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Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue
Author(s) -
Jones J.B.,
Somodi G.C.,
Scott J.W.
Publication year - 1997
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.1997.00251.x
Subject(s) - lysozyme , detection limit , chromatography , extraction (chemistry) , absorbance , xanthomonas campestris , chemistry , phosphate buffered saline , lipopolysaccharide , microbiology and biotechnology , biology , biochemistry , endocrinology , gene
In vitro and in planta sensitivity of an indirect enzyme‐linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10‐foldby using a newextraction buffer (gl of : KH 2 PO 4 , 2; NaHPO 4 , 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10 7 and 1×10 8 cells ml −1 with the conventionalextraction buffer phosphate‐buffered saline (PBS) and less than 1×10 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 lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10 7 cfu ml −1 and 1×10 8 cfu ml −1 with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10 5 cfu ml −1 in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant 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 isthe reacting epitope.