Pseudomonas aeruginosa: Immune Status in Patients with Cystic Fibrosis

In order to have a better understanding of the clinical significance of Pseudomonas aeruginosa, circulating and secretory antibodies were measured. Of 100 patients diagnosed as having cystic fibrosis (CF) and an atypical mucoid P. aeruginosa cultured from their sputum, each possessed serum precipitins. These immunoprecipitates, however, were not detected in the sera of 40 CF patients, some of whom were chronically ill with pulmonary colonization by typically rough-smooth strains of P. aeruginosa. The sera of 46 CF patients and 27 CF patient parents not colonized by P. aeruginosa were negative for the precipitins. The sera from 15 of 45 chronically ill patients not having CF, however, but harboring P. aeruginosa, also possessed serum precipitins. The sera from 85 subjects not having CF and not clinically infected with P. aeruginosa were negative for precipitins. Serum hemagglutination titers as high as 1:4096 were measured in older CF patients having advanced pulmonary disease and who were infected with mucoid P. aeruginosa. Salivary titers ranged from 1:8 to 1:64. Increased levels of both circulating and secretory antibodies of the immunoglobulin A and G classes were demonstrated in patients with CF. Once a patient with CF becomes colonized with P. aeruginosa a process of conversion from the rough and smooth forms to the mucoid form is almost inevitable. Although the mucoid form predominates in the sputum, intermediates of the various colony types are often present. Serum precipitins were demonstrable only after the appearance of mucoid strains in the sputum of patients with CF. Although antibiotics tend to reduce the number of mucoid microorganisms, they are rarely, if ever, eradicated from these patients'' lungs. Recurrent episodes of servere pulmonary infection and the evidence of increasing antibody formation to mucoid strains indicates the invasiveness of these particular strains.     

Rapid enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against

A rapid ELISA was developed for the detection of specific IgG against Micropolyspora faeni and Thermoactinomyces vulgaris and compared with counterimmunoelectrophoresis (CIE) in twenty-seven patients with suspected farmer's lung disease (FLD). Seventeen patients had precipitins to M. faeni or T. vulgaris or both, and ten had no precipitins. The optimum conditions for each step in the ELISA were determined: pre-equilibration of reagents at 37⁰C and vigorous agitation during incubation enabled the total test time required for the procedure to he reduced to 4 hr. A serum dilution of 10-fold produced good differentiation between CIE-positive and -negative sera. Little correlation was seen between CIE and ELISA for either M. faeni or T. vulgaris antigens in tests with sera from patients with precipitins: high readings were often recorded in ELISA where no precipitins had been detected with the same antigen. Positive- negative discrimination of unknown sera in ELISA was achieved through the inclusion of CIE-positive and -negative reference sera in each test run. Thirteen CIE-positive sera were classed as positive in ELISA with the M. faeni antigen while eight of thirteen CIE-positive sera were positive in ELISA with the T. vulgaris antigen. For both antigens, four CIE-negative sera were recorded as positive in ELISA.     

Serological investigations on Indian kala-azar.

Detailed serological investigations were carried out in forty-nine active kala-azar (KA) cases in North Bihar, India. Various classes of immunoglobulin (IgG, IgA, and IgM) and third component of complement (C3) levels were determined in these sera and results were compared with those obtained in normal controls. Antibody titres were determined by the indirect haemagglutination (IHA) method using soluble Leishmania antigen. Immunoglobulin G and M class-specific antibody titres were also determined separately by the enzyme-linked immunosorbent assay (ELISA) method. Polyclonal hypergammaglobulinaemia with marked increase in serum IgG (and to a lesser extent in IgM) level was a rather common feature in the majority of these sera. Much of this immunoglobulin increase, however, appeared to be non-specific in nature and no absolute correlation could be noted between serum IgG or IgM levels and corresponding IgG or IgM antibody titres. Significant decrease in serum C3 level was observed in KA and this decrease was found to be independent of immunoglobulin levels or specific antibody titres. A fairly good correlation between aldehyde test and serum IgG level was evident from this study. Aldehyde-positive KA sera usually gave higher antibody titres than aldehyde-negative ones. Anti-leishmanial antibodies belonged mostly to IgG class although some IgM antibodies were also demonstrable. The latter class of antibodies probably appeared early in KA infection although their serological specificity was poorer to IgG antibodies. Out of forty-nine KA sera examined in this study thirty-six (73.5%) gave positive IHA titres while forty-six (93.9%) were positive by IgG-ELISA which appeared to be a highly specific and sensitive serodiagnostic method particularly for the early detection of KA cases.     

