Determination of the components of immune complexes made in vitro with antigens derived from Pseudomonas aeruginosa (original) (raw)
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Journal of Medical Microbiology, 1992
Monoclonal antibodies (MAbs) to the core antigen region of lipopolysaccharide (LPS) of Pseudomonas aeruginosa were produced from mice immunised with whole cells of heat-killed rough mutants of Pseudomonas aeruginosa expressing partial or complete core LPS. MAbs were screened in an enzyme-linked immunosorbent assay (ELISA) against three different antigen cocktails : S-form LPS from three P . aeruginosa strains, R-form LPS from six P . aeruginosa strains and, as a negative control, R-form LPS from Salmonefla typhimurium and Escherichia coli. Selected MAbs were subsequently screened against a range of extracted LPS and whole cells from both reference strains and clinical isolates of P . aeruginosa. The antibodies were also screened in ELISA against whole-cell antigens from other Pseudomonas spp. as well as strains of Haemophilus influenzae, Neisseria subjlava and Staphylococcus aureus. Five MAbs reacting with the core component of P. aeruginosa LPS were finally selected. Two of these, MAbs 360.7 and 304.1.4, were particularly reactive in immunoblots against unsubstituted core LPS, including that from 0-antigenic serotypes of P. aeruginosa. The MAbs also reacted with some of the other Pseudomonas spp., but not with P. cepacia or Xanthomonas (Pseudomonas) maltophilia. Cross-reactivity with whole cells from other bacterial species was minimal or not observed. Reactivity of MAbs with some Staph. aureus strains was observed, and binding to the protein A component was implicated. The reactivity of the M Abs was investigated further by flow cytometry and immunogold electronmicroscopy. The suitability of the MAbs for an immunological assay for detection of P. aeruginosa in respiratory secretions from C F patients is discussed.
FEMS Microbiology Letters, 1988
It has been observed that each strain of the Pseudomonas aeruginosa species harbours the socalled polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysac-Correspondence to: A. Marx, Max-Planck-Institut fi.ir Immunbiologie, D-7800 Freiburg i. Br., F.R.G. charide also contains the PA which could possibly play an important role in the patient by protecting P, aeruginosa cells against anti-PA antibodies.
European journal of biochemistry, 1987
EJB 86 1041 related immunotype 6 (Fisher classification [l 11) lipopolysacremoved by centrifugation and the supernatant was subjected charides. Two new diN -acyl derivatives of 5,7-diaminoto gel filtration on Sephadex G-50. The fraction B (Fig.4) 3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic acid, which corresponding to small oligosaccharides was rechromawas called by us pseudaminic acid [8], were identified as the tographed in each case on Sephadex G-15 to give trisacconstituents of these polysaccharides. For immunotype 6 0charides I, 2 and 3 with the yields of 25-35%; [KID-63.3", specific polysaccharide see preliminary communication [12].-69.8" and-65.5" (c 1) respectively. MATERIALS AND METHODS Miscellaneous methods 'H and 13C nuclear magnetic resonance spectra were recorded with a WM-250 (Bruker) spectrometer in DzO at 60°C for lipopolysaccharides and at 30 "C for oligosaccharides and monosaccharides with acetone (6, 2.23 ppm) or methanol (6, 50.15 ppm) as internal standards. Optical rotations were measured with a Perkin-Elmer polarimeter (model 141) in water at 20°C. Solutions were freeze-dried or evaporated in vacuo at 40°C. Serological tests were performed as described earlier 1131. Chromatography and electrophoresis Ascending paper chromatography was carried out on FN-11 paper with n-butanollpyridinelwater (61413, v/v, system A) and ethyl acetatelpyridinelacetic acidlwater (5151113, v/v, system B). Paper electrophoresis was performed in 0.025 M pyridine acetate buffer, pH 4.5, at 28 V/cm. Substances were detected on paper using