Diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by enzyme-linked immunosorbent assay.

An easily applicable test for diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by enzyme-linked immunosorbent assay (ELISA) for determination of serum immunoglobulin G to P. aeruginosa was developed. Soluble antigens obtained by ultrasonication of P. aeruginosa, serotypes O:1 to O:17, were used as antigens immobilized to polystyrene microtiter plates. The intraplate, plate-to-plate, and day-to-day variations were 14, 19, and 20%, respectively. Plates coated with the antigens could be stored for at least 64 days at +4 and +22 degrees C without any significant change in activity. Normal values were determined in sera from 164 controls (100 children and 64 adults). The sensitivity and specificity of the ELISA was determined by using serum samples from 243 cystic fibrosis patients and were compared to results with crossed immunoelectrophoresis (CIE). The ELISA could diagnose chronic P. aeruginosa infection with a diagnostic sensitivity of 93% and specificity of 92%. The sensitivity and specificity for the diagnosis of the early stages of chronic P. aeruginosa infection by a single sample were 90 and 100%, respectively, and by using an increased antibody response in paired samples, the sensitivity was 93% and specificity was 87%. There was a statistically significant correlation between antibody levels obtained by ELISA and those obtained by CIE. The sensitivity and specificity of the ELISA were equal to those of CIE, and because of its simplicity, the ELISA is recommended as a routine test in patients with 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 aemginosa 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.     

Mucosal immunity to Pseudomonas aeruginosa alginate in cystic fibrosis.

Patients with cystic fibrosis commonly acquire chronic pulmonary infection with alginate-producing Pseudomonas aeruginosa. The infection remains localized at the mucosal surfaces of the airways. Using enzyme-linked immunosorbent assays immunoglobulin concentrations and titers of specific antibodies to purified P. aeruginosa alginate and to P. aeruginosa sonicated antigens were measured in tears, saliva, sputum and serum. CF patients had significantly higher concentrations of IgG, IgA and SIgA in serum and saliva than controls. They also had significantly higher levels of specific antibodies to alginate and sonicated antigen in secretions and serum. Local production of IgA, IgG and IgM antibodies to P. aeruginosa was demonstrated. Only a minor proportion of specific IgA antibodies were present as secretory IgA in tears, saliva and sputum. The ratio of alginate-specific SIgA to specific monomeric IgA in sputum was significantly lower than the similar ratio in saliva, whereas the same ratio for specific P. aeruginosa sonicate antigens was found in saliva and sputum.     

Detection of antibodies to alphaviruses by enzyme-linked immunosorbent assay.

Cell culture-derived antigens detected antibodies to alphaviruses in human sera with the enzyme-linked immunosorbent assay technique. Results correlated with those from hemagglutination inhibition and neutralization tests.     

Pseudomonas aeruginosa flagellar antibodies in patients with cystic fibrosis.

An enzyme-linked immunosorbent assay specific for flagellum type (a or b) of Pseudomonas aeruginosa was used to detect serum immunoglobulin antibodies in 98 random outpatients and 14 colonized cystic fibrosis patients. Antibodies were detected to both types of flagella in addition to M-2 lipopolysaccharide. Titers to both flagellar antigens (FlAg) were 10 to 100 times higher in cystic fibrosis patients than in random outpatients of a comparable age group. Mean antibody titers against b-type FlAg were 454 for outpatients (ages newborn to 21 years), whereas the mean titer for cystic fibrosis patients (ages 6 to 21 years) was 51,520. Titers against a-type FlAg were generally lower, with mean outpatient titers of 68 and mean cystic fibrosis patient titers of 34,323. Differences were also seen in antibody titer against M-2 lipopolysaccharide, but these differences did not correspond to M-2 FlAg titers. In 98 random outpatients (ages newborn to 86 years), FlAg titers generally increased with age. To demonstrate further specificity of the enzyme-linked immunosorbent assay for flagellum antibody, Western blots were performed with selected high-titer cystic fibrosis patient sera. Sera that had a high titer (greater than 25,600) for b- or a-type FlAg showed a corresponding reactive band. These results demonstrate that flagellum antibodies are produced in humans in response to P. aeruginosa infection.     

