RESUMO
AIMS: Adequately and accurately identifying carbapenemase-producing Enterobacterales (CPE) is vital for selecting appropriate antimicrobial therapy and implementing effective infection control measures. This study aims to optimize the phenotypic detection method of carbapenemase for routine diagnostics in clinical microbiology laboratories. METHODS AND RESULTS: Carbapenemase genes in 2665 non-duplicate CRE clinical strains collected from various regions of China were confirmed through whole-genome sequencing (WGS). The carbapenemase inhibition test (CIT) was conducted and interpreted using different methods and breakpoints, then compared with the NG-Test CARBA 5 for carbapenemase detection. The diagnostic performance of the CIT method was optimal when the carbapenemase types were determined by comparing the inhibition zone diameters of the imipenem disc with 3-aminophenylboronic acid (APB) plus ethylenediaminetetraacetic acid (EDTA) to those of the imipenem disc with either APB or EDTA alone, with a breakpoint of 4 mm. The overall sensitivities of the current CIT, the modified CIT, and NG-Test CARBA 5 were 91.4%, 94.9%, and 99.9%, respectively. For detecting isolates co-producing Klebsiella pneumoniae carbapenemase (KPC) and metallo-ß-lactamases (MBLs), the modified CIT method had higher sensitivity than the current method (70.0% vs. 53.3%), though this difference was not statistically significant (P = 0.063). The NG-Test CARBA 5 showed excellent performance for multi-carbapenemases diagnosis, with sensitivity and specificity of 97.1% and 100%, respectively. CONCLUSIONS: Optimizing and standardizing the CIT method for clinical use is necessary. It has certain advantages in diagnosing multi-carbapenemase and rare carbapenemase production. However, for identifying common carbapenemase types, the NG-Test CARBA 5 demonstrated superior performance.
Assuntos
Proteínas de Bactérias , beta-Lactamases , beta-Lactamases/metabolismo , beta-Lactamases/análise , Proteínas de Bactérias/metabolismo , Humanos , China , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Sensibilidade e Especificidade , Sequenciamento Completo do Genoma , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologiaRESUMO
BACKGROUND: Rapid and accurate identification of carbapenemase-producing organism (CPO) intestinal carriers is essential for infection prevention and control. Molecular diagnostic methods can produce results in as little as 1 h, but require special instrumentation and are expensive. Therefore, it is urgent to find an alternative method. The broth enrichment-multiplex lateral flow immunochromatographic assay was recently reported, but using it to directly detect CPO intestinal carriers in rectal swabs still requires the evaluation of many samples. The aim of this study was to compare the performance of these two methods, and to explore the control measures of CPO infection. METHODS: Through CPO selective culture, PCR and DNA sequencing, 100 rectal swabs confirmed to be CPO-positive and 100 rectal swabs with negative results were collected continuously. After eluting the rectal swabs with saline, three aliquots were used: one for counting, one for detection by Xpert Carba-R, and one for culture in broth for 0 h, 1 h, 2 h, 3 h and 4 h, followed by NG-Test CARBA 5 assessment. The sensitivity and specificity of the NG-Test CARBA 5 method after different incubation times were calculated. The limit of detection (LoD) of this assay after 4 h broth incubation was estimated by examining the bacterial suspensions and simulated faecal suspensions prepared with CPOs producing different types of carbapenemases. RESULTS: Xpert Carba-R demonstrated a combined sensitivity of 99.0% and specificity of 98.0%. The sensitivity and specificity were higher than 90.0% for the different enzyme types. The specificities of five common carbapenemases detected by the broth enrichment NG-Test CARBA 5 combined method after different incubation times were 100%. The sensitivities increased with increasing incubation time. At 4 h, the Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), imipenemase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM), and oxacillinase (OXA) -48 detection sensitivities were 93.0%, 96.3%, 100%, 100% and 85.7%, respectively. The LoDs were between 102 and 104 CFU/mL for all five enzymes after 4 h of incubation. CONCLUSIONS: This investigation highlighted that the broth enrichment-multiplex lateral flow immunochromatographic assay can be used as a new method for screening CPOs in rectal swabs.
