Performance of four different indirect enzyme-linked immunosorbent assays (ELISAs) to detect specific IgG, IgA, and IgM in Legionnaires' disease.

Currently recommended methods in Legionnaires' disease serology are based upon crude whole-cell antigenic preparations. To investigate whether purified antigens would perform better in a given diagnostic test for antibodies against Legionella pneumophila, we compared the performance of three antigenic preparations of L. pneumophila serogroup 1 consisting of outer membrane protein (OMP), flagellin (FLA), and lipopolysaccharide (LPS) to a sonic extract (SON) in indirect immunosorbent assay (ELISA) measuring both IgG, IgA, and IgM. The reactivity of sera from 20 patients with culture-verified Legionnaires' disease and sera from 12 patients with pneumonia and a diagnostic rise in titre by a microagglutination test (MA) was studied. Our results indicated that the SON IgA assay was the most sensitive test in both groups of patients. The LPS IgG and IgM assays, however, were the most specific tests, closely followed by the corresponding SON tests. By combining two individual assays, a maximum nosographic sensitivity of 85% could be obtained. Whereas no benefit of using purified outer membrane protein or flagella instead of a sonic extract in the indirect ELISAs was found, the LPS antigen provided a sensitive and specific alternative to the sonic extract.

Effect of Legionella pneumophila sonicate on killing of Listeria monocytogenes by human polymorphonuclear neutrophils and monocytes.

Legionella pneumophila shares with other intracellular pathogens the ability to resist intracellular killing within phagocytes. An increasing number of cellular components of L. pneumophila are proposed as pathogenic factors of the organism. At the site of infection, the phagocytic cells will be exposed to bacterial components, either expressed on the surface of the organisms or released in the environment upon cell lysis. In this study, we have investigated the effect of water-soluble bacterial components present in L. pneumophila sonicate on the phagocytosis and bactericidal activity of human polymorphonuclear neutrophils and monocytes. Preincubation of neutrophils with L. pneumophila sonicate did not affect phagocytosis of L. monocytogenes, whereas Listeria killing was significantly inhibited at sonicate concentrations of 1 and 2 mg/ml. The phenol phase of a phenol-water extraction, containing most of the lipopolysaccharide (LPS), had no inhibitory effect on the listericidal activity of neutrophils. Killing of Listeria by monocytes was inhibited in a similar manner. The inhibitory activity was mainly recovered in the sonicate fraction above 100 kDa, suggesting that components organized in larger molecular complexes are most likely to represent the inhibitory factors. The inhibitory activity of L. pneumophila sonic extract appears to be related to inhibition of killing mechanisms since uptake of Listeria was not affected by the sonicate. Our observations indicate that as Legionella infection progresses, bacterial components liberated by cell lysis could exert a detrimental effect on the antimicrobial function of phagocytes, stressing the importance of early treatment of Legionnaires' disease to reduce bacterial numbers in the infected tissues.

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, endotoxin) from single bacterial colonies of the 20 recognized Pseudomonas aeruginosa 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 studied. Silver staining of LPS after PAGE showed that 13 of the P. aeruginosa LPSs had high numbers of O-repeating units arranged in 1-4 clusters 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-rough. Corresponding immunoblots using the CF sera showed LPS patterns very similar to the silver-stained appearance, indicating an immune reaction 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 strains (mostly O:3, O:6 and O:9) had been isolated from the patients. Absorption experiments using purified and chemically defined P. aeruginosa rough LPS and smooth LPS suggested these immune reactions were due to antibodies cross-reactive to core/lipid A as well as to lower molecular 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. aeruginosa LPSs seemed also to be caused by cross-reactive antibodies. The described microextraction methods, followed by PAGE and silver staining or immunoblotting, are easy and convenient techniques with which to study antibodies against LPS epitopes and to screen for LPS phenotypic appearance using only a few bacterial colonies from larger numbers of Gram-negative bacterial strains.

