The role of major olive pollen allergens Ole e 1, Ole e 9, and Ole e 10 on mice sensitization.

BACKGROUND: Olive pollen is an important cause of allergy in Mediterranean countries. To date, 10 allergens (Ole e 1 to Ole e 10) have been isolated and characterized. Animal models of olive pollen allergy are suitable tools for testing the efficacy and safety of new forms of immunotherapy. OBJECTIVES: To characterize the immune response in mice sensitized with olive pollen extract and to compare it with that of allergic patients. METHODS: BALB/c mice were sensitized by 4 intraperitoneal injections of olive pollen extract in aluminum hydroxide. The allergic state was proved by measuring serum specific IgG1 and total IgE antibody levels. The IgG1 responses to olive pollen allergens were assayed by immunoblotting and enzyme-linked immunosorbent assay. Competition experiments between human IgE and mouse IgG1 binding to olive pollen allergens were performed. RESULTS: Sensitization with olive pollen extract induced high levels of specific IgG1 and total IgE in all tested animals. Immunoblotting experiments showed that the mouse IgG1 binding pattern to pollen extract was complex and heterogeneous, as occurs with human IgE. High IgG1 antibody levels to the major olive pollen allergens described for humans were detected in serum samples from sensitized mice, whereas minor olive pollen allergens induced no significant IgG1 response. Coincubation of mouse serum samples with a cocktail of Ole e 1, Ole e 9, and Ole e 10 resulted in a significant decrease (60%) in IgG1 binding to olive pollen extract. Specific mouse IgG1 strongly inhibited human IgE binding to olive pollen allergens. CONCLUSIONS: This mouse model of olive pollen sensitization mimics immunologic features of human pollinosis and could be a useful tool for designing novel forms of immunotherapy for olive pollen allergy based on allergen cocktails.     

Determination of mycobacterial antigens in sputum by enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) was examined for its usefulness in detecting mycobacterial antigens in sputum. A double-antibody sandwich procedure was set up by using a commercially available hyperimmune serum directed against Mycobacterium bovis, BCG. The ELISA was able to detect 10 ng of protein per ml of BCG sonic extract. The system also clearly distinguished Mycobacterium tuberculosis organisms from Mycobacterium avium and Mycobacterium kansasii organisms. A total of 68 unknown sputum specimens submitted to the clinical laboratories for examination for tuberculosis were tested by ELISA. Of the 20 specimens that were smear positive and culture positive, 12 (60%) were positive by ELISA; 6 of the 11 (55%) smear-positive culture-negative samples were positive by ELISA; 1 of 2 (50%) of the smear-negative culture-positive samples was positive by ELISA; and only 3 of 35 (9%) of the smear-negative culture-negative samples were positive by ELISA. This approach offers promise as an aid in the presumptive differentiation of nontuberculous mycobacteria from the M. tuberculosis complex.     

Determination of anti-lipid A and lipid A by enzyme immunoassay

Immunizing rabbits and goats with acid-treated Salmonella minnesota R595 bacterial cells, that expose lipid A on their surface, an IgG anti-lipid A antibody response could be obtained. In passive hemolysis test, using lipid-A-coated sheep red blood cells and complement, the highest IgG anti-lipid A antibody activity was found in immunized rabbits, while goats produced a relatively weak antibody response against lipid A. The passive hemolysis test showed that IgG anti-lipid A antibody activity was completely abolished by preceding preincubation with lipid A. No reactivity of IgG anti-lipid A antibodies was found with the tested intact lipopolysaccharides from one Salmonella abortus equi and one Escherichia coli strain. Quantitative analysis of free lipid A was carried out by the enzyme-linked immunosorbent assay (ELISA). In sandwich antigen type ELISA, using goat IgG anti-lipid A antibodies bound to the wells of polystyrene microassay plates, rabbit IgG anti-lipid A antibodies and peroxidase-labeled goat anti-rabbit antibodies (PGRA), the measuring range for free lipid A (0.001 to 1 µg/ml) correlated significantly (2p < 0.01) with absorbance. In sandwich antibody type (SAB) ELISA binding lipid A to the solid phase, and using rabbit IgG anti-lipid A antibodies and PGRA insignificant correlation could be found for the same measuring range (2p > 0.1 ). Comparison of passive hemolysis test and SAB type ELISA to determine IgG anti-lipid A antibody activity showed that ELISA was more sensitive for measuring anti-lipid A antibody activity (from antibody dilution of -log: 2.43 to 3.7) than the passive hemolysis test.     