Assuntos
Proteínas de Bactérias , beta-Lactamases , Humanos , Suspensões , Proteínas de Bactérias/genética , Proteínas de Bactérias/análise , beta-Lactamases/genética , beta-Lactamases/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , ImunoensaioRESUMO
Due to universal contamination and synergistic toxicity of multiple mycotoxins in foodstuff, reliable and high-throughput detection methods for multiple mycotoxins are urgently needed in corn products. In this study, a novel dual-channel immunochromatographic assay (ICA) based on improved up-conversion nanoparticles (IUCNPs) was developed for rapidly detecting aflatoxin B1 (AFB1) and zearalenone (ZEN). The synthesized IUCNPs doped by 30% Lu3+ showed a larger size, more regular structure, and brighter fluorescence intensity than conventional UCNPs. The limits of detection (LODs) of single-channel ICA test strips for AFB1 and ZEN detection were 0.01 and 0.1 ng/mL, respectively. After the optimization, the dual-channel ICA of AFB1 and ZEN in 10 min was conducted, resulting in low detection limits of 0.025 and 0.1 ng/mL, respectively. Moreover, the built assay was revealed to be highly specific for six other food-contaminated mycotoxins, and exhibited excellent accuracy, with corresponding R2 of 0.9931 and 0.9982 in calibration curves, respectively. Long-term storage experiments indicated that the dual-channel test strips had superior stability and precision. The LODs of AFB1 and ZEN in spiked maize were 0.025 and 0.25 µg/kg, demonstrating great sensitivity and matrix tolerance. Furthermore, the IUNCP-ICA was validated by high-performance liquid chromatography (HPLC) analyses, and a satisfactory consistency was obtained in 15 natural maize samples. Thus, the IUCNPs-ICA proposed in this work realized rapid and sensitive detection of AFB1 and ZEN, providing broad application potential in on-site screening for multiple mycotoxins in agricultural products.
Assuntos
Micotoxinas , Nanopartículas , Zearalenona , Zearalenona/análise , Aflatoxina B1/análise , Zea mays/química , Contaminação de Alimentos/análise , Limite de Detecção , Micotoxinas/análiseRESUMO
Vancomycin (VAN) has a narrow therapeutic window and variable pharmacokinetic properties, which require timely monitoring of plasma concentration to adjust the dosage for better effectiveness. A sensitive and quantitative immunochromatographic method was developed to detect VAN in plasma. The linear response range of the method to detect plasma VAN was 1.25-50 µg/mL. The intra- and inter-assay coefficients were 4.68% and 8.81%, respectively, while the recovery was 89.10%-98.32%. The clinical comparative experiment results demonstrated a good correlation (r > 0.90) between the fluorescent immunochromatographic strip test and the mass spectrum. In this study, a simple, rapid, and high-sensitivity immunochromatographic method was established for detecting VAN, which helped establish the basis for further application of monitoring plasma concentration.
Assuntos
Vancomicina , Cromatografia de Afinidade/métodosRESUMO
Lychnis mottle virus (LycMoV; genus Unassigned, family Secoviridae) infection of Angelica sinensis produces mottle and mosaic symptoms, damaging the host. Early detection of relevant pathogens is the most critical step in preventing the potential transmission of infectious disease. Polyclonal antibodies with high potency and high specificity were prepared using the recombinant LycMoV capsid protein as an antigen. Here, we developed and optimized a rapid colloidal gold immunochromatography assay (GICA) detection system for LycMoV using this antibody. Under optimum conditions, GICA specifically detected (up to 10,000-fold) positive LycMoV samples. A real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) system was also established by selecting the primers with high sensitivity and specificity to LycMoV. The RT-LAMP detection threshold was 1.42 fg/µl (291 copies/µl). A GICA-RT-LAMP assay system was further established and optimized. The minimum GICA detection line was calculated at 1.52 × 10-2 ng/µl. Although GICA did not detect positive samples after capturing virus at 2.53 × 10-3 ng/µl, GICA-LAMP and GICA-RT-PCR did, whose sensitivity was comparatively greater than sixfold. This is the first report showing that GICA-RT-LAMP is a cost-effective approach for use in detecting LycMoV without extracting nucleic acids. These sensitive assays will help improve virus disease management in A. sinensis crops.