Lipopolysaccharide is present in immune complexes isolated from sputum in patients with cystic fibrosis and chronic Pseudomonas aeruginosa lung infection.

Sputum samples from seven patients with cystic fibrosis and chronic P. aeruginosa lung infection were investigated for immune complexes by PEG precipitation and in two different complement binding assays. All seven patients were immune complex positive. The components involved in immune complex formation were identified by SDS-PAGE and immunoblotting. We found P. aeruginosa lipopolysaccharide as a major antigen. Both core and O-specific saccharide antigens could be demonstrated. IgG and IgA were the immunoglobulins involved, with IgG2 as the dominating IgG subclass. Lipopolysaccharide has a number of biological activities and its presence in sputum may have consequences for the pathogenesis of lung disease in cystic fibrosis.

Determination of IgG subclass antibodies to Pseudomonas aeruginosa outer membrane proteins in cystic fibrosis lung infection using immunoblotting and enzyme-linked immunosorbent assay.

IgG subclass antibodies to Pseudomonas aeruginosa outer membrane proteins (OMP) were investigated in serum from cystic fibrosis (CF) patients by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Fifteen patients (eight in good and seven in poor clinical condition) have been followed for an average of 13 years with multiple serum samples covering the preinfection, and early and late stages of chronic infection. Laser-scanning densitometry of photographs taken from immunoblots was used to quantify antibody level and compare with ELISA titres. The earliest anti-OMP antibodies to appear were of the IgG1 subclass. There was no significant difference in IgG subclass antibody levels to OMPs between patients in relatively good and poor clinical condition. Data presented indicate a high positive correlation among measurements of IgG subclass antibodies to P. aeruginosa OMPs using ELISA and immunoblotting.

Serum antibodies to Pseudomonas aeruginosa outer-membrane proteins and iron-regulated membrane proteins at different stages of chronic cystic fibrosis lung infection.

Serum samples collected over periods up to 15 years from nine patients with cystic fibrosis (CF) were investigated by immunoblotting and crossed immuno-electrophoresis (CIE) for antibodies to Pseudomonas aeruginosa outer-membrane proteins (OMPs). The earliest antibody response to OMPs was directed against proteins G, H1 and I. Detection by immunoblotting sometimes preceded the CIE response; the appearance of antibodies to the other major OMPs was co-incident with an increase in CIE precipitins. Isolation of the mucoid form of P. aeruginosa was associated with a rapid increase in both precipitin numbers and antibodies detected by immunoblotting. Antibodies to iron-regulated OMPs could be detected in all the serum samples that showed eight or more CIE precipitins but their presence became pronounced only in the advanced stages of disease. The clinical strain used in this study and other isolates from CF patients showed several atypical OMPs, perhaps as a consequence of antibiotic therapy or related to the serum sensitivity of mucoid P. aeruginosa. Their expression in vivo was confirmed by detecting antibodies to them in patients' serum.

Comparison of crossed immunoelectrophoresis, enzyme-linked immunosorbent assays, and tube agglutination for serodiagnosis of Yersinia enterocolitica serotype O:3 infection.

Antibodies against Yersinia enterocolitica serotype O:3 were measured by crossed immunoelectrophoresis (XIE) using whole-cell sonic extract as antigen and by enzyme-linked immunosorbent assays (ELISAs) using either purified lipopolysaccharide or whole formalinized cells expressing virulence plasmid-encoded surface antigens (pYV+ cells). The results were compared with those obtained with the standard tube agglutination method. Sera from three groups of people were examined by using these assays. The first group consisted of healthy blood donors, the second consisted of patients with recent infection due to microorganisms other than Y. enterocolitica O:3, and the third consisted of patients with recent Y. enterocolitica O:3 infection. Sera from the last group were also obtained at regular intervals for 12 months postinfection. Results obtained with XIE and the ELISAs were in good agreement with those obtained with tube agglutination. Variation, diagnostic sensitivity, and diagnostic specificity were satisfactory for all the assays studied. However, the lipopolysaccharide ELISA was less laborious than tube agglutination and XIE and carried a somewhat greater diagnostic specificity than the pYV+ ELISA. XIE and the pYV+ ELISA, on the other hand, also had advantages. XIE enabled simultaneous examination of the individual antibody response against a wide range of chromosome-encoded antigens, and the pYV+ ELISA enabled detection of specific pYV antibodies when sera were adsorbed with formalinized pYV-cured Y. enterocolitica O:3 cells prior to the assay.