Detection of antibodies to Mycoplasma pulmonis by an enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay, which entails the use of antigen-coated tubes and enzyme-labeled anti-immunoglobulins, was applied for the detection of antibodies against Mycoplasma pulmonis in mice. A lysate of M. pulmonis was used as the antigen, and anti-mycoplasmal antibodies of the different immunoglobulin classes were detected by class-specific anti-immunoglobulin labeled with alkaline phosphatase. The optimal conditions for the test were determined, the specificity was evaluated, and the assay was compared with other procedures for detection of mycoplasmal infection. The enzyme-linked immunosorbent assay was found to be a specific, highly sensitive, and reliable procedure for detecting anti-mycoplasmal antibodies in mice.     

Detection by enzyme-linked immunosorbent assays of antibody specific for Pseudomonas proteases and exotoxin A in sera from cystic fibrosis patients.

Enzyme-linked immunosorbent assays were developed to measure serum antibody specific for Pseudomonas elastase, alkaline protease, and exotoxin A. Antibody responses to each Pseudomonas antigen were measured in cystic fibrosis (CF) patients who were not colonized with Pseudomonas aeruginosa, in those who were colonized, in those who were chronically infected with this organism, and in control subjects. Antibody levels for each antigen in the colonized and infected CF patients were higher than levels in uncolonized CF patients or non-CF control subjects. The antibody responses to elastase were similar in patients of the colonized and infected groups. However, infected CF patients had significantly elevated levels of antibody to exotoxin A (P less than 0.01) and alkaline protease (P less than 0.05) when compared with patients simply colonized with P. aeruginosa. These findings confirm that Pseudomonas alkaline protease, elastase, and exotoxin A are produced by Pseudomonas strains which colonize and infect CF patients. As an adjunct to established procedures (X-ray, microbiological culture, etc.), the antitoxin and anti-protease enzyme-linked immunosorbent assays may be clinically useful tests for differentiating colonized CF patients from those who have more severe Pseudomonas pulmonary infections.     

Detection of immunoglobulin G to Pasteurella haemolytica capsular polysaccharide by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of immunoglobulin G (IgG) to the capsular polysaccharide (CP) of Pasteurella haemolytica serotype 1. Purified CP was first covalently coupled to poly-L-lysine and then optimally adsorbed at a concentration of 5 micrograms/ml to microtiter plates in the presence of carbonate-bicarbonate buffer (pH 9.8). The ELISA was used to evaluate and compare the CP-specific IgG response of calves vaccinated with different P. haemolytica-derived experimental vaccines. Elevated levels of ELISA IgG titers were detected in postvaccination sera and lung lavage from calves vaccinated intradermally with live logarithmic-phase organisms or the culture supernatants. The ELISA was found to be a rapid, reproducible, and sensitive technique for the detection of CP-specific antibodies and may be useful to delineate the protective role of these antibodies in bovine pneumonic pasteurellosis.     

Development of enzyme linked immunosorbent assay (ELISA) to detect antibodies to Pseudomonas aeruginosa cell surface antigens in sera of patients with cystic fibrosis.

An enzyme linked immunosorbent assay (ELISA) to measure free serum IgG antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis was developed. Seven strains of P aeruginosa cells, treated with glutaraldehyde and representing the most commonly isolated serotypes in our cystic fibrosis unit, were used. The specificity of the test was confirmed by the absence of cross reacting antibodies to other Gram negative bacteria. The results showed differences in the titres of antibodies at different stages of P aeruginosa infection. Because of its reproducibility, specificity, and sensitivity these preliminary results suggest that this test may be of value in monitoring the progress of P aeruginosa infection in patients with cystic fibrosis.     