Assuntos
Lychnis , Vírus de RNA , Coloide de Ouro , Transcrição Reversa , Cromatografia de AfinidadeRESUMO
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.
Assuntos
Penicilinas , beta-Lactamas , Animais , beta-Lactamas/análise , Penicilinas/análise , Proteínas de Ligação às Penicilinas , Leite/química , Microesferas , Anticorpos/análise , ImunoensaioRESUMO
INTRODUCTION: Miroestrol (Mi) and deoxymiroestrol (Dmi) are trace, yet potent, phytooestrogens found in white Kwao Krua [Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, PM]. However, the analysis of these substances is difficult because of complex matrix effects and their various analogues. In addition, alteration in the cross-reactivity of a gold nanoparticle (AuNP)-based immunochromatographic assay (ICA) resulting from the electrostatic adsorption between antibodies and AuNPs has not yet been evaluated. OBJECTIVES: This study aims to develop, characterise, and validate ICA with a monoclonal antibody exhibiting similar reactivity against Mi and Dmi (MD-mAb). MATERIALS AND METHODS: The ICA performance was validated for cross-reactivity and performance in comparison with those of indirect competitive enzyme-linked immunosorbent assays (icELISAs) with MD-mAb and mAb exhibiting specificity against Mi (Mi-mAb). RESULTS: The ICA showed a limit of detection (LOD) at 1 and 16 µg/mL for Mi and Dmi, respectively. The cross-reactivity of the ICA with Dmi was lower (6.25%) than that observed with the icELISA (120%). Cross-reactivity of ICA against other compounds of the PM was also correlated with those of icELISA; no false-positive/negative results were observed. The repeatability and reproducibility of the ICA were confirmed. The results obtained using ICA in samples of PM are correlated with the concentrations determined through icELISAs. CONCLUSION: An ICA with MD-mAb was constructed and validated. However, direct conjugation via the electrostatic adsorption of mAb-AuNPs was expected to alter the cross-reactivity of ICA, especially that of the analyte analogue Dmi.
Assuntos
Nanopartículas Metálicas , Pueraria , Pueraria/química , Ouro , Reprodutibilidade dos Testes , Anticorpos Monoclonais , Imunoensaio , Ensaio de Imunoadsorção Enzimática/métodosRESUMO
BACKGROUND: Mycotoxins are secondary metabolites produced by fungi, which have serious effects on humans and animals. In this study, we selected the monodispersed polystyrene fluorescent microspheres with good luminescence performance and strong stability as markers to conjugate with four mycotoxins antibodies for preparing fluorescent probes. We have developed a fluorescent microsphere based immunochromatographic assay (FMICA) to detect sensitively and quickly zearalenone (ZEN), aflatoxin B1 (AFB1 ), fumonisin B1 (FB1 ), and ochratoxin A (OTA) in cereal. RESULTS: Under optimal experimental conditions, the procedure of this method can be completed within 10 min. The limit of detection (LOD) of FMICA for ZEN, AFB1 , FB1 , and OTA is 0.072, 0.093, 0.32, and 0.19 µg L-1 , respectively. And FMICA has good specificity and no cross-reactivity with other mycotoxins. Four mycotoxins in naturally contaminated cereal samples (corn, rice, and oat) are detected by this method, and the results are highly consistent with that of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). CONCLUSION: The developed FMICA has good accuracy, high sensitivity, simplicity, convenience, rapidity, and low cost, and it could be employed for sensitive and quantitative detecting of mycotoxins in cereal on-site. © 2022 Society of Chemical Industry.
