APPROPRIATE COATING METHODS AND OTHER CONDITIONS FOR ENZYME-LINKED-IMMUNOSORBENT-ASSAY OF SMOOTH, ROUGH, AND NEUTRAL LIPOPOLYSACCHARIDES OFPSEUDOMONAS-AERUGINOSA

Smooth, rough, and neutral forms of lipopolysaccharide (LPS) from Pseu domonas aeruginosa were used to assess the appropriate conditions for effective enzyme-linked immunosorbent assay (ELISA) of LPS. Each of th ese forms of well-defined LPS was tested for the efficiency of antigen coating by various methods as well as to identify an appropriate type of microtiter plate to use. For smooth LPS, the standard carbonate-bi carbonate buffer method was as efficient as the other sensitivity-enha ncing plate-coating methods compared. The rough LPS, which has an over all hydrophobic characteristic, was shown to adhere effectively, regar dless of the coating method used, to only one type of microtiter plate , CovaLink. This type of plate has secondary amine groups attached on its polystyrene surface by carbon chain spacers, which likely favors h ydrophobic interactions between the rough LPS and the well surfaces. D ehydration methods were effective for coating microtiter plates with t he neutral LPS examined, which is composed predominantly of a D-rhamna n. For the two dehydration procedures, LPS suspended in water or the o rganic solvent chloroform-ethanol was added directly to the wells, and the solvent was allowed to dehydrate or evaporate overnight. Precoati ng of plates with either polymyxin or poly-li-lysine did not give any major improvement in coating with the various forms of LPS. The possib ility of using proteinase K- and sodium dodecyl sulfate-treated LPS pr eparations for ELISAs was also investigated. Smooth LPS prepared by th is method was as effective in ELISA as LPS prepared by the hot water-p henol method, while the rough and neutral LPSs prepared this way were not satisfactory for ELISA.