Serological tests in the diagnosis of pulmonary aspergillosis

Sera from 44 patients with clinically suspected pulmonary aspergillosis (mainly of an allergic type) were examined for antibodies to Aspergillus fumigatus using an enzyme-linked immunosorbent assay (ELISA) and counter-immunoelectrophoresis (CIE). Of the sera, 15 were considered to contain Aspergillus antibodies using ELISA; 11 of these also contained precipitins by CIE. In no instance was a CIE-positive serum negative by ELISA. The serum of 1 patient with autopsy-verified invasive pulmonary aspergillosis was negative in both tests. Protein-enriched antigens derived by ammonium sulphate precipitation of crude hyphal homogenates seemed of most use in ELISAs. In addition, 4 of 8 sera obtained from patients with possibly invasive candidosis also revealed significant levels of Aspergillus antibody by ELISA (but not CIE). All of the 8 sera contained readily detectable Candida precipitins by CIE. Problems of potential cross-reactivity obviously need careful consideration with ELISAs. Our results suggest that ELISA procedures under appropriately controlled conditions are more sensitive than CIE for detecting Aspergillus antibodies. However it seems that some patients with invasive aspergillosis will be antibody-negative even with sensitive tests such as ELISA.     

Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection. I. Measurement of serum antibodies by radioimmunoassay.

Serum antibody responses to the lipopolysaccharide and protein antigens of S. typhi in typhoid patients were studied using a solid-phase radioimmunoassay technique. Sera from 24 adult typhoid patients and 20 non-typhoid adult controls were compared. As a group, sera from typhoid patients showed increased IgA, IgG and IgM immunoglobulin levels and gave significantly higher anti-LPS and anti-protein antibody titres in all three major immunoglobulin classes than did non-typhoid controls. Levels of antibodies against LPS or protein in sera of typhoid patients were highly variable with a skew distribution. A good correlation was found between antibody titres to the LPS antigen and those to a protein antigen. No correlation, however, was found between the anti-LPS antibody titres measured by radioimmunoassay and the anti-O antibody titres measured by the Widal agglutination test. Titration of anti-LPS or anti-protein antibodies by radioimmunoassay was found to be more sensitive and specific than Widal test for the serological diagnosis of typhoid fever. The advantages of measuring antibody response by radioimmunoassay over conventional Widal test are discussed.     

Antibody response toShigella sonnei infection determined by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for determination of the IgA, IgM and IgG antibody responses against a lipopolysaccharide antigen representingShigella sonnei phase I bacteria. Two or more sera from 33 patients infected withShigella sonnei were collected during a 12 month period after onset of the disease. Convalescent sera from 56 patients with other enteric infections (salmonellosis, yersiniosis, campylobacteriosis) and sera drawn from 40 healthy blood donors served as controls. Twenty-eight of the 33 patients (85 %) had at least one serum specimen where two or three of the immunoglobulin titres were classified as positive (>+2SD above mean titres seen in healthy blood donors), whereas only ten of 56 patients (18%) with other enteric infections had similarly elevated titres (p<0.001). TheShigella sonnei ELISA using purified liopolysaccharide as antigen is considered more sensitive and specific than the formerly used agglutination tests.     

Serological studies of Actinomyces israelii by crossed immunoelectrophoresis: taxonomic and diagnostic applications.