Validation of respiratory syncytial virus enzyme immunoassay and shell vial assay results.

The Pathfinder respiratory syncytial virus (RSV) enzyme immunoassay (EIA) (Kallestad), the shell vial (SV) technique, and conventional cell culture (CC) were compared for detection of RSV in nasopharyngeal aspirates. We found sensitivities, specificities, and positive and negative predictive values of 58.4, 100, 100, and 68.2%; 80.7, 97.2, 97.0, and 81.9%; and 77.6, 97.2, 96.9, and 79.5% for the CC, EIA, and SV methods, respectively. The SV and EIA techniques were both more sensitive than CC (P < 0.001). Finally, 29 respiratory viruses other than RSV were identified by CC.     

Detection of Chlamydia trachomatis antigen by enzyme immunoassay: importance of confirmatory testing.

AIM--To determine when a fluorescence assay for Chlamydia trachomatis elementary bodies in the specimen buffer is of most value as a verification test for genital specimens reactive on screening enzyme immunoassay (EIA). METHOD--Genital swabs from high and medium prevalence populations were tested using EIA. Samples with absorbance values greater than the positive threshold and those within the range of 30% below this value were verified by the MicroTrak direct fluorescence assay (DFA) test. Quotients derived from the threshold value and specimen absorbances were used to establish confidence limits for the EIA. RESULTS--Of 13,283 swabs tested, 474 from the high risk group and 236 from the medium risk group were reactive on EIA and confirmed by DFA. Thirty six (5.9%) patients with confirmed reactive samples would have been missed if the kit criteria alone were followed. When confidence limits were applied to the calculated quotients, only those samples with an EIA quotient of > or = 4.0 were universally confirmed by the DFA. CONCLUSION--A scheme of testing which uses the DFA to verify EIA reactive specimens over a specified range was found to improve the sensitivity and specificity of the EIA screening test.     

Evaluation of repeat Clostridium difficile enzyme immunoassay testing

Clostridium difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis, which have significant morbidity and mortality. Accurate and timely diagnosis is critical. Repeat enzyme immunoassay testing for C. difficile toxin has been recommended because of <100% sensitivity. All C. difficile tests between 1 January 2006 and 31 December 2006 were retrospectively analyzed for results and testing patterns. The Wampole C. difficile Tox A/B II enzyme immunoassay kit was used. There were a total of 8,256 tests from 3,112 patients; 49% of tests were repeated. Of the 3,749 initially negative patient tests, 96 were positive upon repeat testing within 10 days of the first test. Of repeat tests, 0.9% repeated on day 0 (same day as the first test), 1.8% on day 1, 3.8% on day 2, 2.6% on day 3, 5.4% on days 4 to 6, and 10.6% on days 7 to 10 were positive. Thirty-eight patients had a positive test within 48 h of an initial negative test, and based on chart review, 18 patients were treated empirically while 16 were treated following the new result. None had evidence of medical complications. Of initially positive patients, 91% were positive upon repeat testing on day 0, 75% on day 1, and 58% on day 2, to a low of 14% on days 7 to 10. Depending on the clinical setting, these data support not repeating C. difficile tests within 2 days of a negative result and limiting repeat testing to ≥1 week of a positive result.     

Comparison of skin-prick test assay and reverse enzyme immunoassay competition (REINA-C) for biological activity of allergens

An in vitro technique for measuring the relative potency of allergenic extracts has been developed and compared with the dose-response skin-prick test, The in vitro technique is a modification of an EL1SA based on the method of antigen capture using polystyrene microtitre wells as solid phase (REINA-compelition) and is also based on a doseresponse. Both methods have been applied to allergen extracts of Dermatophagoides pteronyssinus. Phleum pratense, Secale cereale, Lolium perenne, Plantigo lanceolata, Artemisia vulgaris, Salsola kali and Parietaria judaica. For each allergen, the slope of the regression log-log dose response lines displayed by skin-prick test and REINA-C does not show a statistically significant difference, being parallel (P < 0.001), and indicating that the allergen ligand binding kinetics are identical in both methods.     