Assuntos
Micotoxinas , Zearalenona , Humanos , Animais , Micotoxinas/análise , Grão Comestível/química , Microesferas , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Zearalenona/análise , Imunoensaio/métodos , Contaminação de Alimentos/análiseRESUMO
BACKGROUND: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. RESULTS: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. CONCLUSIONS: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.
Assuntos
Infecções por Circoviridae , Circovirus , Doenças dos Suínos , Sulfato de Amônio , Animais , Anticorpos Antivirais , Proteínas do Capsídeo , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/veterinária , Colódio , Coloide de Ouro/química , Imunoensaio/veterinária , Coelhos , SuínosRESUMO
African swine fever (ASF), a highly contagious and lethal disease, poses a tremendous threat and burden to the swine industry worldwide. Lack of available vaccines or treatments leaves rapid diagnosis as the key tool to control the disease. Quantum dots (QDs) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In this study, we developed a quantum dots-based fluorescent immunochromatographic assay (QDs-FICA) using CD2v as the diagnosis antigen to detect ASFV antibodies. The titre of the test strip was 1 : 5·12 × 105 . In addition, the strip was highly specific to anti-ASFV serum and had no cross-reaction with CSFV, PPV, PRRSV, PCV-2, PRV and FMDV. Moreover, a comparative test of 71 clinical samples showed that the coincidence rate was 85·92% between the test strip and the commercial ELISA kit (coated with p30, p62 and p72). The QDs-FICA can be used to detect ASFV antibodies, which is meaningful for the surveillance, control and purification of ASF.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Pontos Quânticos , Febre Suína Africana/diagnóstico , Febre Suína Africana/prevenção & controle , Animais , Diagnóstico Diferencial , Imunoensaio , SuínosRESUMO
Staphylococcal enterotoxin A (SEA) is an important biotoxin, produced by Staphylococcus aureus under appropriate conditions, and often contaminates milk and dairy products. Herein, an anti-SEA monoclonal antibody (anti-SEA mAb) was prepared by injecting the SEA protein in BALB/c mice, and a novel immunochromatographic assay (ICA) was developed for the rapid and sensitive determination of SEA in pasteurized milk by using highly luminescent quantum dot beads (QB) as signal amplification probe. Given the 1020-fold enhancement in the photoluminescence intensity of QB to the original quantum dot, the proposed QB-ICA exhibits high sensitivity for SEA determination in real milk samples with a limit of detection of 1.89 ng/mL, and shows good dynamic linearity for SEA quantitative detection from 2 to 150 ng/mL within 15 min of test time. The proposed QB-ICA also shows good selectivity to SEA detection with a negligible cross-reaction to common analogs, including staphylococcal enterotoxins B, C, D, and E. In addition, the accuracy and precision of QB-ICA were assessed by analyzing SEA-fortified milk samples. The average recoveries of intra- and interassays range from 85.5 to 128.1%, and the coefficients of variation range from 4.6 to 14.2%, indicating an acceptable accuracy for the quantitative detection of SEA in real milk samples. In summary, this work provides a powerful and rapid analysis tool for the sensitive monitoring of SEA contamination in pasteurized milk samples.
Assuntos
Pontos Quânticos , Animais , Cromatografia de Afinidade/métodos , Cromatografia de Afinidade/veterinária , Enterotoxinas/análise , Imunoensaio/veterinária , Camundongos , Leite/química , Pontos Quânticos/químicaRESUMO
A method is described to achieve accurate quantitative detection of atrazine (ATZ) in maize by using lateral flow strips based on gold nanoparticles (GNPs) and a handheld scanning reader. GNPs of 15 nm in diameter were applied as label, and a lateral flow immune assay strip was prepared. The linear range was 5.01-95.86 ng mL-1 with a detection limit of 4.92 ng mL-1 in phosphate buffer, 4 times better than the readout by the naked eye. ATZ-spiked corn samples were also analysed. The accuracy of results of spiked samples was confirmed by ELISA and liquid chromatography-tandem mass spectrometry (HPLC), which proved the reliability of the proposed method. A handhold device with an optical scanning system was designed for on-site quantitative detection. Combined with the pretreatment, the assay could be completed in less than 20 min.
