RESUMEN
Herein, a simple and rapid method is described for detection of L-ascorbic acid by ascorbate oxidase immobilized onto polycarbonate strip pre-activated by 1-fluoro-2-nitro-4-azidobenzene in photochemical reaction. Covalent attachment of ascorbate oxidase was confirmed by XPS studies. The immobilized-ascorbate oxidase shows higher pH, thermal and storage stability in comparison to free enzyme.
Asunto(s)
Ascorbato Oxidasa/química , Ácido Ascórbico/análisis , Técnicas de Química Analítica/métodos , Enzimas Inmovilizadas/química , Azidas/metabolismo , Nitrobencenos/metabolismo , Cemento de Policarboxilato/metabolismoRESUMEN
Here we demonstrate a novel microwave-mediated enzyme-linked immunosorbent assay (MELISA) method that has dramatically reduced the enzyme-linked immunosorbent assay (ELISA) timing to less than 5 min with a result comparable to that obtained by 18-h conventional ELISA. Efficacy of the MELISA procedure is demonstrated by detecting human immunoglobulin G (IgG), rabbit IgG, human immunoglobulin E (IgE), human interleuken 1ß (IL-1ß), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. MELISA could be an excellent substitute for time-consuming conventional ELISA for rapid diagnosis of diseases in cases of medical urgency, outbreak of infectious diseases, and screening of samples in blood banks or emigration counters.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Microondas , Animales , Humanos , Inmunoglobulinas/análisis , ConejosRESUMEN
Herein, we describe a non-conventional method for immobilization of enzymes onto different solid surfaces using ultrasound as a source of energy. When horseradish peroxidase (HRP) was taken on the surface of an activated support and allowed to float on a sonicator bath operating at a frequency of 40 KHz, it readily started binding itself to the surface. Maximum binding was observed in 10 min whereas a control experiment carried out similarly without ultrasound waves showed insignificant immobilization. Ultrasound wave-mediated immobilization is rapid and reproducible and is better suitable for versatile applications in different fields, including fabrication of enzyme-based biosensors or bioreactors.
Asunto(s)
Biotecnología/métodos , Enzimas Inmovilizadas/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Sonicación/métodos , Azidas/química , Colorimetría , Enzimas Inmovilizadas/química , Ondas de Choque de Alta Energía , Peroxidasa de Rábano Silvestre/química , Nitrobencenos/química , Poliestirenos/química , Unión ProteicaRESUMEN
The enzyme-linked immunosorbent assay (ELISA) technique is the backbone of the diagnostic assay for detection of infectious and allergic diseases. Here, we demonstrate a unique ELISA method for the detection of an antigen or antibody, in which ELISA steps are carried out under pressure instead of conventional thermal incubation. Pressure-mediated ELISA (PELISA), carried out in 1 h shows more than a 2-fold increase in absorbance value than the control experiment carried out at the same time and temperature without applying pressure. Estimation of total IgE by the 1-h PELISA method gives similar absorbance value (1.081+/-0.031, 823.12 IU) to that obtained by 3-h heat-mediated ELISA on an activated surface (HELISA) (1.165+/-0.037, 810.96 IU). Since PELISA is sensitive, specific, and reproducible (intra- and interassay CVs were 6.47% and 9.65%, respectively), it could be an excellent alternative to HELISA or conventional ELISA procedures.