Determination of lymphocyte subpopulations by enzyme immunoassay. Comparison with conventional fluorescence microscopy

An enzyme immunoassay for measuring lymphocyte subpopulations was evaluated and the results compared with those obtained with conventional fluorescence microscopy, using two different panels of antibodies. The enzyme immunoassay is a photometric method which expresses the results using a standard curve with known amounts of cells. The method was reproducible and accurate. The intra-assay variation for the standard curve ranged from 3.2 to 5.7% and the interassay variation from 9.5 to 13.8%. The intra-assay variation for clinical samples ranged from 3.3 to 8.2%. Results obtained with the enzyme immunoassay and with conventional fluorescence microscopy showed a significant correlation (P less than 0.05) for all the subclasses of lymphocytes tested using two different panels of monoclonal antibodies. We conclude that the choice of method should be related to the particular needs and manpower in the laboratory.     

Monoclonal antibody-based method for measuring olive pollen major allergen Ole e 1.

BACKGROUND: Olive tree pollen is an important cause of inhalant allergy in Mediterranean countries. The major allergen of this pollen, Ole e 1, has caused reactions in the sera of >80% of olive-sensitive patients. Accurate standardization of allergenic products for diagnosis and immunotherapy is essential to guarantee their quality, and measurement of the major allergen content is becoming an important aspect of standardization procedures. OBJECTIVE: To develop a two-site enzyme-linked immunoadsorbent assay (ELISA) for the quantification of Ole e 1. METHODS: BALB/c mice were immunized with purified natural Ole e 1. After fusion and screening by direct ELISA, one of the monoclonal antibodies (5A3) was selected as the capture antibody in an ELISA for Ole e 1 quantification. Bound allergens were detected by a combination of biotinylated Ole e 1-specific polyclonal rabbit antibody and peroxidase-conjugated streptavidin. This ELISA was subsequently evaluated and compared with other techniques. RESULTS: The developed ELISA was highly reproducible and sensitive, with a detection limit of 0.5 ng/mL and a practical range of 1 to 10 ng/mL. The Ole e 1 content ranged from 3 to 50% of the total protein among the nine Olea europaea pollen extracts studied. The assay also detected Ole e 1-like proteins in pollen from other Oleaceae. Correlation was good between the Ole e 1 content determined by ELISA and scanning densitometry and the immunoglobulin E-binding activity of the extracts. CONCLUSION: The described Ole e 1 ELISA is sensitive, reproducible, specific, and reliable, and therefore, can be helpful for standardization of olive pollen extracts intended for clinical use.     

Determination of amylase by measurement of enzymatic activity and by enzyme immunoassay and radioimmunoassay

Alpha-amylase content was determined by enzyme immunoassay and radioimmunoassay and enzyme activity by enzymatic test in some pig tissues and serum during ontogenetic development. Among the studied organs, the highest enzyme content and activity were found in the pancreas. In this organ, amylase ratio in fetuses, newborns and adults was 1:13:1500. The ratio of the adult amylase content in pancreas, parotid gland and kidney was 4000:5:1, respectively. Close correlation was shown between the results obtained with the immunological and enzymatic methods.     

Analytic sensitivity and specificity of enzyme immunoassay results in testing for human immunodeficiency virus type 1 antibody.

In 1986, a performance evaluation program at the Centers for Disease Control was implemented to assess the quality of performance of laboratories testing for human immunodeficiency virus type 1 antibody and to identify problems that occur during the testing process. Laboratories participating in the Centers for Disease Control Model Performance Evaluation Program for human immunodeficiency virus type 1 antibody testing furnished enzyme immunoassay results after they tested performance evaluation panels that were sent to them in August and November 1989. The panels consisted of 10 individual samples containing antibody-negative and antibody-positive samples, some of which were duplicates. Not all laboratories received the same panel of samples. Low false-negative and false-positive rates, as well as high intrashipment and intershipment reproducibility, indicate that most laboratories did not experience difficulty in testing performance evaluation samples sent to them in August and November 1989.     

Predicting human immunodeficiency virus type 1-positive sera by using two enzyme immunoassay kits in a parallel testing format.