Rapid emergence of resistance in Pseudomonas aeruginosa in cystic fibrosis patients due to in-vivo selection of stable partially derepressed beta-lactamase producing strains.

The development of significant mechanisms of resistance to beta-lactam antibiotics in Pseudomonas aeruginosa in cystic fibrosis (CF) patients have been studied in ten CF patients during a two week course of anti-pseudomonal beta-lactam antibiotic therapy. Sputum samples were collected on days 1, 7 and 15. Entire homogenized sputum samples were examined directly for the number of bacteria resistant to different levels of antibiotics. This allowed the detection of pre-existing resistant subpopulations of bacteria as well as following the changes in beta-lactam antibiotic susceptibility during treatment. P. aeruginosa isolates were characterized by means of sero-grouping, phage- and pyocin-typing. Outer membrane proteins of paired sensitive and resistant strains were characterized. Sonicated extracts of cells were assayed for basal and induced beta-lactamase activity. Beta-lactamase activity was further characterized by isoelectric focusing and inhibition profiles. Our observations were in accordance with the hypothesis that the sensitive inducible population was overrun by the pre-existing resistant subpopulation, during treatment. The resistant in-vivo selected P. aeruginosa population exhibited stable partially derepression but the beta-lactamase inhibitor tazobactam restored beta-lactam antibiotic activity.

Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres.

Alginate-producing, mucoid P. aeruginosa is frequently found in the lungs of patients with cystic fibrosis (CF), where it causes a chronic infection. The importance of alginate in the pathogenesis was demonstrated by the ability to establish chronic P. aeruginosa lung infection in rats if P. aeruginosa entrapped in minute alginate-beads were inoculated transtracheally. Alginate beads containing P. aeruginosa were formed by nebulizing a suspension of seaweed sodium-alginate and P. aeruginosa into a calcium solution. The alginate bead method of establishing infection was compared to an agar-bead method and proved to be quantitatively similar after 4 weeks. The ability of the two methods to induce formation of precipitins, IgA and IgG antibodies against P. aeruginosa antigens, including outer membrane proteins, flagella, exoenzymes and alginate, was assessed by crossed immunoelectrophoresis, enzyme-linked immunosorbent assay and immunoblotting. The two methods of inducing infection were comparable and infected rats had significantly higher antibody response than rats inoculated with sterile beads. We suggest that the alginate bead model closely resembles the later stages of CF-lung infection and that it offers the theoretical advantage of using a substance which is chemically similar to the alginate produced in vivo by P. aeruginosa.

Immunology of Pseudomonas aeruginosa infection in cystic fibrosis.

Adherence of P. aeruginosa to the lining of the respiratory tract is probably mediated by interaction of pili or alginate with lactosyl and sialosyl residues on the respiratory cells. The toxins produced by P. aeruginosa (for example, elastase and alkaline protease) may play a key role during the initial persistent colonization as they interfere with important defense mechanisms. Neutralizing antibodies are eventually produced, and a poor prognosis is correlated to a pronounced antibody response. The bacteria are protected against the host's defense mechanisms by production of alginate which encapsulates microcolonies of P. aeruginosa in the lungs. During the chronic infection, toxins of P. aeruginosa probably play little if any direct pathogenic role, however, immune complexes seem to be a major trigger of chronic inflammation in the lungs. Proteolytic enzymes and oxygen radicals released from the abundance of neutrophils in the lungs are probably responsible for most of the tissue damage. The individual course of the chronic infection may be explained by regulatory mechanisms, such as cleavage of immune complexes by neutrophil elastase, and by the balance between the different IgG subclass-specific antibody responses.