Assuntos
Atrazina , Nanopartículas Metálicas , Atrazina/análise , Ouro/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Sistemas Automatizados de Assistência Junto ao Leito , Reprodutibilidade dos TestesRESUMO
Mycotoxins are secondary metabolites of fungi, which seriously threaten human health. Among them, ochratoxin A (OTA) and deoxynivalenol (DON) have become the main factors that pollute cereals and by-products. In order to achieve simultaneous detection of OTA and DON quantitatively, a novel dual-flux immunochromatographic assay (dICA) was established. The dual-flux assay is based on upconversion nanoparticles (UCNPs) as fluorescence tags to label antigens and gold nanoparticles (AuNPs) as fluorescence quencher to label monoclonal antibodies (mAbs). The intensity of the green fluorescence (540 nm) of UCNPs can be used as an analytical signal, indicating the formation of antigen-antibody immune complexes, thereby indicating the presence or absence of the target analyte. The intensity of the red fluorescence (660 nm) of UCNPs is not affected and can be used as a quality control signal, and the dual-flux bidirectional single-line labeling mode allows for the simultaneous detection of two different mycotoxins on two test lines. This work indicated that the developed dICA provided a sensitive, rapid, and reliable on-site simultaneous detection of multiple mycotoxins.
Assuntos
Nanopartículas Metálicas , Micotoxinas , Humanos , Ouro/química , Luminescência , Nanopartículas Metálicas/química , Transferência Ressonante de Energia de Fluorescência/métodos , Micotoxinas/análiseRESUMO
A multicolor immunochromatographic assay platform based on quantum dot nanobeads (QBs) for the rapid and simultaneous detection of nitrofuran metabolites in different aquatic products is documented. These metabolites include 3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin (AHD), semicarbazide (SEM), and 3-amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ). QBs with emission colors of red, yellow, green, and orange were employed and functionalized with the corresponding antibodies to each analyte to develop a multicolor channel. The visual detection limits (cutoff values) of our method for AOZ, AHD, SEM, and AMOZ reached up to 50 ng/mL, which were 2, 20, 20, and 20 times lower than those of traditional colloidal gold test strips, respectively. The test strip is capable of detection within 10 min in real samples while still achieving good stability and specificity. These results demonstrate that the developed multicolor immunochromatographic assay platform is a promising technique for multiplex, highly sensitive, and on-site detection of nitrofuran metabolites.
Assuntos
Nitrofuranos , Pontos Quânticos , Contaminação de Alimentos/análise , Nitrofuranos/química , Cromatografia de Afinidade/métodos , SemicarbazidasRESUMO
5-Fluorouracil (5-FU) is an effective anticancer drug widely used in the world. To improve therapy efficiency and reduce side effects, it is very important to frequently detect the concentration of 5-FU in blood samples of patients. In this work, a new type of lateral flow immunochromatographic assay (LFIA) based on surface-enhanced Raman scattering (SERS) for ultrasensitive and specific detection of 5-FU in blood samples was developed. Au@Ag/Au nanoparticles (NPs) employing Au particles as the core and Ag/Au alloy as the shell were synthesized, characterized and used as the substrate in SERS-LFIA due to their high SERS enhancement and biocompatibility. The immunoprobe was made in the form of AuMBA@Ag/Au-Ab in which mercaptobenzoic acid (MBA, a common Raman active reporter) was embedded in the core-shell layer and the monoclonal antibody (mAb) against 5-FU was immobilized on the surface. The performance of SERS-LFIA was similar to that in colloidal gold based-LFIA, and the entire assay time was within 20 min. According to the color intensity on the testing (T) lines of LFIA strips visualized by eyes, the contents of 5-FU in the samples could be qualitatively or semi-quantitatively identified. Furthermore, by measuring the characteristic Raman intensities of MBA on T lines, quantitative detection of 5-FU in the samples were achieved. The IC50 and limit of detection (LOD) of the LFIA for 5-FU were found to be 20.9 pg mL-1 and 4.4 pg mL-1, respectively. There was no cross-reactivity (CR) of the LFIA with nine relative compounds, and the CR with cytosine, tegafur and carmofur were less than 4.5%. The recoveries of 5-FU from spiked blood samples were in the range of 78.6~86.4% with the relative standard deviation (RSD) of 2.69~4.42%. Five blood samples containing 5-FU collected from the Cancer Hospital were measured by SERS-LFIA, and the results were confirmed by LC-MS/MS. It was proven that the proposed method was able to simply and rapidly detect 5-FU in blood samples with high sensitivity, specificity, accuracy and precision.
