Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue.
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.
Jones
J B
JB
University of Florida, Gulf Coast Research and Education Center, Bradenton 34203, USA.
Somodi
G C
GC
Scott
J W
JW
D000942:Antigens, Bacterial / Q000276:immunology; D002021:Buffers; D004492:Edetic Acid / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008070:Lipopolysaccharides / Q000276:immunology; D009113:Muramidase / Q000737:chemistry; D018515:Plant Leaves / Q000382:microbiology; D012680:Sensitivity and Specificity; D016959:Xanthomonas campestris / Q000276:immunology / Q000302:isolation & purification*
D000942:Antigens, Bacterial; D002021:Buffers; D008070:Lipopolysaccharides; D004492:Edetic Acid; D009113:Muramidase