Crossed immunoelectrophoresis (CIE) with intermediate gel was applied to the serological analysis of Actinomyces israelii to develop a test with high efficiency in the laboratory diagnosis of human actinomycosis and classification of A. israelii. Recently developed standard antigen-antibody systems for A. israelii by CIE were used as reference. The reference systems were based on standard preparations of cytoplasmic and whole cell-associated antigens of A. israelii and a standard immunoglobulin G pool purified from rabbit antisera to formalin-treated whole cells and cell lysates of A. israelii. The specificity of the standard antigens for A. israelii was evaluated in CIE studies by screening for antibodies to components of the antigens in rabbit antisera raised against related bacteria. The standard system for A. israelii based on cytoplasmic antigens formed species-specific precipitins whereas antisera raised against A. naeslundii and/or Propionibacterium acnes precipitated components of the other standard antigens. As a result of these analyses, the standard system for A. israelii based on 10 cytoplasmic antigens was used as reference for CIE studies to detect humoral antibodies to A. israelii in sera from nine patients with actinomycosis. All the sera from the patients formed at the time of diagnosis one or more precipitins in terms of the 10 reference precipitins. Up to five precipitins were found in single sera. Follow-up studies covering a period of one-half year after treatment showed a gradually decreased precipitin response in the course of time. In control sera from patients with newly diagnosed tuberculosis, nocardiosis, deep Candida infection, and aspergillosis, and in sera from healthy blood donors, no antibodies were detected with specificity for the reference antigens.     

Determination of rotavirus serotype-specific antibodies in sera by competitive enhanced enzyme immunoassay

A method is described for the specific detection of antibody to individual rotavirus serotypes in sera. A competitive enzyme immunoassay (EIA) was developed in which rotavirus serotype-specific monoclonal antibodies against VP7 compete with antibodies in test sera for rotavirus serotype-specific antigen bound to a solid phase. There was an excellent correlation between serotype-specific EIA results and serotype-specific neutralization titres (r = 0.915, P = less than 0.001). The value of this method for rotavirus epidemiology and vaccine trials is discussed.     

Agar-Gel Precipitating Antibody in Pseudomonas aeruginosa Infections

The human immune response to Pseudomonas aeruginosa infection was studied by using the double diffusion in agar-gel technique. Antigens from Fisher-Devlin-Gnabasik immunotypes were prepared by both trichloroacetic acid extraction and ultrasonic disruption. Serum from 72 of 168 patients (43%) from whom P. aeruginosa was isolated formed from one to eight precipitin bands. Precipitins were demonstrated in the sera of 60 of 66 (91%) patients recovering from bacteremia and deep infections; however, they were usually absent when Pseudomonas infection was fatal or when there was no clinical evidence of significant infection. Precipitating antibody was detectable at serum dilutions as high as 1:32, and appearance of single bands correlated with hemagglutinating antibody titers of ≥1:128. Antigen from sonically disrupted organisms usually resulted in stronger precipitin bands than trichloroacetic acid extracts, and antigen from the homologous infecting strain occasionally increased test sensitivity. None of 50 normal controls had Pseudomonas precipitins as was the case in patients convalescing from Escherichia coli (15 patients), Klebsiella-Enterobacter-Serratia (18), and Proteus (14) bacteremias. Measurement of agar-gel precipitins was useful and specific in evaluating the circulating antibody response to P. aeruginosa infections.     

A study of immunoglobulin M antibody to measles, canine distemper, and rinderpest viruses in sera of patients with subacute sclerosing panencephalitis.

Seven boys were studied who had the clinical features of subacute sclerosing panencephalitis (SSPE) and whose brain histology was consistent with SSPE. Measles antigen was detected in the seven brains by the direct fluorescent antibody method. Three out of the seven boys had in their sera measles specific immunoglobulin M (IgM) which was detected by the indirect fluorescent antibody method, and the cell receptors for it were acetone stable. A prozone effect was noted in the sera of two patients. The absorption of one patient's serum with Staphylococcus aureus to reduce the titre of immunoglobulin G (IgG) removed the prozone effect. Two of the boys who had high titres of measles specific IgM also had serum IgM which reacted with canine distemper virus antigen but the titres were eightfold lower. None of the boys had detectable rinderpest specific IgM in their sera.     