Determination of the components of immune complexes made in vitro with antigens derived from Pseudomonas aeruginosa.

A method for identification of the components of immune complexes would increase our understanding of the pathogenesis of chronic diseases such as Pseudomonas aeruginosa lung infection in cystic fibrosis. Capillary tube, gel diffusion and turbidimetric methods of determining immune complex formation were investigated with antigens from P. aeruginosa and the homologous rabbit antisera. Visible complexes were formed in the first two methods with flagella antigens. Purified lipopolysaccharide from P. aeruginosa would not form visible precipitates and a rapid and economical turbidimetric method was developed with 96-well microtiter plates. Larger quantities of immune complexes were formed in vitro with antigen/antibody ratios determined by the above methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting (Western blotting) were evaluated and found to be useful in determining the antigen and antibody components of these immune complexes.

Antilipopolysaccharide antibodies and differential diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Chronic lung infection in cystic fibrosis is characteristically associated with polyagglutinable, serum-sensitive, mucoid strains of Pseudomonas aeruginosa. Enzyme-linked immunosorbent assay (ELISA) methods for standard-free quantitation of immunoglobulin G (IgG) and IgM antibodies to P. aeruginosa lipopolysaccharides (LPSs) have been developed. We now report the development of assays for quantitation of monomer and dimer total IgA and IgA anti-LPS antibodies. Use of these methods in diagnosis of early chronic P. aeruginosa lung infection was assessed. IgG and IgA anti-LPS levels increased significantly at the onset of chronic infection and continued to increase to very high levels in the later stages of infection. IgM anti-LPS levels also rose at the onset of chronic infection but did not increase further. The function of true- and false-positive rates was illustrated by using various concentrations of IgG, IgA, and IgM anti-LPS for discrimination of patients. Values that gave optimum separations were used for statistical evaluation of the diagnostic sensitivities and specificities of anti-LPS antibody concentrations. The results obtained in these assays were compared with a diagnosis, based on the number of precipitins in crossed immunoelectrophoresis, of serum samples from cystic fibrosis patients. In 64 paired serum samples taken before and immediately after the onset of chronic infection, as defined by crossed immunoelectrophoresis precipitins, the predictive values of a positive ELISA were 86% for IgG and 89% for IgA. The predictive values for a negative ELISA were 98% for IgG and 97% for IgA. Results of the IgM anti-LPS ELISA had a lower predictive value. Immunoblotting and absorption studies showed that IgG anti-LPS antibodies were directed specifically against LPS of P. aeuruginosa. ELISAs were developed to determine the specific IgG sublclasses involved. The increase in IgG anti-LPS involved all four subclasses. Highest anti-LPS titers were seen with IgG1 and IgG4, but the largest relative increases were seen with IgG2 and IgG3.

Purification, characterization, and immunological cross-reactivity of alginates produced by mucoid Pseudomonas aeruginosa from patients with cystic fibrosis.

Alginates from nine mucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis were purified by repeated ethanol precipitation, nuclease digestion, anion-exchange chromatography, dialysis, and lyophilization. Uronic acid constituted 72% of the dry weight when mannuronolactone was used as the internal standard in the carbazole-borate assay for uronic acids. The average degree of acetylation was 16%, and the ratio of mannuronic acid to gluluronic acid was 4.7. No homopolymeric blocks of guluronic acid were found when analyzed by nuclear magnetic resonance spectroscopy. Contaminating proteins were denatured by heating, and during purification the content of protein relative to alginate fell from 566 to 0.9%. The content of lipopolysaccharide was 0.012%. No immunological or biological activity was attributable to the protein or lipopolysaccharide content as estimated by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and a neutrophil chemotaxis assay. Rabbits were hyperimmunized with P. aeruginosa alginates and alginate from the seaweed Laminaria hyperborea, and an ELISA that detected alginate-specific antibodies was developed. Antibodies to P. aeruginosa alginate were detected by ELISA in 1:4,000 dilutions of serum from patients with cystic fibrosis with chronic P. aeruginosa lung infection. The serological cross-reactions between serum from the nine patients with cystic fibrosis and the corresponding P. aeruginosa alginates were investigated and showed considerable heterogeneity. This finding indicates that P. aeruginosa alginate from more than one P. aeruginosa strain should be used in serological tests. There was no serological cross-reactivity between P. aeruginosa and Laminaria hyperborea alginate in either rabbits or patients with cystic fibrosis.

