Analysis by flow cytometry of surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa.

Antisera were produced in mice immunized with 18 synthetic peptide conjugates representing various regions throughout the length of the outer membrane protein F molecule of Pseudomonas aeruginosa and analysed by flow cytometry to identify those antisera capable of binding to the surface of whole cells of P. aeruginosa. Antibodies to peptides 9, 18, 10, and 4 were significantly cell-surface reactive. The maximum median percentage of antibody-binding cells in this assay was 36.6%. Over six different determinations, peptide 9 antisera binding to the cells ranged from 16.9 to 57.0% of the cell population. We propose that the surface accessibility of protein F epitopes varies during the cell cycle.

Ability of synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa to afford protection against P. aeruginosa infection in a murine acute pneumonia model.

Three synthetic peptides (Nos 9, 10 and 18) representing surface-exposed, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa were each conjugated to the carriers keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), with the conjugates being used to immunize mice intranasally. Mice were also immunized intranasally with a KLH/BSA carrier control or with a peptide No. 8 conjugate as a negative control. An immunoglobulin G response reactive with P. aeruginosa whole cells was demonstrated by enzyme-linked immunosorbent assay (ELISA) of sera from mice immunized with peptide 9, 10 or 18, whereas no whole-cell reactivity by ELISA was detected in sera from mice immunized with peptide 8. Upon pulmonary challenge of immunized mice with a Fisher-Devlin immunotype 4 strain of P. aeruginosa, only those mice immunized with peptide 9 or peptide 10 had a significantly greater survival rate compared to control mice immunized with the carriers alone. Peptides 9 (TDAYNQKLSERRAN) and 10 (NATAEGRAINRRVE) have potential for further development as a protective vaccine against P. aeruginosa infections.

Use of synthetic peptides to identify surface-exposed, linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa.

In a previous study (Hughes EE, Gilleland LB, Gilleland HE Jr. [1992] Infect Immun 60:3497-3503), ten synthetic peptides were used to test for surface-exposed antigenic regions located throughout the length of outer membrane protein F of Pseudomonas aeruginosa. An additional nine peptides of 11-21 amino acid residues in length were synthesized. Antisera collected from mice immunized with each of the 19 synthetic peptides conjugated to keyhole limpet hemocyanin were used to determine which of the peptides had elicited antibodies capable of reacting with the surface of whole cells of the various heterologous Fisher-Devlin immunotypes of P. aeruginosa. Cell surface reactivity was measured by an enzyme-linked immunosorbent assay (ELISA) with whole cells of the various immunotypes as the ELISA antigens and by opsonophagocytic uptake assays with the various peptide-directed antisera, immunotype 2 P. aeruginosa cells, and polymorphonuclear leukocytes of human and murine origin. Three peptides located in the carboxy-terminal portion of protein F elicited antibodies with the greatest cell-surface reactivity. Peptide 9 (TDAYNQKLSERRAN), peptide 10 (NATAEGRAINRRVE), and peptide 18 (NEYGVEGGRVNAVG) appear to have sufficient potential for further development as vaccine candidates for immunoprophylaxis against infections caused by P. aeruginosa. A topological model for the arrangement of protein F within the outer membrane of P. aeruginosa is presented.

Synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa that elicit antibodies reactive with whole cells of heterologous immunotype strains of P. aeruginosa.

By using the published amino acid sequence for mature outer membrane protein F of Pseudomonas aeruginosa, a computer-assisted analysis was performed to identify sites with potential as surface-exposed, antigenic regions located throughout the length of the protein molecule. Synthetic peptides 13 to 15 amino acid residues in length were synthesized for 10 such regions. Mice were immunized with each of the 10 synthetic peptides conjugated to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) of the antisera was performed by using each of the synthetic peptides as the ELISA antigen to verify that immunoglobulin G (IgG) antibodies capable of reacting with the peptide used as immunogen were elicited by each peptide. Each of the antipeptide antisera was screened for the presence of IgG antibodies that could bind to the surface of intact cells of strains representing the seven heterologous Fisher-Devlin immunotypes of P. aeruginosa by use of an ELISA with whole cells of the various strains as the ELISA antigen. Three peptides elicited antibodies capable of reacting with whole cells of all seven immunotype strains. Peptide 10, corresponding to amino acid residues 305 to 318, elicited whole-cell-reactive antibodies at high titers. Peptide 9, corresponding to amino acid residues 261 to 274, elicited whole-cell-reactive antibodies at more intermediate titers. Peptide 7, corresponding to amino acid residues 219 to 232, elicited such antibodies only at low titers. The carboxy-terminal portion of the mature protein appears to be the immunodominant portion. In particular, peptides 10 (NATAEGRAINRRVE) and 9 (TDAYNQKLSERRAN) appear to have potential for use as immunogens in a synthetic vaccine for immunoprophylaxis against infections caused by P. aeruginosa. Antisera from mice immunized with either peptide 9 or 10 mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells of P. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant of P. aeruginosa.

A calibration system for producing known concentrations of mercury vapor in air.

This paper describes the construction and evaluation of a system capable of producing well-defined test mixtures of mercury in air, or other diluent gas, at mercury concentrations between zero and 16 mg/M3. The various parameters that affect the generation system and their interactions are discussed and data is given for the calibration of several different mercury monitors.

Development of gas-blending systems for calibration: application to hydrogen fluoride, arsine and phosgene in air.

Preparation of samples of known concentration of hydrogen fluoride, arsine and phosgene in air from 0.5 to 10 times the TLV is described. These are required to evaluate and calibrate analytical monitoring devices used for measuring these gases in work atmospheres. Dynamic gas-blending systems have been constructed and evaluated at the National Bureau of Standards for preparing these samples. Measurements to estimate the long term concentration stability of these gases are also included.

Development of standard reference materials for air quality measurement.

The National Bureau of Standards is engaged in a continuing program involving gaseous Standard Reference Materials for air pollution measurements. Preparation of such materials requires definition of the stability, homogeneity, and accuracy of the samples. This information is obtained by long term studies of the gas systems, by development of absolute methods of analysis, and by analysis of large numbers of samples prepared in bulk. The results of studies, extending over several years, of low concentration of carbon monoxide in nitrogen and nitric oxide in nitrogen are reported. Over one thousand samples of these materials have been analyzed and the stability with time and the within-batch homogeneity have been characterized. Accuracy is achieved by use of gravimetric standards and with dynamic dilution systems. Accuracy attainable by either method is described. The use of permeation tubes of sulfur dioxide and nitrogen dioxide is necessary in some situations because of the reactivity of the gases. Data covering the stability and accuracy of these devices has been collected over a period of several years.