Immunological Methods (immunological + methods)

Distribution by Scientific Domains

Selected Abstracts

Serological detection and immunogold localization of cross-reactive antigens shared by Camellia sinensis and Exobasidium vexans

B.N. Chakraborty
Abstract Aims:, Pathogenicity of Exobasidium vexans, causal agent of blister blight of tea, was studied in 30 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen using immunological techniques. Methods and Results:, Whole plant inoculation of tea varieties with E. vexans showed that T-78 and T-17/1/54 were most susceptible and most resistant respectively. Antigen preparations from tea varieties, pathogen, nonpathogen (Fusarium oxysporum) and of nonhosts (Glycine max, Leucaena leucocephala and Oryza sativa) were compared by indirect enzyme-linked immunosorbent assay and dot-immunobinding assay using polyclonal antibodies raised against the pathogen, nonpathogen, susceptible and resistant tea varieties. Cross-reactive antigens (CRA) were found among susceptible varieties and E. vexans isolates but not in resistant varieties, nonhosts or nonpathogen. Indirect staining of antibodies using fluorescein isothiocyanate indicated CRA were concentrated mainly around epidermal and mesophyll cells in compatible host (T-78). This was substantiated by ultrastructural studies using gold-labelled antibodies through transmission electron microscopy which showed specific localization in the chloroplasts and host cytoplasm. Conclusion:, Pathogenicity of E. vexans to different tea varieties is therefore related to the level of antigenic similarity between host and pathogen. Significance and Impact of the Study:, Immunological methods proved to be valuable in screening commercially cultivated tea varieties against E. vexans. [source]

Identification of the rat adrenal zona fasciculata/reticularis specific protein, inner zone antigen (IZAg), as the putative membrane progesterone receptor

FEBS JOURNAL, Issue 7 2001
Farah S. Raza
Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55,60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism. [source]

Simultaneous detection of two verotoxin genes using dual-label time-resolved fluorescence immunoassay with duplex PCR

Kazuyuki Watanabe
Abstract Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans. Two types of verotoxin are known. Conventional diagnosis of verotoxin-producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs. This method is complicated and time-consuming. Recently, rapid, direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these techniques continue to suffer from limited sensitivity and a lack of specificity. These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non-O157 antigens, such as O26, O103 and O111 antigens, exist. These VTEC groups did not react with anti-O157 antibody. Consequently, it is necessary to diagnose the VT gene in these bacteria. Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time-resolved fluorescence immunoassay (TRFIA). Copyright 2002 John Wiley & Sons, Ltd. [source]

Standardization of allergen products: 1.

ALLERGY, Issue 7 2009
Detailed characterization of GMP-produced recombinant Bet v 1.0101 as biological reference preparation
Background:, Standardization of allergen extracts requires the availability of well-characterized recombinant allergens, which can be used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101, which shall be used in a ring trial within the framework of the Biological Standardization Programme BSP090 of the European Directorate for Quality of Medicines and Healthcare. Methods:, Recombinant Bet v 1.0101 Y0487 was produced under good manufacturing practice conditions and analysed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, as well as biological activity. Results:, Batch Y0487 was shown to contain monomeric and well-folded protein being identical with rBet v 1.0101, as determined by mass spectrometry. SDS-PAGE, isoelectric focusing, deamidation analysis and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4C batch Y0487 retained the monomeric state up to 3 months. Protein quantification determined by amino acid analysis was found coinciding with half-maximal inhibition of serum IgE in ELISA. Biological activity of batch Y0487 was shown to be comparable to natural Bet v 1 by IgG and IgE immunoblotting, as well as basophil and T-cell activation. Conclusion:, Recombinant Bet v 1.0101 Y0487 was characterized extensively by physicochemical and immunological methods. It was shown highly stable, monomeric and immunologically equivalent to its natural counterpart. Thus, it represents an appropriate candidate reference standard for Bet v 1. [source]

Localization of arginine decarboxylase in tobacco plants

Cristina Bortolotti
The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types. [source]