Immunochemical and biological reactivity of human anti-lipopolysaccharide IgG obtained by screening of blood donors.

An anti-lipopolysaccharide (LPS) preparation for the intravenous treatment of septic endotoxic shock was prepared by purifying immunoglobulin G (IgG) from pooled serum from Danish blood donors. The sera were selected by the use of an enzyme-linked immunosorbent assay (ELISA) to screen blood donors for high concentrations of antibodies to a mixture of LPS from 11 different Gram-negative bacteria. ELISA was also used for indirect quantification of IgG antibodies to lipid A, and to rough LPS from Escherichia coli Ra and Salmonella minnesota R60 (Ra). The concentration of human antibodies to the LPS mixture correlated with the concentration of antibodies to the E. coli and S. minnesota rough LPS and to lipid A. The specificity of sera with high concentrations of anti-LPS IgG was investigated by immunoblotting. Sera from individual donors reacted with LPS from different bacteria and recognized different sites on the LPS molecules. The range of specificities to different LPS was increased by the pooling of selected sera. The IgG fraction from the high titre donor pool neutralized biological activities of LPS such as activation of the Limulus amoebocyte lysate reaction and induction of tumour necrosis factor secretion from human monocytes.

Outer membrane proteins of polymyxin resistant Pseudomonas aeruginosa: effect of magnesium depletion.

Pseudomonas aeruginosa strain PA01, which is normally sensitive to 10 units/ml of polymyxin, was adapted in eight successive steps to be resistant to polymyxin at 6000 units/ml. The polymyxin-resistant variant was very sensitive to all other antibiotics with which it was challenged, including hydrophobic antibiotics such as erythromycin. This increased sensitivity implies that the acquired polymyxin resistance had disrupted the permeability barrier of the outer membrane. Changes in the outer membrane protein profile of PA01 were studied at each adaption step using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At the third step (resistant to 80 units/ml) protein H1 was absent. At succeeding steps up to 6000 units/ml of polymyxin, protein H1 was still absent and the levels of OM proteins D, E, F (major porin) and G were all considerably reduced compared with the wild type. Overproduction of protein H1 by wild type P. aeruginosa under conditions of magnesium depletion has been proposed as a mechanism to explain polymyxin resistance. However, protein H1 remained absent from the outer membrane of the polymyxin-resistant variants even under magnesium depletion. Furthermore, protein H1 could not be induced by magnesium depletion when the resistant variant was repeatedly grown in concentrations of polymyxin as low as 10 units/ml yet, at this low concentration, the variant retained its resistance to 6000 units/ml. These observations make it unlikely that induction of protein H1 alone is a mechanism of polymyxin resistance in P. aeruginosa.

Use of immunoblot detection of serum antibodies in the diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Antibodies to Pseudomonas aeruginosa were investigated in serum from cystic fibrosis (CF) patients by immunoblotting (Western blotting). The results were compared with determinations of precipitating antibodies in serum by crossed immune electrophoresis (CIE). The number of CIE precipitins is a sensitive and specific indication of infection and is used, with sputum bacteriology, to distinguish between colonisation and invasive lung infection. Immunoblotting was considerably more sensitive than CIE for detecting antibodies to P. aeruginosa. Paired serum samples from 64 CF patients, taken before a diagnosis of P. aeruginosa lung infection and immediately afterwards, showed a marked increase in the number of serum antibodies with the onset of infection. The intensity of the reaction, as shown by the density of blotted bands, was also increased. Laser scanning densitometry of immunoblots, and of photographic negatives taken from them, was used to quantify the increases. Differences in the number and intensity of blotted bands were highly significant between the two groups. The reproducibility of the method was good. An immunoblot assay may be a sensitive and useful method for routine diagnosis of early P. aeruginosa lung infection in CF.

