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 immuno assay technique, using a monoclonal antibody specific for the lipopoly saccharide (LPS) of Xanthomonas campestris pv. vesicatoria, was increa sed 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) c ells ml(-1) with the conventional extraction buffer phosphate-buffered saline (PBS) and less than 1 x 10(6) cells ml(-1) when lysozyme extra ction buffer was substituted for PBS. In comparing 22 X. c. vesicatori a strains, absorbance readings were increased close to three-fold with the lysozyme extraction buffer as opposed to PBS. When leaf tissue ex tract 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 syst em, 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 phos phate buffer resulted in release of a significant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure, terme d lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA react ions where LPS is the reacting epitope.