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An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies.

ABSTRACT: Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.

SUBMITTER: Dutoit-Lefevre V

PROVIDER: S-EPMC4489013 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies.

Dutoit-Lefèvre Virginie V Dubucquoi Sylvain S Launay David D Sobanski Vincent V Dussart Patricia P Chafey Philippe P Broussard Cédric C Duban-Deweer Sophie S Vermersch Patrick P Prin Lionel L Lefranc Didier D

PloS one 20150701 7

Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The supe ...[more]

PMID: 26132557

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Project description:BackgroundThe emergence and spread of drug resistance in Trichomonas vaginalis parasites has become an important concern in trichomoniasis treatment. Fast and reliable growth assessment is critical for validating in vitro drug susceptibility and high-throughput screening of newly developed drugs.MethodsModified media without yeast extract were evaluated for their ability to support the growth of T. vaginalis parasites. The potential of the nucleic acid-binding dye SYBR Green I for detecting T. vaginalis drug resistance was characterized, and seeding parasite concentration and incubation time were optimized. The fluorescence assay based on SYBR Green I was further validated in four T. vaginalis isolates with different susceptibilities to the antibiotics metronidazole, tinidazole, ornidazole and secnidazole, and compared with the traditional method that detects minimum lethal concentrations (MLCs).ResultsA modified medium consisting of RPMI 1640 and Tryptone Plus as replacements for yeast extract and tryptone, respectively, in traditional trypticase-yeast extract-maltose (TYM) medium exhibited similar performance as TYM medium in maintaining T. vaginalis growth, while it showed much lower background fluorescent signals. The T. vaginalis SYBR Green I-based fluorescence (TSF) drug assay was found to have to satisfy one of two conditions to demonstrate the 50% inhibitory concentration of metronidazole for the sensitive isolate TV-334: (i) a seeding density of 3 × 104 parasites/ml and an incubation time of 48 h; or (ii) a seeding density of 1 × 104 parasites/ml and an incubation time of 72 h. Subsequent validation experiments revealed that the 48-h incubation/3 × 104 parasites/ml seeding density condition had a greater sensitivity to detect drug resistance than the 72-h condition. The TSF assay also exhibited high efficiency in identifying parasite drug resistance, as evidenced by its strong correlation with the standard MLC assay results (P = 0.003).ConclusionsThis study presents a robust TSF assay that has the potential to facilitate high-throughput, automated in vitro anti-trichomoniasis susceptibility testing for drug resistance monitoring and drug development. In comparison to the standard MLC method, this assay offers the advantages of reduced labor and elimination of subjective examination.

Dataset Information

An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies.

Publications

An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies.

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