RESUMEN
Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p'-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions, the standard curves ranged from 0.137 to 100 ng/mL with the quantification limit of 0.41 ng/mL. The coefficients of variation (CV%) were 5.42-10.53% for intra-assay and 6.04-7.26% for inter-assay. Cross-reactivities with DDT metabolites (DDTs, including o, p'-DDT, p, p'-DDD, o, p'-DDD, p, p'-DDE, o, p'-DDE, p, p'-dichlorobenzophenone (DCBP), o, p'-DCBP) were investigated. The polyclonal antibody showed relatively low and/or no cross-reactivity with these compounds, and the assay was seen to be highly selective for p, p'-DDT. Moreover, the DDTs could be ranked by their reactivity: DDT > DDD > DDE > DCBP. In addition, the characterization of the polyclonal antibody indicated that the antiserum possesses a high specificity for p, p'-isomers. The results indicated that the developed EIA using this antibody could be a convenient and supplemental analytical tool for monitoring DDT.
Asunto(s)
DDT/análisis , Monitoreo del Ambiente/métodos , Contaminación Ambiental/análisis , Técnicas para Inmunoenzimas , Residuos de Plaguicidas/análisis , Plaguicidas/análisis , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Carpas/metabolismo , Reacciones Cruzadas , Técnicas para Inmunoenzimas/métodos , Técnicas para Inmunoenzimas/normas , Isomerismo , Conejos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Contaminantes del Suelo/análisisRESUMEN
A simple and sensitive spectrophotometric method for the determination of human serum albumin (HSA) was established based on the ternary complex-formation reaction of HSA with o-sulfophenylfluorone (SPF) as a xanthene dye and metal ion (niobium(V) and bismuth(III)) in the presence of a dispersion agent. This new method enabled the determination of HSA in the range of 1 - 15 microg/ml HSA by measuring the difference of the absorbance at 530 nm between HSA-SPF-metal ion and SPF-metal ion solutions. In the determination of HSA, this method is about 2-times more sensitive than the Pyrogallol Red-molybdenum(VI) method (PR method), which accounts for more than 80% of the quantification methods for urinary protein assays in Japan. There was no significant difference between the results obtained by the present method and the PR method for human urine samples. The binding process between the SPF-metal complex and HSA was studied by determining the binding parameters and the thermodynamic parameters.
Asunto(s)
Albuminuria/diagnóstico , Albuminuria/orina , Bismuto/química , Fluoresceínas/química , Niobio/química , Compuestos Organometálicos/química , Albúmina Sérica/análisis , Bismuto/metabolismo , Colorantes/química , Humanos , Japón , Niobio/metabolismo , Compuestos Organometálicos/metabolismo , Sensibilidad y Especificidad , Albúmina Sérica/química , Espectrofotometría , Xantenos/químicaRESUMEN
We developed a sensitive and specific immunoassay system for human neuronal nitric oxide synthase (hnNOS) using synthetic hnNOS(998-1024) peptide and anti-hnNOS(998-1024) antibody. The novel antibody and radioimmunoassay system revealed a typical nNOS protein in human neuroblastoma NB-OK-1 cell (160 kDa, 180 fmol/10(6) cells). The kinetic parameters of the enzyme were K(m)=4.88 microM and V(max)=4.34 pmol/min/mg protein for L-arginine. On incubation of NB-OK-1 cell for 24 h, betamethasone phosphate decreased both nNOS-immunoreactivity (nNOS-IR) and enzymatic activity in the cell dose-dependently. On the other hand, pituitary adenylate cyclase activating polypeptide(1-38) (PACAP38) increased both nNOS-IR and enzymatic activity at concentrations of 10(-10) and 10(-9) M, but inversely decreased both at 10(-7) M. These suggest the positive and negative implications of endogenous NO in proliferation and differentiation of the cell, which support mitogenic activity of NO generated by nNOS in the cell. The present findings also provided evidence that the quantitative change of nNOS protein controls the integrated activity of the enzyme in the cell and, in turn, substantiate the validity and reliability of the present immunoassay system for hnNOS and its practical usefulness.