alkaline silver nitrate or benzidine with potassium iodide after treatment with chlorine [14]. Gel filtration was performed on columns of Sephadex G-50 (70 x 3.7 cm) and Sephadex G-15 (75 x 1.5 cm) in pyridine acetate buffer, and of Fractogel TSK 40 (90 x 2 cm) in water. Ion-exchange chromatography was carried out on a column (20 x 1 cm) of DEAE-Trisacryl M. Elution profiles were recorded by using a Technicon sugar analyzer. Monosaccharides were determined with a Technicon sugar analyzer and a BC-200 amino acid analyzer as described earlier [15]. Gas-Liquid chromatography was carried out on a Pye-Unicam 104 instrument (model 64) using a column (150 x 0.1 cm) packed with 5% OV-1 on Diatomite CQ (100-200 mesh) at 190-270°C; carrier gas nitrogen, flow rate 30 ml/min. Combined gas-liquid chromatography/mass spectrometry was performed on a Varian MAT Gnom 111 instrument using the same phase. Bacterial cultures and isolation of lipopolysaccharides Bacterial cultures of P. aeruginosa OSa,b,c, OSa,b,d, 05a,d and immunotype 6 (strains 170011, 170012, 170013 and 170046 respectively) were kindly provided by Dr Lhnyi (Institute of Hygiene, Budapest). Cultures were grown as described previously [ 131. Acetone-dried cells (30 g each strain) were extracted with 45% aqueous phenol [16], nucleic acids precipitated with Cetavlon and lipopolysaccharides recovered from the aqueous solution by the addition of ethanol (10 volumes) followed by dialysis and freeze-drying [16]. The yield of the lipopolysaccharides was 10-3 5% of the dry cell weight. Mild acid degradation Lipopolysaccharides (1 g of each) were heated with 1% acetic acid (100 ml, lOO"C, 1.5 h), the lipid precipitate was Solvolysis with hydrogen fluoride Trisaccharide 1 (50 mg) was treated with hydrogen fluoride (10 ml, 20"C, 3 h) freshly distilled over cobalt trifluoride [17]; hydrogen fluoride was removed in vacuo by absorption with solid sodium hydroxide. The residue was dissolved in water (10 ml), evaporated, again dissolved in water and passed through a column (3 x 1 cm) with Amberlite CG 120 resin (H+-form). The column was washed with water, the eluate was evaporated and D-xylose (8 mg), [ .
Journal of clinical …, 1992
Enzyme-linked immunosorbent assays were developed separately for the three main parts of the Pseudomonas aeruginosa lipopolysaccharide (LPS) molecule, namely, lipid A, core, and 0 polysaccharide. Anti-lipid A, anticore, and anti-O polysaccharide antibodies were measured in serum samples from 12 patients with cystic fibrosis (CF) in a longitudinal study covering the period before P. aeruginosa infection was established through at least 5 years of chronic infection. The serum antibody response to all parts of the P. aeruginosa LPS molecule increased during the course of chronic infection. The increase in anti-lipid A antibodies was specific for P. aeruginosa lipid A, since no increase in anti-Escherichia coli lipid A antibodies was seen. Immunoglobulin G, A, and M (IgG, IgA and IgM) antibodies were all involved in the specific systemic response to P. aeruginosa lipid A, core, and the 0 polysaccharides. IgG and IgA levels in particular increased during the course of infection and were significantly higher than the antibody increase seen with age in a healthy control group. The local immune response in the lungs was investigated by measuring IgG, IgA, and IgM antibodies to the separate parts of the P. aeruginosa LPS molecule in sputum samples from 18 CF patients with at least a 5-year history of chronic P. aeruginosa infection. Antibodies detected in sputum were mainly anti-lipid A and anti-O polysaccharide antibodies of the IgG and IgA isotypes. Very high IgA anti-lipid A titers were detected in sputum samples from some CF patients. ) " The same CF patients were tested before P. aeniginosa lung infection was established (preinfection), at 0 to 2 years after chronic P. aenrginosa lung infection was diagnosed (early stage), and at the late stage of chronic lung infection (>5 years of infection).
