The lipopolysaccharide (LPS) of Pseudomonas aeruginosa PAO1 contains two species of O polysaccharide termed A and B bands. The high-molecular-weight B-band LPS determines the O specificity of the bacterium, while the antigenically distinct A-band LPS consists of only shorter-chain polysaccharides. Seven hybridomas secreting A-band-specific monoclonal antibodies were produced and used to study the LPS of standard and clinical strains. Although A-band antibodies did not agglutinate any of the serotype strains presently in the International Antigenic Typing Scheme, Western immunoblots revealed that Il of the 17 serotype strains possessed A-band LPS. In a group of 250 clinical isolates from patients with cystic fibrosis, 170 (68%) had A-band LPS on the basis of agglutination tests, but in silver-stained gels all were shown to be deficient in O-antigen-containing B band. Investigation of serial isolates from a single patient revealed a pattern of antigenic variation. During the course of the infection, serotypeable isolates became nontypeable, and the O antigen was replaced with A band as the major LPS antigen. These results suggest that A-band LPS may be the major LPS antigen in nontypeable clinical isolates and a common antigen among other P. aeruginosa strains.

Monoclonal antibodies against 12 of the 17 IATS serotype strains ofPseudomonas aeruginosa were produced. Eighty-seven hybridoma clones were isolated, and the antibodies secreted were found to be reactive with both Formalin-fixed whole cells and purified lipopolysaccharide of homologous strains in enzyme-linked immunosorbent assays. Among these monoclonal antibodies, the predominant antibody class was immunoglobulin M (IgM) (76%), although antibodies of the IgG2a and IgG3 isotypes were also produced. The monoclonal antibodies could further be divided intQ two groups based on their ability to agglutinate whole cells of homologous strains. The agglutinating monoclonal antibodies were found to immunoblot with the 0 side chains of homologous lipopolysaccharide, while the nonagglutinating monoclonal antibodies were found to be reactive with outer membrane protein-associated lipopolysaccharide. The applicability of monoclonal antibodies for serotyping was examined, and several antibodies were found to agglutinate whole cells and immunoblot with 1051

Two lipopolysaccharide 0-antigen-specific monoclonal antibodies, MA1-8 (an immunoglobulin Gl [IgGl]) and MF15-4 (an IgM), were used to localize the 0 antigen of the lipopolysaccharide of Pseudomonas aeruginosa PAO1. A protein A-dextran-gold conjugate with an average particle diameter of 12.5 nm was used to label bacterial cells treated-with MA1-8, while a second antibody (goat anti-mouse IgM) was required before the same probe could interact with cells treated with the IgM antibody MF15-4. Both antibodies resulted in exclusive labeling of the surface of P. aeruginosa PAO1 but not that of an isogenic 0-antigen-lacking rough mutant. When the monoclonal antibodies became attached to the cell surface of P. aeruginosa PAO1, resulting in an even coating, the foldings and other topographic details could not be discerned by negative staining. In thin sections of monoclonal-antibody-treated bacteria, a 20-and a 30-to 40-nm thick amorphous layer was observed around the outside of the outer membrane when MA1-8 (IgG) and MF15-4 (IgM) plus goat anti-mouse IgM antibodies were used, respectively. This amorphous layer presumably resulted from the stabilization of the lipopolysaccharide structure by the monoclonal antibodies which prevented the long 0-antigen chains from collapsing owing to dehydration.Bacterial cell surface studies were initiated to generate knowledge of how microorganisms function as well as how they interact with an animal host or with the environment. In the past, much effort has been spent to improve highresolution electron microscopy (20) and the techniques for surface antigen localization. Some of these localization methods for studying microorganisms involve the use of an enzyme and its end-product depositions (1, 28) or the use of polyclonal antibodies conjugated to an electron-dense marker, such as ferritin (28,34,40,41). More recently, monodispersed colloidal gold has emerged as the most versatile marker for cell-labeling studies. The advantages of using colloidal gold versus the use of other markers have been described in detail in several recent reviews (14,21,38). With the development of hybridoma technology by Kohler and Milstein (23), monoclonal antibodies have been rapidly replacing polyclonal antibodies as the immunological reagent in in situ antigen detection studies because of their qualities of specificity, availability, and reproducibility.Lipopolysaccharide (LPS) plays an important role as a structural component of the cell walls of gram-negative bacteria. This macromolecule has also been implicated as a potential virulence factor of some organisms (9). In the past two decades, many LPS localization studies involving Escherichia coli and Salmonella typhimurium have been reported (3,(32)(33)(34)40). Although data from these studies provided information on the distribution of LPS in the outer membrane, the architectural relationship between the LPS molecules and the cell surface has not been thoroughly examined. We produced monoclonal antibodies of both immunoglobulin G (IgG) and IgM isotype...

A panel of 22 monoclonal antibodies against 8 of the 17 International Antigenic Typing Scheme (IATS) serotypes of Pseudomonas aeruginosa was produced. The antibodies were characterized for cross-reactivities, isotypes, titers, and epitope specificities. The results complemented those of our previous study and marked the completion of a set of monoclonal antibodies for serotyping P. aeruginosa.

Coliphage K30, a bacteriophage specific for strains bearing the Escherichia coli serotype K30 capsular polysaccharide, produced plaques surrounded by extensive haloes, a characteristic of phage which produce capsule depolymerase (glycanase) enzymes. Klebsiella K20, a strain producing a capsular polysaccharide chemically identical to that of E. coli K30, was not lysed by coliphage K30, although the bacteriophage encoded glycanase enzyme did degrade the K20 polysaccharide. Morphologically, coliphage K30 belonged to Bradley group C. The coliphage K30 particle comprised 20 structural polypeptides which varied from 9.5–136 kDa and genomic DNA of 38.7 ± 1.0 kb.