Counterimmunoelectrophoretic Detection of a High Incidence of Precipitin Reactions in Normal Human Sera Against Staphylococcal Teichoic Acids and Protein A

The use of counterimmunoelectrophoresis (CIE) for detection of serum antibodies to staphylococcal teichoic acids was evaluated against teichoic acids prepared by sonic treatment or lysostaphin extraction of Staphylococcus aureus (Lafferty strain). Of 54 patient sera from suspected cases of staphylococcal endocarditis, osteomyelitis, or septicemia, 33 (61.1%) were positive by CIE analysis; however, 128 of 291 sera (44.0%) from normal adult donors were also positive. Selected CIE-positive sera from patient and control groups were titered by Ouchterlony gel diffusion. In the control group of normal sera, 65% were also positive by gel diffusion, but only 15% had titers of >/=1:2. Of the patient sera, 44.4% had gel diffusion titers of >/=1:2. In addition to the specific teichoic acid band, a second precipitation band could be demonstrated with both patient or normal sera by CIE or gel diffusion. This second precipitin band was shown to involve interactions of test sera with staphylococcal protein A present in the teichoic acid extracts. The protein A precipitins were detected at high concentrations of the antigen extracts, whereas the anti-teichoic acid precipitins were optimally detected at lower antigen concentrations. The formation of protein A precipitin bands did not correlate with the presence of anti-teichoic acid antibodies, as most sera tested were positive for protein A regardless of anti-teichoic acid activity. This study suggests that a high incidence of normal people have levels of antibodies to teichoic acids which are detectable by the highly sensitive, but nonspecific, technique of CIE.     

Cytomegalovirus-specific antibody responses in renal transplant patients with primary and recurrent CMV infections

Cytomegalovirus (CMV) specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody responses were measured before and after renal transplantation in 20 patients with primary CMV infection and in 16 patients with recurrent CMV infection. In primary CMV infection IgG antibody titres to late antigen (IgG-LA) measured by indirect fluorescence (IFA) were approximately seven times higher than those obtained by the complement fixation test (CFT). In contrast, in recurrent CMV infection this difference was found to be about twofold. Virus-specific IgM antibody to late antigen (IgM-LA) was detected in 100 percent of patients with primary CMV infection and in only 50 percent of patients with recurrent CMV infection. The IgM-LA titres were highest in primary CMV infection and reached peak levels at approximately 10 weeks post transplantation, whereas in recurrent CMV infection the IgM-LA titres were lower and reached peak levels at three months post transplantation. Moreover, IgM-LA was found to persist in patients from both groups at nine months post transplantation. IgM antibody to early antigen (IgM-EA) was not detected in any patient in this study. However, significant fourfold titre rises in IgG antibody to EA (IgG-EA) were detected in 100 percent of patients with recurrent CMV infection and in 50 percent of patients with primary CMV infection. These results clearly show the difference in antibody responses to the various antigens of CMV in patients with primary and recurrent CMV infection.     

PrecipitatingPseudomonas aeruginosa antibodies and antimicrobial therapy in cystic fibrosis patients

Forty patients with cystic fibrosis were studied bacteriologically and serologically. PrecipitatingPseudomonas aeruginosa antibodies were monitored by crossed-immunoelectrophoresis (CIE) in order to evaluate the possibility of preventing chronic colonization byPseudomonas aeruginosa by cycles of antimicrobial therapy. Sputum or pharyngeal aspirate and serum samples from all patients were analyzed by means of spread on selective media and CIE, respectively. Significant differences in the number of precipitins were obtained: noncolonized and intermittently colonized patients had no precipitins, whereas the number of precipitins in the chronically colonized patients varied from 11 to 44. An increase in the number of precipitins could be a good marker for initiation of therapy with antimicrobial agents that are either active againstPseudomonas aeruginosa or able to inhibit the release of virulence factors.     

