Use Of Counterimmunoelectrophoresis Research Articles

Detection of antibodies against avian antigens in bronchoalveolar lavage from patients with pigeon breeder's disease: Usefulness of enzyme‐linked immunosorbent assay and enzyme immunotransfer blotting

The study reported here evaluated the usefulness of the enzyme-linked immunosorbent assay (ELISA) in the detection of antibodies against pigeon antigens in the serum and bronchoalveolar lavage (BAL) of patients with clinical, radiological, and functional evidence of interstitial lung disease (ILD) with and without pigeon breeder's disease (PBD). The results were compared with those obtained by the simultaneous use of counterimmunoelectrophoresis (CIE) in the same patients. In PBD, ELISA detected antibodies against pigeon's sera in both serum and BAL in 100% of patients, while CIE failed to detect the antibodies in the serum of one patient and in most of the samples of BAL. In addition, we used enzyme immunotransfer blotting to determine the number of epitopes in pigeon serum recognized by antibodies present in serum and BAL. There was a heterogeneous response in both fluids, but the reaction pattern demonstrated that patient's sera recognize to-25 different pigeon epitopes. We conclude that ELISA is a highly sensitive and specific method for the detection of antibodies against pigeon antigens in the serum and BAL of patients with PBD and that the host response involves a great number of avian antigens.

Use of counter Immunoelectrophoresis in detection of antibodies to tickborne fever

A micromethod using the counter immunoelectrophoresis technique for the detection of antibodies to Cytoecetes phagocytophila, the causative agent of tickborne fever (TBF) is described. Antibodies were first detected nine to 11 days after experimental infection of lambs with TBF and persisted for six to 10 weeks after infection. The test was also applied to approximately 440 field samples of ovine sera collected from tick infested farms in Scotland and the north of England, 16 per cent of which proved positive and showed a marked seasonal variation.

Routine use of counterimmunoelectrophoresis for the detection of pneumococcal antigen in sputum.

Sputum samples obtained routinely for culture from patients at a thoracic department were also examined for pneumococcal antigen by means of counterimmunoelectrophoresis (CIE), using a polyvalent antipneumococcal type serum (omniserum). Pneumococci were found in 1.3% of the 880 cultures, whereas pneumococcal antigen was detected with CIE in 6.5%. The validity of these findings was tested by correlating them with the presence of clinical symptoms in those with positive tests and also by antigen detection in ELISA using monoclonal antibodies specific for the C-polysaccharide common to all types of pneumococci. Clinical findings corresponding to confirmed or probable current chest infection were found in 36 of the 48 patients with positive CIE. ELISA was positive in 33 of the 38 patients with positive CIE who were tested. Although the study deals with an unselected material of chest patients, it indicates that CIE is a sensitive method and that it is independent of current antibiotic treatment. Pneumococcal infection is probably of importance in exacerbations of chronic obstructive lung disease, but the clinical usefulness of detecting pneumococcal and other antigens in this patient group needs to be studied further.

Diagnosis of Johne's disease in sheep by counter-immunoelectrophoresis.

The use of counter-immunoelectrophoresis (CIEP) in the diagnosis of Johne's disease was investigated in flocks of sheep in Iran and Libya. In 50 sheep with no history of paratuberculosis none had antibodies to Mycobacterium paratuberculosis. Animals vaccinated as neonates were either negative or had reciprocal titres (RT) of less than 4 at 18 months of age unless exposed to infection. Among 105 non-vaccinated adults in an infected flock 91% had antibodies. Twenty-one animals that either died or were slaughtered were autopsied. Eight animals with RT of 8 or above and one with RT of 4 had gross lesions with demonstrable acid-fast organisms. Two animals with RT of 2 had clinical disease while three with RT of 4 and one with RT of 2 had histopathological lesions only. All animals found negative for M. paratuberculosis infection had RT of 2 or below. The critical interpretation of the test is discussed.

Bacteremic infections caused by nontypable Haemophilus influenzae in patients with dysgammaglobulinemia.

Two patients with dysgammaglobulinemia had bacteremic infections due to nontypable Haemophilus influenzae. The regular use of counterimmunoelectrophoresis or the more careful use of existing agglutination techniques to serotype Haemophilus isolates may identify nontypable H. influenzae in this clinical setting more frequently than has been though possible.

Counterimmunoelectrophoresis in the Diagnosis of Hemophilus influenzae Pleural Effusion

A child with a sterile pleural effusion resulting from an infection with Hemophilus influenzae type b (Hi b) is described. The diagnosis was established by use of counterimmunoelectrophoresis (CIE). The alarming increase in incidence of pneumonia due to Hi b is noted, as is the large number of associated pleural effusions. CIE provides a rapid, reliable, and sensitive means by which to establish the exact etiology of such bacterial infections so that optimal antibiotic therapy can be started promptly.

Detection of candida antigenemia by counterimmunoelectrophoresis in patients with invasive candidiasis.