A Study on the Serological Relationships of Various Fractions of Pseudomonas aeruginosa
1973
The purpose of this research was to determine the relationship of the slime layer antigen(s) to the "101" or LPS antigens and to attempt to evaluate the role of antibodies against the latter in protection against experimental infections in mice with the homologous strain of Pseudomonas aeruginosa. Results from agglutination tests, chromatographic separations, passive protection tests, and characterizations of the antigens by gel double diffusion do not support the concept that LPS is a necessary portion of the immunogenic material. The immunogenicity of LPS can be attributed to co-purification of residual amounts of slime layer antigens on the washed cells from which LPS was extracted.
Classification of Pseudomonas Aeruginosa O Antigens by Immunoelectrophoresis
Journal of Medical Microbiology, 1975
IN a study reported previously (LBnyi and AdBm, 1973), it was shown that double agar-gel precipitation and agglutination give, as a rule, identical crossreaction patterns for the 0 antigens of Pseudomonas aeruginosa. On the basis of precipitation of different kinds of bacterial extract, the antigens were classified into the following groups: (1) those of which saline extracts, supernates of phenol-water extracts (L1 fractions) and purified lipopolysaccharide (LPS) precipitated readily with homologous antisera; (2) those in which saline extracts and LPS precipitated but L1 fractions did not; and (3) two partial antigens, none of the extracts of which precipitated with absorbed antisera, although the presence of the antigen was demonstrable by means of agglutination tests. In the experiments described in this paper, a further classification of P. aeruginosa 0 antigens was attempted. MATERIALS AND METHODS Bacterial strains. Twenty-three P. aeruginosa cultures represented Unyi's 0-antigen type strains (Lanyi, 1966-67 and 1970); in addition, 53 strains of different serogroups were chosen from our collection. Of 13 strains received from Professor P. V. Liu, Louisville, Kentucky, USA, as type strains of the provisional international P. aeruginosa antigenic scheme, 12 originated from Habs (1957) and one from Sandvik (1960). Saline extracts were made from all these cultures, but only Lanyi's 23 0-antigen type strains were used for the preparation of L1, LPS, trichloracetic-acid (TCA) and alkali extracts. Antigens. Extracts in physiological saline (" saline ") were prepared by heating the bacterial suspension at 100°C for 23 h; each ml of the supernate obtained after centrifugation represented c. 30 mg of bacteria (moist weight). L1 fractions were the freeze-dried supernates
European journal of biochemistry, 1987
EJB 86 1041 related immunotype 6 (Fisher classification [l 11) lipopolysacremoved by centrifugation and the supernatant was subjected charides. Two new diN -acyl derivatives of 5,7-diaminoto gel filtration on Sephadex G-50. The fraction B (Fig.4) 3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic acid, which corresponding to small oligosaccharides was rechromawas called by us pseudaminic acid [8], were identified as the tographed in each case on Sephadex G-15 to give trisacconstituents of these polysaccharides. For immunotype 6 0charides I, 2 and 3 with the yields of 25-35%; [KID-63.3", specific polysaccharide see preliminary communication [12].-69.8" and-65.5" (c 1) respectively. MATERIALS AND METHODS Miscellaneous methods 'H and 13C nuclear magnetic resonance spectra were recorded with a WM-250 (Bruker) spectrometer in DzO at 60°C for lipopolysaccharides and at 30 "C for oligosaccharides and monosaccharides with acetone (6, 2.23 ppm) or methanol (6, 50.15 ppm) as internal standards. Optical rotations were measured with a Perkin-Elmer polarimeter (model 141) in water at 20°C. Solutions were freeze-dried or evaporated in vacuo at 40°C. Serological tests were performed as described earlier 1131. Chromatography and electrophoresis Ascending paper chromatography was carried out on FN-11 paper with n-butanollpyridinelwater (61413, v/v, system A) and ethyl acetatelpyridinelacetic acidlwater (5151113, v/v, system