RESUMEN
Multiplex arrays designed for enzyme-linked immunosorbent assays (ELISAs) are robust and cost-effective for profiling biomarkers. Identification of relevant biomarkers in biological matrices or fluids helps in the understanding of disease pathogenesis. Here, we describe a sandwich ELISA-based multiplex assay to assess growth factor and cytokine levels in cerebrospinal fluid (CSF) samples derived from multiple sclerosis patients, amyotrophic lateral sclerosis patients, and control subjects without any neurological disorder. Results indicate that multiplex assay designed for the sandwich ELISA method is a unique, robust, and cost-effective method for profiling growth factors and cytokines present in CSF samples.
Asunto(s)
Esclerosis Amiotrófica Lateral , Citocinas , Humanos , Citocinas/líquido cefalorraquídeo , Péptidos y Proteínas de Señalización Intercelular , Ensayo de Inmunoadsorción Enzimática/métodos , Biomarcadores/líquido cefalorraquídeoRESUMEN
This work describes the development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method that uses disposable pipette extraction (DPX-UHPLC-MS/MS) to determine the endocannabinoid anandamide (AEA) in cerebrospinal fluid samples (CSF). The DPX parameters sorption equilibrium time, sample volume, number of draw-eject cycles, washing solvent volume, and elution solvent volume were optimized by design of experiments (DOE) techniques. The simple DPX protocol proposed herein required a reduced amount of CSF sample and organic solvent. The DPX-UHPLC-MS/MS method presented linear range from 0.10â¯ngâ¯mL-1 (LLOQ) to 3.0â¯ngâ¯mL-1, inter- and intra-assay accuracy with EPR values varying from -8.2% to 9.6%, inter- and intra-assay precision with CV values ranging from 1.3% to 14.8% (except for the LLOQ), and no significant matrix effect. The innovative DPX-UHPLC-MS/MS method was successfully applied to determine AEA in CSF samples from Parkinson's disease (PD) patients and should therefore be used in clinical studies.
Asunto(s)
Ácidos Araquidónicos/líquido cefalorraquídeo , Cromatografía Líquida de Alta Presión/métodos , Endocannabinoides/líquido cefalorraquídeo , Alcamidas Poliinsaturadas/líquido cefalorraquídeo , Espectrometría de Masas en Tándem/métodos , Ácidos Araquidónicos/aislamiento & purificación , Endocannabinoides/aislamiento & purificación , Humanos , Modelos Lineales , Alcamidas Poliinsaturadas/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Considering the great lethality and sequels caused by meningitis, rapid diagnosis and prompt treatment initiation have a great impact on patient outcome. Here, we developed a multiplex-PCR for simultaneous detection of the four most prevalent bacterial pathogens directly in CSF samples. The multiplex-PCR was designed to detect the following genes: fbsA (Streptococcus agalactiae), lytA (Streptococcus pneumoniae), crtA (Neisseria meningitidis), p6 (Haemophilus influenzae), and 16S rRNA (any bacterial agent). The multiplex-PCR showed a DNA detection limit of 1 pg/µL. Among 447 CSF samples tested, 40 were multiplex-PCR positive, in which 27 and 13 had positive and negative bacterial culture, respectively. Our multiplex-PCR is fast, reliable, and easily implementable into a laboratory routine for bacterial meningitis confirmation, especially for patients who previously started antimicrobial therapy. Our molecular approach can substantially improve clinical diagnosis and epidemiological measures of meningitis disease burden.
Asunto(s)
Líquido Cefalorraquídeo/microbiología , Haemophilus influenzae/genética , Meningitis Bacterianas/diagnóstico , Neisseria meningitidis/genética , Streptococcus agalactiae/genética , Streptococcus pneumoniae/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Haemophilus influenzae/aislamiento & purificación , Humanos , Lactante , Recién Nacido , Masculino , Meningitis Bacterianas/microbiología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Neisseria meningitidis/aislamiento & purificación , Streptococcus agalactiae/aislamiento & purificación , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
BACKGROUND: Enterovirus (EV) and herpes simplex virus 1 and 2 (HSV1 and HSV2) are the main etiologic agents of central nervous system infections. Early laboratory confirmation of these infections is performed by viral culture of the cerebrospinal fluid (CSF), or the detection of specific antibodies in serum (e.g., HSV). The sensitivity of viral culture ranges from 65 to 75%, with a recovery time varying from 3 to 10 days. Serological tests are faster and easy to carry out, but they exhibit cross-reactivity between HSV1 and HSV2. Although molecular techniques are more sensitive (sensitivity >95%), they are more expensive and highly susceptible to cross-contamination. METHODS: A real-time RT-PCR for the detection of EV, HSV1, and HSV2 was compared with end-point nested PCR. RESULTS: We tested 87 CSF samples of patients with a clinical diagnosis of viral meningitis or encephalitis. Fourteen samples were found to be positive by RT-PCR, but only 8 were positive by end-point PCR. The RT-PCR showed a specificity range of 94-100%, the negative predictive value was 100%, and the positive predictive value was 62, 100, and 28% for HSV1, HSV2, and EV, respectively. CONCLUSION: Real-time RT-PCR detected EV, HSV1, and HSV2 with a higher sensitivity and specificity than end-point nested RT-PCR.
Asunto(s)
Líquido Cefalorraquídeo/virología , Infecciones por Enterovirus/diagnóstico , Herpes Simple/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enterovirus/genética , Humanos , México , Sensibilidad y EspecificidadRESUMEN
A new electrochemical sensor for simultaneous determination of norepinephrine (NE) and serotonin (5-HT) was fabricated. The electrochemical behavior of NE and 5-HT were investigated using CV and SWV at the MWNTs-ZnO/chitosan composites modified screen-printed electrode (MWNTs-ZnO/chitosan SPE). The results showed that the current responses of NE and 5-HT greatly enhanced due to the high catalytic activity of composites. The peak potentials of NE and 5-HT were separated at about 90mV and 280mV, respectively. The peak currents of NE and 5-HT were linearly dependent on their concentrations in the range of 0.5-30µM and 0.05-1µM, with the limit of detection of 0.2µM and 0.01µM, respectively. The modified electrode can be stored stably for at least 3 month at 4°C in a refrigerator. Furthermore, the modified electrode was successfully applied to detect the level of NE and 5-HT in rat cerebrospinal fluid (CSF) with excellent selectivity and sensitivity.