Because of the difficulty in diagnosis of invasive candidiasis, an assay that makes use of counterimmunoelectrophoresis (CIE) was developed to detect candida antigen. A cell-wall polysaccharide of Candida was extracted with hot formamide, ethanol precipitation, and gel filtration. Rabbit antiserum was tested by CIE against sera from 20 healthy individuals, 15 patients with mucosal candidiasis, and 48 compromised patients with cultures positive for Candida. Antigen was detected in sera from 13 patients by CIE; eight of these patients were eventually proven to have invasive disease. None of the antigen-negative patients for whom autopsy was performed had evidence of invasive candidiasis. Healthy individuals and patients with superficial mucosal candidiasis were also antigen-negative. The detection of candida antigenemia by CIE is specific for invasive disease and serves as a guide in initiation and follow through of antifungal therapy.

Diagnostic value of candida precipitins determined by counterimmunoelectrophoresis in patients with acute leukemia: a prospective study.

Thirty-nine patients receiving chemotherapy for acute leukemia had weekly determinations of Candida precipitins by use of counterimmunoelectrophoresis. The only two patients who had proven disseminated candidiasis both had diagnostic (greater than or equal to 1/8) titers. However, most patients with "possible" disseminated candidiasis did not have diagnostic titers. The test's specificity was high (90%), and only one of five patients infected with other fungi had diagnostic titers. However, because there were so few "true positives" in this small series, the relatively small number of "false positives" resulted in a low predictive value for this test. Based on a review of the literature and the authors' experience, the value of this test for patients who have acute leukemia does not appear to be well established.

Characteristics of ascitic fluid in the alcoholic cirrhotic

A prospective study was conducted to define the characteristics of ascitic fluid in alcoholic cirrhotics with and without spontaneous bacterial peritonitis (SBP); to correlate these with findings in the peripheral blood; and to determine whether the use of counterimmunoelectrophoresis (CIE) for bacterial antigens will aid in the early diagnosis of SBP. Fifty-one alcoholic cirrhotics had simultaneous determination of their blood or serum and ascitic fluid for the following: WBC and differential count, RBC, LDH, amylase, glucose, total protein, and protein electrophoresis, CIE for pneumococcal andKlebsiella antigens, culture for aerobic and anaerobic bacteria and mycobacteria, and cytology. Of the 51 patients, 2 had SBP (4%). In the other 49 patients (54 sera and ascitic fluids), CIE was positive for pneumococcal antigen in 4/54 sera and in 3/54 ascitic fluids. The mean WBC count in the ascitic fluid was 349. In 4% the count was above 1000, in 18% between 501–1000, and in 32% between 301–500; polymorphs were >30% in 19/54 (32%). Specific gravity was >1.020 in 10/54 (22%), and ascitic fluid total protein of 3.0g/100 ml or above was noted in 24% (12/54). Mean ascitic fluid/serum ratios of total protein, albumin, and globulin were 0.31, 0.33, and 0.30 respectively, and mean ascitic fluid/serum ratios of LDH, amylase, and glucose were 0.54, 0.79, and 1.04. All cultures (except those with SBP) and cytology were negative. Our study confirmed the observation of others, that a significant number of noninfected cirrhotics have increased ascitic fluid WBC, % polymorphs, specific gravity, and total protein concentration. CIE was not helpful in the early diagnosis of SBP.

Detection by Counterimmunoelectrophoresis of Anti-Candida Precipitins in Sera from Cancer Patients

Five antigen preparations from yeast-phase cells and hyphalphase cells of Candida albicans were used to detect antibodies in sera from cancer patients by use of counterimmunoelectrophoresis (CIE). Sera from uninfected patients, patients with superficial infections, and patients who had invasive or systemic candidiasis were analyzed for antibodies. A high incidence (27%) of false-negative data was obtained for patients who had systemic candidiasis. Serum titers of all patient groups were below 1:10. Of the antigens tested, cytoplasmic antigen from yeast-phase cells provided the greatest sensitivity and diagnostic value.

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.

Direct use of counterimmunoelectrophoresis in detection of group B streptococci in specimens containing mixed flora

Isolation procedures for the group B streptococci were bypassed by applying counterimmunoelectrophoresis (CIE) directly to the broth containing the initial inoculum. A total of 458 tests were performed on 389 specimens received from 93 mothers and 81 babies. Of all tests, 19.4% were positive group B streptococci by the Lancefield precipitin method. Twenty hours of incubation were required to produce a positive CIE result in 54% of those specimens which were eventually proven a contain group B streptococci by Lancefield technique. There were no false positive CIE results. It is recommended that the initial broth culture be sampled and tested with CIE after an overnight incubation in those cases where another 24 h may be critical in terms of identifying the group B streptococci. A positive result with CIE can be clinically relied upon, but a negative result is indeterminate, and routine isolation procedures would need to be followed in that case.

