| Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue. | |
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MedLine Citation:
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PMID: 9351221 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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In vitro and in planta sensitivity of an indirect enzyme-linked immunoassay technique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria, was increased 10-fold by using a new extraction buffer (gl of: KH2PO4, 2; NaHPO4, 11.5; EDTA disodium, 0.14; thimerosal, 0.02; and lysozyme, 0.2). The procedure improved sensitivity without increasing background levels. In vitro, the limit of detection was between 1 x 10(7) and 1 x 10(8) cells ml-1 with the conventional extraction buffer phosphate-buffered saline (PBS) and less than 1 x 10(6) cells ml-1 when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c. vesicatoria strains, absorbance readings were increased close to three-fold with the lysozyme extraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, the limit of detection was 1 x 10(7) cfu ml-1 and 1 x 10(8) cfu ml-1 with the lysozyme solution and PBS, respectively, as the extraction buffers. When using the lysozyme extraction buffer in combination with a commercial amplification system, the limit of detection was decreased to less than 1 x 10(5) cfu ml-1 in leaf tissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of a significant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure, termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS is the reacting epitope. |
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Authors:
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J B Jones; G C Somodi; J W Scott |
Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, Non-P.H.S. |
Journal Detail:
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Title: Journal of applied microbiology Volume: 83 ISSN: 1364-5072 ISO Abbreviation: J. Appl. Microbiol. Publication Date: 1997 Oct |
Date Detail:
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Created Date: 1998-02-10 Completed Date: 1998-02-10 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 9706280 Medline TA: J Appl Microbiol Country: ENGLAND |
Other Details:
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Languages: eng Pagination: 397-401 Citation Subset: IM |
Affiliation:
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University of Florida, Gulf Coast Research and Education Center, Bradenton 34203, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Antigens, Bacterial
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immunology Buffers Edetic Acid / chemistry Enzyme-Linked Immunosorbent Assay / methods* Lipopolysaccharides / immunology Muramidase / chemistry Plant Leaves / microbiology Sensitivity and Specificity Xanthomonas campestris / immunology, isolation & purification* |
| Chemical | |
Reg. No./Substance:
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0/Antigens, Bacterial; 0/Buffers; 0/Lipopolysaccharides; 60-00-4/Edetic Acid; EC 3.2.1.17/Muramidase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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