Longitudinal study of antibody response to lipopolysaccharides during chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Antibodies to Pseudomonas aeruginosa from 10 cystic fibrosis patients with chronic P. aeruginosa lung infections were quantitatively and qualitatively analyzed. The development of specific antibodies in patient serum was evaluated in a longitudinal study (1972 to 1987). The concentrations and specificities of immunoglobulin G (IgG) and IgM antibodies to purified lipopolysaccharides (LPS) from clinical isolates of P. aeruginosa and to a variety of other gram-negative bacteria were studied by immunoblotting and enzyme-linked immunosorbent assay techniques. Results were compared with the number of immunoprecipitates to P. aeruginosa whole-cell extracts detected by crossed immunoelectrophoresis. IgG, but not IgM, anti-Pseudomonas LPS concentrations increased significantly at the onset of chronic infection and continued to increase during the course of the infection. There was a good positive correlation between the concentration of IgG anti-Pseudomonas LPS antibodies and the number of crossed-immunoelectrophoresis precipitins. The increases in IgG anti-LPS antibody concentrations were much higher to Pseudomonas LPS than to other LPSs. Binding studies demonstrated an increase in binding of IgG anti-Pseudomonas LPS during infection, whereas the binding of other anti-LPS antibodies decreased. Immunoblotting studies confirmed that antibodies reacted strongly with Pseudomonas LPS and weakly with Escherichia coli core-lipid A. The specificity of the reaction with Pseudomonas LPS increased with the duration of infection. It is concluded that anti-LPS response in cystic fibrosis patients during chronic P. aeruginosa infection demonstrates a marked increase in IgG anti-Pseudomonas LPS antibody concentration, specificity, and affinity. The anti-LPS enzyme-linked immunosorbent assay is proposed as a routine test to diagnose and to follow the course of chronic P. aeruginosa lung infection in patients with cystic fibrosis.

Comparative immunochemistry of lipopolysaccharides from typable and polyagglutinable Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis.

Lipopolysaccharide (LPS) was extracted and purified from three Pseudomonas aeruginosa strains isolated from the infected lungs of patients with cystic fibrosis. Two of the strains could be typed by O-specific antibody (O:3 and O:9), and the third was polyagglutinable (O:3/9). The separated LPS was characterized by chemical and serological methods. The main neutral sugar constituents (glucose, rhamnose, and heptose) were found in various proportions in the three strains, whereas the amounts of glucosamine, galactosamine, ketodeoxyoctonate, and phosphate were more constant. Ester-bound C12, C16, 3-OH-C10, and 2-OH-C12, together with amide-bound 3-OH-C12, fatty acids were present in equimolar proportions in all three strains. Considerable amounts of LPS were liberated in the culture supernatant of the O:3 bacteria but not in those from the other two strains. This free LPS was shown to be immunologically identical to the cell-bound LPS and the extracted LPS. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, O:3 and O:9 LPS showed a ladder pattern characteristic of smooth LPS, while O:3/9 LPS appeared rough. Rabbit antisera used for O-typing were found by enzyme-linked immunosorbent assay to contain anti-LPS antibodies that reacted strongly with homologous LPS, moderately with O:3/9 LPS, and slightly with heterologous LPS. Immunoblotting showed that common antigenic determinants in the core-lipid A part were involved in the observed cross-reaction. The polyagglutinability of P. aeruginosa may be explained by the antibodies to these common determinants that arose from the partial absence of O polysaccharides.