Assuntos
Antineoplásicos , Nanopartículas Metálicas , Cromatografia Líquida , Fluoruracila , Ouro , Humanos , Imunoensaio/métodos , Nanopartículas Metálicas/química , Prata/química , Análise Espectral Raman/métodos , Espectrometria de Massas em TandemRESUMO
The paper present the results of a survey of people who have undergone immunization with a combined vector vaccine for the prevention of coronavirus infection COVID-19 «Sputnik V - Gam-COVID-Vac¼, as well as COVID-19 recovalents. Using a quantitative enzyme-linked immunosorbent assay, the levels of specific IgG were determined in persons who had had different degrees of severity before vaccination, in persons who were immuno-negative before immunization, as well as in convalescents who had undergone coronavirus infection of varying severity. The immunological targeting of antibodies against various SARS-CoV-2 proteins is considered.
Assuntos
COVID-19 , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunização , SARS-CoV-2 , VacinaçãoRESUMO
The paper presents the results of the development of a technology for the quantitative determination of D-dimer in blood with an immunochromatographic (LFIA) kit of reagents «LFIA-D-dimer¼ and instrument accounting of the results. Registration and processing of the digitized indicator of the intensity of staining of the LFIA-test strip using the LFIA-analyzer software allows quantifying the D-dimer content in the sample (in ng DDU/ml). The effectiveness of the proposed approach was evaluated on 258 clinical samples examined in the LFIA with visual and instrument accounting of the results, in comparison with the indicators of D-dimer determination in ELISA. The high reproducibility of the digitized LFIA results was shown - the coefficient of variation (CV) for samples in the range of 100-300 ng DDU/ml (near-threshold in relation to pathological values) was 2.5-5.1%; a tendency to increase CV with a further increase in the concentration of D-dimer was traced. A high correlation of the digitized LFIA result with the research data in the ELISA has been established, which makes it possible to recommend the technology for widespread use in urgent medicine.
Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio/métodos , Reprodutibilidade dos TestesRESUMO
This study assessed the diagnostic performance of the new COVID19SEROSpeed IgM/IgG rapid test (BioSpeedia, a spinoff of the Pasteur Institute of Paris) for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in comparison to other commercial antibody assays through a large cross-European investigation. The clinical specificity was assessed on 215 prepandemic sera (including some from patients with viral infections or autoimmune disorders). The clinical sensitivity was evaluated on 710 sera from 564 patients whose SARS-CoV-2 infection was confirmed by quantitative reverse transcription-PCR (qRT-PCR) and whose antibody response was compared to that measured by five other commercial tests. The kinetics of the antibody response were also analyzed in seven symptomatic patients. The specificity of the test (BioS) on prepandemic specimens was 98.1% (95% confidence interval [CI], 96.2% to 99.4%). When tested on the 710 pandemic specimens, BioS showed an overall clinical sensitivity of 86.0% (95% CI, 0.83 to 0.89), with good concordance with the Euroimmun assay (overall concordance of 0.91; Cohen's kappa coefficient of 0.62). Due in part to simultaneous detection of IgM and IgG for both S1 and N proteins, BioS exhibited the highest positive percent agreement at ≥11 days post-symptom onset (PSO). In conclusion, the BioS IgM/IgG rapid test was highly specific and demonstrated a higher positive percentage of agreement than all the enzyme-linked immunosorbent assay/chemiluminescence immunoassay (ELISA/CLIA) commercial tests considered in this study. Moreover, by detecting the presence of antibodies prior to 11 days PSO in 78.2% of the patients, the BioS test increased the efficiency of the diagnosis of SARS-CoV-2 infection in the early stages of the disease.
Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoensaio/métodos , SARS-CoV-2/isolamento & purificação , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , COVID-19/sangue , COVID-19/patologia , Cromatografia de Afinidade , Europa (Continente) , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Cinética , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Ochratoxin A (OTA) is a toxic metabolite produced mainly by Aspergillus and Penicillium species. A quantitative method was developed for the rapid, simple, and sensitive detection of OTA in corn by quantum dots-based fluorescent immunochromatographic assay (QDs-ICA). The CdSe/ZnS QDs-labelled anti-OTA monoclonal antibody (mAb) conjugates were designed as the fluorescent signal probe. The QDs-ICA included the designation of test line (T line) and control line (C line), which were sprayed with optimal concentrations of the OTA-OVA and staphylococcal protein-A (SPA), respectively. Under the optimal experimental conditions, the QDs-ICA exhibited excellent specificity and good accuracy and precision. For qualitative detection, the cut-off value for the T line of the visual detection method was 2.5 ng/mL. For quantitative detection, the linear regression equation of the standard curve was y = 0.366x + 0.514 with a reliable correlation coefficient (R2 = 0.992). Moreover, the 50% inhibition value (IC50) of the QDs-ICA was 0.91 ng/mL, the limit of detection (LOD) was 0.07 ng/mL, and the detection range was 0.05 to 10 ng/mL. In addition, the recovery rates ranged from 91.82 to 100.35% with a coefficient of variation (CV) below 3% for intra-assay, whereas the recovery rates for inter-assay changed from 94.29 to 104.62% with a CV below 10%. These results indicate that the QDs-ICA can serve as a potential large-scale preliminary device for rapid determination of OTA. Using CdSe/ZnS QDs as the fluorescent signal for quantum dots-based fluorescent immunochromatographic assay, the QDs-ICA provided a novel method for the rapid simultaneous qualitative and quantitative determination of OTA.
Assuntos
Carcinógenos/análise , Cromatografia de Afinidade/métodos , Corantes Fluorescentes/química , Ocratoxinas/análise , Pontos Quânticos/química , Compostos de Cádmio/química , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Contaminação de Alimentos/análise , Limite de Detecção , Reprodutibilidade dos Testes , Compostos de Selênio/química , Sulfetos/química , Compostos de Zinco/químicaRESUMO
The recent Zika virus (ZIKV) epidemic poses a serious threat to global health due to its association with microcephaly and congenital diseases in newborns and neurological complications and Guillain-Barré syndrome in adults. However, the majority of people infected with ZIKV do not develop symptoms. The platforms aimed to specifically diagnose ZIKV infection are needed for patient care and public health surveillance. In the study, four ZIKV envelope (E) protein-specific monoclonal antibodies (mAbs) (A1, B1, C1, and 9E-1) have been developed by using the conventional mAb technology. The binding epitopes of mAbs A1, B1, C1, and 9E-1 are located at E(238-257), E(410-431), E(258-277), and E(340-356), respectively. mAb 9E-1 performs 1.4- to 47-fold strong affinity to ZIKV E protein compared to another three mAbs. mAbs A1, C1, and 9E-1 do not have cross-reactivity against the recombinant E proteins of dengue virus serotypes 2, 3, and 4. Although these four mAbs do not have ZIKV neutralizing activity, mAbs B1 and 9E-1 have been developed as the lateral flow immunochromatographic assay for specific detection of ZIKV E protein and virions. KEY POINTS: ⢠The mAbs targeting to the regions of E(238-257), E(410-431), E(258-277), and E(340-356) do not have ZIKV neutralizing activity. ⢠The binding epitope of mAb 9E-1 is highly specific to ZIKV E protein. ⢠mAbs B1 and 9E-1 can bind to ZIKV virions and have been developed as the lateral flow immunochromatographic assay.