Detection and quantitation of antibodies against Rhizopus by enzyme-linked immunosorbent assay.

In sawmills high concentrations of spores from the mould Rhizopus microsporus may occur, causing allergic alveolitis in exposed workers. Both symptomatic and asymptomatic exposed workers may develop antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Rhizopus has been developed in order to study the relationship between antibody levels and exposure levels. The precision of the measurements of Rhizopus antibodies by ELISA carried out on the same microtiter plate was estimated to be 11%. It is therefore possible to detect changes in antibody levels of approximately 25% or more. Antibodies were studied longitudinally by ELISA in 60 wood trimmers. The observed changes in antibody levels exceeded the precision of the ELISA method substantially, indicating significant variability in antibody levels in wood trimmers. The ELISA test was compared with the double immunodiffusion test (DID). Sera from 67 wood trimmers were analyzed by both methods. Antibodies were detected by ELISA in 70% and by DID in 28% of the workers in this group, clearly demonstrating that the ELISA test is the most sensitive method for the detection of antibodies to Rhizopus.     

Detection of rotavirus-specific IgG antibodies by immunoperoxidase assay and enzyme-linked immunosorbent assay

An indirect immunoperoxidase assay (IPA) has been developed for determination of IgG antibodies to rotavirus. The technique employed as antigen, SA-11 infected MA 104 cells, which were air-dried on glass slides and acetone-fixed. In parallel, rota-specific IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA). Specific IgG antibodies to rotavirus were determined in sera of healthy children and in sera of patients suffering from gastroenteritis. A good correlation (r = 0.92) and (r = 0.98) for healthy children and patients, respectively, was found between IPA and ELISA techniques. The IPA technique is rapid and simple and positive results, because of the intensive staining, are easily read by low-power light microscope. The potential application of IPA and ELISA methods in serodiagnosis of rotavirus infections is discussed.     

Detection of influenza virus neuraminidase-specific antibodies by an enzyme-linked immunosorbent assay.

We analyzed sera from 28 patients with various types of malignancies for the occurrence of antibodies against exotoxin A of Pseudomonas aeruginosa and two Pseudomonas proteases. A total of 27 of these individuals were colonized or infected with P. aeruginosa at one time or another during the study, whereas the remaining patient was colonized with four non-P. aeruginosa species of Pseudomonas. Sera were obtained from several of these patients before P. aeruginosa colonization or infection of these individuals was detected, which provided an opportunity to evaluate their responsiveness to pseudomonal exoproducts as they acquired the organism. Exotoxin A was purified from culture supernatant fluids of strain PA-103, and the two proteases were purified from an isolate of strain JR3, a highly proteolytic strain originally recovered from the sputum of a cystic fibrosis patient. Antibodies to the exotoxin A and the two proteases were detected in these sera, and sera which contained relatively high antibody levels to exotoxin A afforded mice complete protection against lethal challenges with this substance. Statistical analyses showed that patients infected with P. aeruginosa had consistently higher antibody levels (P less than 0.005) to the exoproducts than patients who were colonized with this organism. Also, patients colonized with P. aeruginosa possessed significantly higher antibody levels (P less than 0.003) to these three exoproducts than uninfected, hospitalized patients. Parke-Davis type 1 was the strain most commonly isolated from these patients (46%), but colonization or infection due to this organism usually resulted in the production of low levels of antibody to Pseudomonas exoproducts. However, infections with Parke-David type 7 organisms were always associated with intermediate- and high-responder sera to exotoxin A. These results indicated that potentially toxic products were elaborated during the course of cancer-related colonization and infection with P. aeruginosa.     

Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response

Alginate, a viscous polysaccharide from mucoid Pseudomonas aeruginosa, may interfere with the host defenses in patients with cystic fibrosis and chronic P. aeruginosa lung infection. The alginate concentration in the sol phase of expectorated sputum was quantitated by a biochemical method and a newly developed enzyme-linked immunosorbent assay. There was a high degree of correlation between the methods, and the concentration of alginate ranged from 4 to 101 micrograms/ml with a median of 35.5 micrograms/ml when measured by enzyme-linked immunosorbent assay. Alginate could not be detected in the bronchial secretions from patients without P. aeruginosa infection. In vitro investigation of alginate did not show any activation of the alternative pathway of complement, as determined by a hemolytic kinetic assay and by testing for neutrophil chemotaxis. At a high concentration, P. aeruginosa alginate caused a slight activation of the classical pathway of complement. Alginate did not cause neutrophil chemotaxis by itself but was able to reduce the neutrophil chemotactic response to N-formylmethionylleucylphenylalanine and for zymosan-activated serum. P. aeruginosa and seaweed alginates were able to prime neutrophils for increased N-formylmethionylleucylphenylalanine-induced neutrophil oxidative burst, as determined by chemiluminescence. Because of its ability to prevent attraction of neutrophils to the site of infection, lack of complement activation, and ability to enhance neutrophil oxidative burst, alginate from P. aeruginosa may contribute to the persistence and pathogenesis of chronic P. aeruginosa infection in cystic fibrosis.     

Detection of antibodies to influenza virus M protein by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay test system was developed in which purified influenza virus M protein was used for the detection of M antibody in human sera. Antibody levels to influenza A virus M protein were monitored in sera from a vaccine study population by using an enzyme-linked immunosorbent assay technique with purified M protein as the adsorbent antigen. A 10-fold variation in titers of preexisting M antibody was observed in this population of young adults. Increases of anti-M titer of 7- to 24-fold were observed upon immunization with Formalin-inactivated vaccine or after natural infection. The antibody response to M protein was dissociated from the response to the hemagglutinin or neuraminidase antigens. The M antibody response preceded or was coincident with the antibody response to H1 hemagglutinin upon natural exposure to circulating virus.     

Detection of immunoglobulin M antibodies to hepatitis A virus by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay for the detection of immunoglobulin M (IgM) antibodies to hepatitis A virus is described. The test uses the principle of binding of IgM antibodies to anti-IgM-coated microtiter plates to determine whether the IgM antibodies attached have specificities for hepatitis A virus. In three patients with hepatitis type A followed up to 12 months, IgM antibodies to hepatitis A virus could be demonstrated from the onset of illness and during the following 2 to 3 months. When acute-phase sera from 48 patients with acute hepatitis were tested, IgM antibodies to hepatitis A virus could only be demonstrated in 18 patients previously classified as type A, whereas 30 patients with type B and non-A non-B hepatitis were negative. IgM antibodies to hepatitis A virus could not be demonstrated in 108 normal sera nor in 55 sera containing rheumatoid factor. These results indicate that the enzyme-linked immunosorbent assay for IgM antibodies to hepatitis A virus is useful in the serodiagnosis of acute hepatitis type A on a single serum sample taken during the acute phase of illness.     

Nosocomial acquisition of Pseudomonas aeruginosa by cystic fibrosis patients.

During a 4-year period, at least 12 of 40 patients with cystic fibrosis (CF) who were newly colonized with Pseudomonas aeruginosa had acquired it at CF recreation camps, clinics, or rehabilitation centers. After introduction of hygienic precautions at the CF clinic, only a single episode of nosocomial transmission of P. aeruginosa was detected at the CF ward during the subsequent 2 years.     

Detection of adenovirus by enzyme-linked immunosorbent assay.