Detection of group B streptococcal antigens in body fluids of neonates

CONSIDERABLE ATTENTION has been focused on neonatal diseases caused by the group B beta hemolytic streptococcus; early diagnosis and differentiation from other infectious and noninfectious etiologies have been stressed.' There have been few reports of the use of counterimmunoelectrophoresis for identification and serotyping of group B organisms and for detection of antigen in body fluids of infants with group B streptococcal 7 We report our experience with burro type-specific antisera for groups Bra, Bn, and Bm streptococci compared to that with a rabbit Brn antiserum and a commercially available group-specific antiserum . Antigen was detected in blood, urine, cerebrospinal fluid, pleural fluid, or cord blood of21 infants, 20of whom had culture proven group B streptococcal disease.

Screening Test for Complement Activation by Counterimmunoelectrophoresis

This report describes the use of counterimmunoelectrophoresis (CIE) as a method of detecting activation of the complement system. With this technic small amounts of C3 split products (C3c/d) can be detected in plasma samples by specific precipitation with antiserum in less than two hours. The CIE technic is a highly sensitive, rapid method for detecting activation of the complement system in the presence of normal concentrations of C3 measured hemolytically or by radial immunodiffusion (RID) in human disease. A clinical investigation was carried out in 40 patients with systemic rheumatic diseases and 116 normal healthy individuals. The following observations were made: (1) plasmas and sera from normal individuals had normal total complement hemolytic activity (CH50), hemolytic active C4 (C4H50) and C3 (C3H50); (2) in 30% of the serum samples it was possible to identify the presence of C3 split products, in contrast to only 2.5% of the plasma samples obtained simultaneously; (3) in the specimens from patients who had rheumatic disease activity, C3 split products were identified by CIE in all cases except one in the presence of normal C3 protein measured by RID.

Detection of candida serum precipitins by counterimmunoelectrophoresis: an adjunct in determining significant candidiasis

We report our experience with the use of counterimmunoelectrophoresis for the detection and quantitation of candida serum precipitins in 164 patients. Group I consisted of 24 patients with significant candidiasis; group II consisted of 97 patients with either colonization or transient candidemia; and group III consisted of 43 subjects with noncandida systemic mycoses, bacterial infections, and normal controls. Prospective studies were done in ten patients. Double immunodiffusion was performed in all cases. Ratios of counterimmunoelectrophoresis precipitin detection were significantly different between groups I and II and groups I and III (P less than 0.001). Precipitin titers of 1:8 or greater were found more often in group I as compared to group II or III (P less than 0.001). All prospectively studied patients who developed significant candidiasis had peak precipitin titers of greater than or equal to 1:8(greater than fourfold titer increase) during the period of observation. Quantitation of candida serum precipitins by counterimmunoelectrophoresis is helpful in diagnosing significant candidiasis.

Detection of circulating carcinoembryonic antigen (CEA) by counter-immunoelectrophoresis

A sensitive method is described for detecting circulating carcinoembryonic antigen (CEA) by combined use of counter-immunoelectrophoresis and intensifiers. Perchloric acid extraction of serum was followed by counter-immunoelectrophoresis against monospecific antiserum to CEA. A precipitin line formed was intensified further by electrophoretic binding of anti-γG γ-globulin and Amido Black staining. Less than 10 ng CEA/ml was detectable. Using this method, a total of 84 sera from patients with or without malignancy was examined. The results obtained were in general agreement with those reported by radioimmunoassay. The method appears to be useful as a diagnostic aid, since it requires neither labeled preparations of purified CEA nor special equipment for assay.

Application of counterimmunoelectrophoresis in the identification of Streptococcus pneumoniae in clinical isolates

The use of counterimmunoelectrophoresis (CIE) as a diagnostic tool in infectious diseases is becoming more widespread. This study was undertaken to determine the possible use of CIE in the more rapid identification of Streptococcus pneumoniae in clinical isolates. Typing sera were obtained from the Statens Seruminstitut, Denmark. Sixty-seven out of 68 pneumococcal isolates that were optochin sensitive and bile soluble were typed by CIE. One isolate was nontypable even after mouse passage. An additional three isolates that were optochin resistant were considered to be pneumococci on the basis of the bile solubility test and their typability by CIE. Seventy-seven alpha-streptococci were tested for the presence of cross-reacting capsular antigens. Fifty-three alpha-streptococci showed no cross-reactions using the omniserum. Precipitin bands were obtained with the omniserum with 10 of the isolates, but these did not react with type-specific antisera. However, 14 isolates did react with the type-specific pneumococcal antisera. The sensitivity of the test was increased by sonicating whole-cell suspensions before electrophoresis was carried out. Mueller-Hinton broths were inoculated with presumptive pneumococcal colonies that formed the predominant or only colony type on primary blood agar plates. These cultures required a 4-h incubation period with an initial inoculum of 10(6) viable organisms/ml before a precipitin band could be detected. Precipitin bands were observed in 54 out of 56 (97%) broth cultures of pneumococci that had been incubated for 4 h at 37 C. These data suggest that CIE could be a useful tool in the identification of S. pneumoniae isolated from clinical specimens.