Immunological studies in ulcerative colitis. VII. Anti-colon antibodies of different immunoglobulin classes

Ten out of sixty-three sera from patients with ulcerative colitis gave positive staining of goblet cells in the indirect immunofluorescence test, applied to acetone–ethanol fixed sections of colon from germ-free rats. Nine of these sera were also positive in triple layer tests in which the second layer consisted of rabbit antibodies to human immunoglobulin chains and the third layer of fluorescein conjugated sheep anti-rabbit immunoglobulin. All sera contained anti-colon antibodies having light chains of both κ- and λ-type. Heavy chains of both γ-, μ- and α-type occurred at almost equal frequency and only three of the nine positive sera contained antibodies of a single immunoglobulin class. One out of four tested sera which contained antibody of the IgA-class also gave positive reactions for secretory-piece determinants. The staining pattern obtained with antibodies of different classes was similar. Anti-colon antibodies belonging to the IgD or IgE classes were not detected. The triple layer test was negative when the patients'' sera were replaced by normal human sera.     

The reactivity between rickettsiae and Weil-Felix test antigens against sera of rickettsial disease patients.

Of the sera which were positive to Rickettsia tsutsugamushi by indirect immunoperoxidase test, approximately 80% sera were positive to a Proteus OXK antigen by Weil-Felix test at 10 or more days after the onset of fever, while only 10% sera were positive within 9 days from the onset of fever. In ELISA using the OXK antigen, almost all of the paired sera of tsutsugamushi disease (TD) patients increased on the IgM antibody titres with the rise of their titres by Weil-Felix test, whereas the IgG antibody titres of these sera were unrelated with the titres of Weil-Felix test. We suspect that the reactivity of TD patients sera to the OXK antigen in Weil-Felix test was derived from the reactivity of the IgM antibody against the OXK antigen common with R. tsutsugamushi. The patient sera infected with a Japanese isolate of spotted fever group rickettsia (SFGR) cross-reacted with the Thai Tick Typhus (TTT) strain of SFGR by indirect immunoperoxidase test. In Weil-Felix test, the reactivity of these sera to OX2 antigen were higher than that to OX19 antigen, like the sera infected with other SFGR, except of R. rickettsii. These sera also reacted with TTT and OX2 antigens by ELISA. The titres of IgM antibody against OX2 antigen in the sera in ELISA were in parallel with the titres of the sera against OX2 antigen in Weil-Felix test, but not the titres of IgG antibody. We suggest that the reactivity of the patient sera infected with SFGR to OX2 antigen of Weil-Felix test is dependent on the IgM antibody.     

Radial counter-immunoelectrophoresis for rapid sero-diagnosis of typhoid fever

Serum samples were collected from 24 confirmed cases of typhoid fever and 23 normal healthy controls. Convalescent sera from the patients were obtained, wherever possible, one week after the first sample. In all, 13 paired sera, 11 acute phase only and 23 normal serum samples were tested for ability to elicit precipitins to Salmonella typhi by radial counter-immunoelectrophoresis using cellulose acetate membranes. In addition, conventional counter-immunoelectrophoresis (CIE) was performed using agar-gel layer for comparison. One of 24 acute phase sera gave positive results whereas all 13 convalescent sera were positive by both methods. The radial CIE test may be useful for rapid sero-diagnosis of typhoid fever as it takes only 4 min and can be used to screen large numbers of serum samples from patients suspected of typhoid fever.     

Precipitins to candida albicans in chronic mucocutaneous candidiasis studied by crossed immunoelectrophoresis with intermediate gel. Correlation with clinical and immunological findings

The precipitating antibodies in the sera of fifteen patients with chronic mucocutaneous candidiasis were examined by crossed immunoelectrophoresis with intermediate gel. The method permitted identification and quantitation of precipitins against thirty-four of the seventy-eight known antigenic components of Candida albicans.

