ANTIBODIES FROM CHRONICALLY INFECTED CYSTIC-FIBROSIS PATIENTS REACT WITH LIPOPOLYSACCHARIDES EXTRACTED BY NEW MICROMETHODS FROM ALL SEROTYPES OF PSEUDOMONAS-AERUGINOSA

Micromethods were developed to extract lipopolysaccharide (LPS, endoto xin) from single bacterial colonies of the 20 recognized Pseudomonas a eruginosa type strains. The appearance of these LPSs in polyacrylamide gel electrophoresis (PAGE) and their reactivity with serum of cystic fibrosis (CF) patients chronically infected with P. aeruginosa was stu died. Silver staining of LPS after PAGE showed that 13 of the P aerugi nosa LPSs had high numbers of O-repeating units arranged in 1-4 cluste rs of banding. Low-molecular-weight LPS fractions were more prominent in six of the serotype strains, of which O:7 and O:14 appeared semi-ro ugh. Corresponding immunoblots using the CF sera showed LPS patterns v ery similar to the silver-stained appearance, indicating an immune rea ction to all P aeruginosa LPS including that from the newly discovered O:18, O:19 and O:20. This was unexpected since only a few serotype st rains (mostly O:3, O:6 and O:9) had been isolated from the patients. A bsorption experiments using purified and chemically defined P aerugino sa rough LPS and smooth LPS suggested these immune reactions were due to antibodies cross-reactive to core / lipid A as well as to lower mol ecular weight O-polysaccharides or ''A-bands''. However, in some cases O:3, O:6, and O:9 LPSs were also found to contain additional distinct O-epitopes. Immune recognition of various polyagglutinable P. aerugin osa LPSs seemed also to be caused by cross-reactive antibodies. The de scribed microextraction methods, followed by PAGE and silver staining or immunoblotting, are easy and convenient techniques with which to st udy antibodies against LPS epitopes and to screen for LPS phenotypic a ppearance using only a few bacterial colonies from larger numbers of G ram-negative bacterial strains.