RESUMO
Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS-CoV-2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free assay efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5-96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry-based diagnosis.
Assuntos
Anticorpos Antivirais , COVID-19 , SARS-CoV-2 , Anticorpos Antivirais/sangue , COVID-19/diagnóstico , Citometria de Fluxo , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Proteínas do Nucleocapsídeo , SARS-CoV-2/imunologia , Sensibilidade e EspecificidadeRESUMO
Type 1 diabetes (T1D) is an autoimmune disease with a strong genetic component that has been associated with several genetic loci. Interleukin 18 (IL-18) is a potent proinflammatory cytokine, which is involved in the innate and adaptive immune responses, and in the pathogenesis of various diseases including T1D. Glucose transporter 4 (GLUT4) is known to be an insulin-responsive glucose transporter and has been associated with various diseases, including diabetes mellitus. We investigated the association of the polymorphisms rs187238 (IL-18) and rs5435 (GLUT4) in a case-control study in Euro-Brazilians with T1D (N = 136) and healthy subjects (N = 144). Real-time PCR with TaqMan® fluorescent probes were applied for genotyping. All polymorphisms were in Hardy-Weinberg equilibrium. The minor allele frequencies for the G-allele (rs187238; IL-18) in healthy and T1D groups were 28.5% [95%CI = 23-34%] vs 31.6% [95%CI = 26-37%], P = 0.416, and for the T-allele (rs5435, GLUT4) were 33% [95%CI = 28-39] vs 27% [95%CI = 23-33%], P = 0.167, respectively. Genotype comparisons for both polymorphisms showed no significant differences (P > 0.05). The polymorphisms rs187238 and rs5435 were not associated with T1D in the studied population. The minor allele frequencies for both polymorphisms were similar to those of other Caucasian populations.
Assuntos
Diabetes Mellitus Tipo 1/genética , Transportador de Glucose Tipo 4/genética , Interleucina-18/genética , Polimorfismo de Nucleotídeo Único , Adulto , Brasil , Estudos de Casos e Controles , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , População BrancaRESUMO
The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.
Assuntos
Azospirillum brasilense/metabolismo , Citometria de Fluxo/métodos , Herbaspirillum/metabolismo , Hidroxibutiratos/metabolismo , Raízes de Plantas/microbiologia , Poliésteres/metabolismo , Microscopia de FluorescênciaRESUMO
The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.
Assuntos
Azospirillum brasilense/metabolismo , Citometria de Fluxo/métodos , Herbaspirillum/metabolismo , Hidroxibutiratos/metabolismo , Raízes de Plantas/microbiologia , Poliésteres/metabolismo , Microscopia de FluorescênciaRESUMO
UNLABELLED: To understand the mechanism of plant-bacterium interaction, it is critical to enumerate epiphytic bacteria colonizing the roots of the host. We developed a new approach, based on flow cytometry, for enumerating these bacteria and used it with rice plants, 7 and 20 days after colonization with Herbaspirillum rubrisubalbicans and Azospirillum brasilense. The results were compared with those obtained with the traditional plate count method. Both methods gave similar numbers of H. rubrisubalbicans associated with rice roots (c. 10(9) CFU g(-1) ). However, flow cytometry gave a number of viable cells of rice-associated A. brasilense that was approx. 10-fold greater than that obtained with the plate count method. These results suggest that the plate count method can underestimate epiphytic populations. Flow cytometry has the additional advantage that it is more precise and much faster than the plate count method. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of precise number of root-associated bacteria is critical for plant-bacteria interaction studies. We developed a flow cytometry approach for counting bacteria and compared it with the plate count method. Our flow cytometry assay solves two major limitations of the plate count method, namely that requires long incubation times of up to 48 h and only determines culturable cells. This flow cytometry assay provides an efficient, precise and fast tool for enumerating epiphytic cells.