A solid-phase direct enzyme-linked immunosorbent assay (ELISA) was developed for the detection of adenovirus antigen in extracts of infected cells by using antihexon serum. Results with simulated clinical specimens consisting of normal nasal wash specimens seeded with varying concentrations of adenovirus type 5 showed that antigen could be detected in extracts of HEp-2 cell cultures inoculated with 10(2.5) 50% tissue culture infective doses (TCID50) and 10(1.5) TCID50 after 2 and 4 days of incubation, respectively. Fifty-three clinical nasal wash specimens containing adenovirus type 5 (stored for 5 years at -70 degrees C) were used to evaluate antigen detection by ELISA in HEp-2 cell extracts and by manifestation of cytopathic effect in human embryonic kidney cells. After 2 days of incubation, 62% were positive by ELISA, whereas none was positive for cytopathic effect. After 4 days of incubation, 76% were ELISA positive and 47% were positive for cytopathic effect. The results according to infectivity titers indicated that clinical specimens containing 10(3.0) TCID50 or greater were all positive by ELISA after 2 days of incubation in HEp-2 cells, and by 4 days all but one specimen containing 10(2.0) TCID50 or greater were ELISA positive. ELISA and immunofluorescent methods for antigen detection were compared using 24 of the 53 clinical specimens containing adenovirus type 5. Nearly equivalent sensitivities were demonstrated. These results suggest that ELISA may provide an alternative method of detecting and identifying adenoviral infections in humans.     

Prospective study of serum antibodies to Pseudomonas aeruginosa exoproteins in cystic fibrosis.

Serum immunoglobulin G to four purified antigens from Pseudomonas aeruginosa, phospholipase C, alkaline protease, exotoxin A, and elastase, were determined in 62 patients with cystic fibrosis by enzyme-linked immunosorbent assay. The patients were followed for 12 to 24 months in a prospective study. Increased titers, i.e., titers more than 2 standard deviations above those of normal controls, were exclusively found in patients chronically colonized with P. aeruginosa and not in patients harboring only Staphylococcus aureus. The frequencies of elevated titers of antibody to the different antigens varied from 100% (phospholipase C) to 58% (alkaline protease and exotoxin A) to 15% (elastase) in the chronically colonized patients. Mean serum titer levels, expressed as multiples of the age-correlated upper normal limit (=1), were significantly higher to phospholipase C in patients with dual colonization with P. aeruginosa and S. aureus than in those colonized only with P. aeruginosa (P less than 0.001). Conversely, the other three antigens showed significantly higher serum antibody titer levels in patients harboring only P. aeruginosa (P less than 0.001). In five patients who became colonized with P. aeruginosa during the study period, serum antibodies to phospholipase C and exotoxin A increased first. Exceptions to the general pattern of antibody responses were found in three patients chronically colonized with Escherichia coli. They showed a delayed enhancement of anti-phospholipase C titers after the chronic P. aeruginosa colonization. Serum titers were not influenced by exacerbations of pulmonary infection or by antimicrobial therapy. The determination of titers of serum antibody to phospholipase C seems to be a valuable indicator of a chronic colonization with P. aeruginosa. The results further suggest that bacterial metabolism and interactions may influence the antibody response.     

Detection of Clostridium botulinum type A toxin by enzyme-linked immunosorbent assay with antibodies produced in immunologically tolerant animals.

Immunological tolerance is a state of unresponsiveness to foreign substances (antigens) which can develop in human and animal species as the result of continued exposure to antigens early in life. We utilized this principle for the preparation of antibodies against Clostridium botulinum type A toxin. By selective suppression of the immunological response of rabbits to unwanted antigens and subsequent immunization with a toxoid, we were able to produce a specific type A antitoxin without the need to purify the toxin. Despite cross-reactivity with C. botulinum type B, our type A antitoxin was otherwise specific since it did not react with culture filtrates of nontoxigenic variants of type B, any other C. botulinum type (C, D, E, F, and G), nor with 18 other Clostridium species, including Clostridium sporogenes. Using this antitoxin, we developed a sensitive enzyme-linked immunosorbent assay for detection of C. botulinum type A toxin.     

Detection of antibodies to Sendai virus by enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Sendai virus, a paramyxovirus, is described. The assay was found to be about 20-fold more sensitive than the hemagglutination inhibition assay. Differentiation between virus specific IgG and IgM is possible. The test appears to be especially useful in the study of early events in antibody formation in vivo as well as in vitro.