The sera from every patient contained precipitins and the number of reactivities per serum ranged from two to thirty-nine. All patients had antibodies to antigen 78, a mannan–protein complex. Many sera also possessed antibodies to many other components of the organism, suggesting that some of the yeast cells had been disrupted in the patients' tissues. However, there were no precipitin profiles that characterized patients with specific forms of chronic candidiasis. Instead, in two cases, the antibody profiles appeared to be related to the patients' ability to develop humoral immune responses. Serial studies of patients during remissions and exacerbations showed that there were no consistent changes in antibody activities.

The role of Candida precipitins in chronic candidiasis remains uncertain. Possible functions include prevention of dissemination of the infection from superficial sites, formation of immune complexes in superficial sites and suppression of cell-mediated immunity as suggested by in vitro tests.

     

Immunoradiometric assay for cytomegalovirus-specific IgG antibodies: assay development and evaluation in blood transfusion practice

An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specificity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT), in that 53% of these sera were positive by RIST and 48% positive by CFT. There were 1303 concordant results, 88 sera positive only by RIST and 19 sera were only positive by CFT. These discrepant results remained after an attempt to exclude false positive reactivity; their significance is discussed. Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate.     

Enzyme-linked immunosorbent assay detection of immunoglobulins G and M to Venezuelan equine encephalomyelitis virus in vaccinated and naturally infected humans.

Enzyme-linked immunosorbent assays (ELISAs) were developed for detection of immunoglobulin G (IgG) and IgM antibodies to Venezuelan equine encephalomyelitis (VEE) virus in vaccinated and naturally infected humans. A total of 441 sera found negative for VEE antibodies by plaque reduction neutralization were examined by IgG ELISA and gave a 1.0% false-positive rate; no false-positives were found in the IgM ELISA. Sera with neutralizing antibody to western or eastern equine encephalomyelitis virus did not react with VEE antigen in the IgG ELISA. Sensitivity of the IgG ELISA was determined by testing 100 coded pre- and postvaccination human sera. Sixty-two were positive by ELISA; 58 of these 62 were also positive by neutralization tests, and 38 were negative by both tests. No neutralization-positive, ELISA-negative sera were found. Comparison of titers obtained by ELISA and neutralization tests indicated that 88% varied randomly by a fourfold dilution factor or less, while 61% were identical or varied only twofold. In sera obtained sequentially from 10 vaccinees and 5 naturally infected patients, both IgG and IgM antibodies appeared between 2 and 3 weeks after vaccination or onset of symptoms. The IgG and IgM antibody ELISAs described are rapid, specific, and sensitive indicators of VEE antibody status in vaccinated and naturally infected individuals.     

Evaluation of viral-lysate enzyme-linked immunosorbent assay kits for detecting human immunodeficiency virus (type 1) infections using human sera standardized by quantitative western blotting.

The first generation of proprietary reagents for detecting antibodies to the Human Immunodeficiency Virus Type 1 (HIV-1) by enzyme-linked immunosorbent assay (ELISA) used as antigen partially purified virus from cell culture lysates. These tests, which are still in use, may vary in their antibody measurement capabilities if different proportions of the viral polypeptides are present in the viral lysate mixtures. We determined the quantities of antibodies in the serum of persons infected with HIV-1 by dilution analysis using 3 ELISA kits: Abbott [A], Du Pont [D], Genetic Systems [G]. The proportionate antibody titres of each serum to p24gag and gp160env/120env were established by quantitative Western blotting. Serum antibody titres were high, frequently over 1:10,000, a result observed both by ELISA and Western blot. For Kit D, sera with high proportions of antibody to p24gag produced antibody titration curves with steep slopes whereas shallower slopes were found in sera with high proportions of antibody to gp160env. In contrast, Kit A gave steeper slopes with sera enriched for gp160env antibodies. Kit G gave results with slopes intermediate between Kits A and D. Serum antibody titres differed between kits depending upon the proportion and concentration of antibodies in a given serum to gp160env and p24gag. The findings that both the concentration and proportion of antibodies to specific viral polypeptides in human sera markedly affect the signal intensity produced by proprietary ELISAs suggest the need for several control sera which reflect the diversity of human serum responses. Standardization of human reference sera by quantitative Western blotting will assist in evaluation and quality control of ELISA tests.