B). Paper electrophoresis was performed in 0.025 M pyridine acetate buffer, pH 4.5, at 28 V/cm. Substances were detected on paper using alkaline silver nitrate or benzidine with potassium iodide after treatment with chlorine [14]. Gel filtration was performed on columns of Sephadex G-50 (70 x 3.7 cm) and Sephadex G-15 (75 x 1.5 cm) in pyridine acetate buffer, and of Fractogel TSK 40 (90 x 2 cm) in water. Ion-exchange chromatography was carried out on a column (20 x 1 cm) of DEAE-Trisacryl M. Elution profiles were recorded by using a Technicon sugar analyzer. Monosaccharides were determined with a Technicon sugar analyzer and a BC-200 amino acid analyzer as described earlier [15]. Gas-Liquid chromatography was carried out on a Pye-Unicam 104 instrument (model 64) using a column (150 x 0.1 cm) packed with 5% OV-1 on Diatomite CQ (100-200 mesh) at 190-270°C; carrier gas nitrogen, flow rate 30 ml/min. Combined gas-liquid chromatography/mass spectrometry was performed on a Varian MAT Gnom 111 instrument using the same phase. Bacterial cultures and isolation of lipopolysaccharides Bacterial cultures of P. aeruginosa OSa,b,c, OSa,b,d, 05a,d and immunotype 6 (strains 170011, 170012, 170013 and 170046 respectively) were kindly provided by Dr Lhnyi (Institute of Hygiene, Budapest). Cultures were grown as described previously [ 131. Acetone-dried cells (30 g each strain) were extracted with 45% aqueous phenol [16], nucleic acids precipitated with Cetavlon and lipopolysaccharides recovered from the aqueous solution by the addition of ethanol (10 volumes) followed by dialysis and freeze-drying [16]. The yield of the lipopolysaccharides was 10-3 5% of the dry cell weight. Mild acid degradation Lipopolysaccharides (1 g of each) were heated with 1% acetic acid (100 ml, lOO"C, 1.5 h), the lipid precipitate was Solvolysis with hydrogen fluoride Trisaccharide 1 (50 mg) was treated with hydrogen fluoride (10 ml, 20"C, 3 h) freshly distilled over cobalt trifluoride [17]; hydrogen fluoride was removed in vacuo by absorption with solid sodium hydroxide. The residue was dissolved in water (10 ml), evaporated, again dissolved in water and passed through a column (3 x 1 cm) with Amberlite CG 120 resin (H+-form). The column was washed with water, the eluate was evaporated and D-xylose (8 mg), [ .
European journal of biochemistry, 1980
A loosely bound lipopolysaccharide-protein complex was extracted from cells of Pseudomonar aeruginosa strain 17001 5 (0 : 7ab ; Lanyi classification) by saline solution and purified from contaminant nucleic acid by Cetavlon treatment followed by precipitation in an ultracentrifuge. The saline-treated cells were re-extracted with hot aqueous phenol to give firmly bound lipopolysaccharide which was isolated from the phenol layer and purified by ultracentrifugation. The identity of both lipopolysaccharide preparations was proved by serological and chemical evidence. Mild acid degradation of the lipopolysaccharide resulted in the splitting off of a lipid component and led to polysaccharide which was purified by gel-filtration on a Sephadex G-50 column. The polysaccharide consisted of' N-acetyl-D-fucosamine, N-acetyl-L-fucosamine and D-glucose in the ratio 1 : 1 : 1. On the basis of nuclear magnetic resonance spectra, results of methylation analysis and two sequential Smith degradations, the following structure can be assigned to the repeating unit of the polysaccharide :-3)~FucNAc(al-3)~FucNAc(Bl-~) D G~C (P~-. The polysaccharide did not show serological activity whereas alkali-treated lipopolysaccharide readily sensitised sheep erythrocytes and inhibited the passive haemagglutination reaction with anti-(0 : 7a,b)serum. Evidence is presented that the oligosaccharide repeating units of the polysaccharide and alkali-treated lipopolysaccharide are indistinguishable. ps. aeruginosa strain 170016 (0 : 7a,c) was shown to have the 0-specific lipopolysaccharide identical with that from strain 170015. The presented data show that subfactors 7b.and 7c in the Lanyi classification of Ps. uevuginosa 0-antigens seem to relate to components of the bacterial surface other than lipopolysaccharides.