estudio tuvo como objetivo determinar la seroprevalencia de Toxoplasma gondii y factores de riesgo asociados en operarios de una planta de beneficio animal del Bío-Bío, Chile. Se muestrearon 39 sueros de operarios y se estudiaron mediante análisis quimioluminiscente para la detección de anticuerpos inmunoglobulina G (IgG) e inmunoglobulina M (IgM), con una sensibilidad y especificidad del 93 y 96 %, respectivamente. Se aplicó una encuesta epidemiológica y se calculó el odds ratio para el análisis de las variables higiénicas, alimenticias y de exposición. Se evidenciaron 24 personas con anticuerpos IgG para una seroprevalencia aparente de 61,5 %, mientras que para IgM esta fue del 0 %. Se determinó además la seropositividad más alta en los operarios que no usaron mascarillas (64 %) y no desinfectaron el material de trabajo (100 %), así como en aquellos que consumieron carne poco cocida (62,5 %). Respecto al tiempo de exposición, se obtuvo un 72,7 % para el grupo mayor de 10 años y 62,2 % de seropositivos expuestos entre cuatro y siete días a la semana. No existieron diferencias significativas para ninguna de las variables analizadas (p > 0,05). Se concluye que existe una seroprevalencia elevada de Toxoplasma gondii en operarios con riesgo ocupacional en la planta de beneficio animal del Bío-Bío.

Resumo: A toxoplasmose é uma zoonose de distribuição mundial causada por Toxoplasma gondii, protozoário que tem como hospedeiros definitivos os felinos, entre estes o gato doméstico, que pode transmitir a infecção ao ser humano. No Chile não existem estudos publicados de soro prevalência em pessoas com risco ocupacional. Por isso o objetivo deste estudo foi determinar a soro prevalência de Toxoplasma gondii e fatores de risco associados em operários de uma planta de beneficiamento animal de Bío Bío, Chile. Foram coletadas amostras de 39 soros de operários estudados por meio de análise quimioluminiscente para a detecção de anticorpos IgG e IgM, com uma sensibilidade e especificidade do 93 e 96 %, respectivamente. Se aplicou uma enquete epidemiológica e se calculou o odds ratio para a análise das variáveis higiênicas, alimentícias e de exposição. Se evidenciaram 24 pessoas com anticorpos IgG para uma soro prevalência aparente do 61,5 %, em quanto que para IgM esta foi do 0 %. Pôde-se determinar além do mais, a soro positividade mais alta nos operários que não usaram máscaras (64 %) e não desinfetaram o material de trabalho (100 %), assim como naqueles que consumiram carne pouco cozida (62,5 %). No que se refere ao tempo de exposição, se obteve um 72,7 % para o grupo maior de 10 anos e 62,2 % de soro positivos expostos entre quatro e sete dias por semana. Não existiram diferenças significativas para nenhuma das variáveis analisadas (p > 0,05). Conclui-se que existe uma soro prevalência elevada de Toxoplasma gondii em operários com risco ocupacional na planta de beneficiamento animal de Bío Bío.

[The immune-enzyme techniques of analysis in diagnostic of cholera.]

The optimal conditions of arrangement of direct alternative of flatbed enzyme-linked immunosorbent assay and dot-immunoanalysis with application of monoclonal peroxidase conjugates for express identification of comma bacillus of serogroups O1 and O139 both in hospital and field conditions without device support. The direct technique enzyme-linked immunosorbent assay in flatbed alternative shortens time of analysis up to 70-80 minutes and in case of dot enzyme-linked immunosorbent assay on membrane - up to 70-90 minutes. It is established that in case of analysis in conditions of room temperature (20-25 oC) sensitivity of techniques remains at initial level.

Application value of MINI-VIDAS automated multiparametric analyzer for immunoanalysis of acute chest pain in military hospital / 医疗卫生装备

Objective To apply MINI-VIDAS automated multiparametric analyzer to auxiliary diagnosis of the serviceman in order to evaluate its clinical value.Methods Totally 1 528 servicemen complaining of acute chest pain were divided into group A and group B equally; the ones in group A went through conventional examination, and the ones in group B underwent conventional examination combined with instant examination with MINI-VIDAS automated multiparametric analyzer. The differences between the two groups were compared for the interval from hospitalization to definite diagnosis, diagnosis correctness, hospitalization rate, length of stay, recovery rate, improvement rate and etc, and then statistical analysis was carried out.Results The two groups had significant differences in the interval from hospitalization to definite diagnosis and diagnosis correctness (P0.05).Conclusion MINI-VIDAS automated multiparametric analyzer can be used for the instant examination of cardiac marker to improve the diagnosis of the acute chest pain, and thus can be popularized in military hospital.

Monoclonal antibodies recognizing larval- and pupal-specific cuticular proteins of Tenebrio molitor (Insecta, Coleoptera).

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type 2, a 26 kDa polypeptide, and type 3, three polypeptides of 18.5, 19.5 and 21.5 kDa. They did not immunoreact with any protein of fat bodies or haemolymph from pharate pupae, suggesting that the antigens originate from the epidermis. The stage-specificity was confirmed by electron microscopic immunogold labelling. Type 1 and 3 MABs recognized antigens characterizing larval and pupal preecdysial sclerotized cuticles, while the antigens recognized by type 2 were localized in the first few lamellae of unsclerotized postecdysial cuticle. When the expression of the adult programme was inhibited by application of a juvenile hormone analogue, the larval-/pupal-specific CPs were detected in the supernumerary pupal cuticle. These results suggest that the genes encoding these proteins are juvenile hormone dependent. These MABs should be useful tools to isolate pupal-specific genes whose regulation sems to be different from that of the adult-specific ones.