Two algorithms for screening sera for antibody to human immunodeficiency virus type 1 were compared for their efficiency in identifying a true-positive sample in a population with heterogeneous risk factors, using the criteria of specificity and positive predictive value (PPV). In the first algorithm, all sera were screened by using a single enzyme immunoassay (EIA) kit, and a specificity of 98.6% and a PPV of 69.3% was calculated for true-positive sera. The second algorithm employed two different EIA kits in parallel to screen each sample. In the first instance, a specificity and a PPV of 100% was calculated if a positive sample was defined as reactive by both EIA kits; in the second, a specificity of 99.97% and a PPV of 99.4% was obtained if this criterion was extended to include a combination of one reactive and one equivocal result obtained with the two EIA kits.     

Detection of poliovirus antigen by enzyme immunoassay.

A solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Rabbit and guinea pig antisera for the assay were raised against purified poliovirus type 3/Fin (strain 3/Fin/K) isolated from a fecal specimen from a meningitis patient during an outbreak of poliomyelitis in Finland in 1984. The EIA was highly specific for poliovirus type 3, and it was about 30 times more sensitive for strain 3/Fin/K than for strain 3/Saukett used in the inactivated poliovirus vaccine. The sensitivity of the EIA was 2 to 5 ng of purified strain 3/Fin/K per ml, whereas disrupted viruses and soluble viral proteins were almost undetectable by the assay. Only 5 of 51 (10%) stool specimens containing poliovirus type 3/Fin detected by virus isolation were positive by the EIA. Quantitation by the EIA, using purified poliovirus 3/Fin/K as a standard, revealed that concentrations of poliovirus type 3 in undiluted fecal specimens of patients with natural poliovirus infection were only 50 ng/ml or less. In conclusion, owing to the small amount of poliovirus in feces, the EIA is not suitable for the diagnosis of poliovirus infections directly from clinical specimens, but it can be used to detect, type, and quantitate poliovirus antigen in infected cells.     

Validation of an enzyme immunoassay for serodiagnosis of acute Q fever

An enzyme immunoassay was validated for the serodiagnosis of acute Q fever. Minimum positive tests were determined for both serial dilutions and a single dilution of patient sera. To establish the specificity of the test, 152 serum samples were tested from individuals with no evidence of pastCoxiella burnetii infection. Diagnostic titers were set at 128 for the IgM and IgG responses to phase I, at 512 for the IgM response to phase II and at 1,024 for the IgG response to phase IICoxiella burnetii. These titers gave a falsepositive rate of 1 %. Alternatively, testing a single dilution of sera (1:128) gave specificities ranging from 97.3 to 98.7 %. Tests with the greatest sensitivities, using serially diluted early convalescent-phase sera, were the IgM (84 %) and IgG (80 %) responses to phase IICoxiella burnetii. At a single serum dilution, 92 % of early convalescent sera had a positive IgG response to phase IICoxiella burnetii. With a high specificity and good sensitivity, the EIA can be used to diagnose acute Q fever with a single convalescent serum specimen. The duration of a positive response was greater than five years.     

Comparison of antinuclear antibody testing methods: immunofluorescence assay versus enzyme immunoassay.

Performances of anti-nuclear antibody testing by immunofluorescence assay (ANA-IFA) and enzyme immunoassay (ANA-EIA) were compared in relation to patient diagnosis. A total of 467 patient serum samples were tested by ANA-IFA (Kallestad; Sanofi) and ANA-EIA (RADIAS; Bio-Rad), and their age, sex, diagnosis, disease status, and medications were obtained through chart review. Reference ranges were established by testing 98 healthy blood donor samples. Eighty-six samples came from patients with diffuse connective tissue diseases, including systemic lupus erythematosus, discoid lupus erythematosus, or drug-induced lupus (n = 71); systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility abnormalities, sclerodactyly, and telangiectasia), or Raynaud's syndrome (n = 8); Sjögren's syndrome (n = 5); mixed connective tissue disease (n = 5); and polymyositis or dermatomyositis (n = 3). The sensitivity, specificity, positive predictive value, and negative predictive value for ANA-IFA were 87.2, 48.0, 29.1, and 93.9%, respectively, for the reference range of < 1:160. For ANA-EIA, they were 90.7, 60.2, 35.8, and 96.4%, respectively, for the reference range of < 0.9. ANA-EIA offers equivalent sensitivity and higher specificity compared to ANA-IFA.     

Toxocariasis: serological diagnosis by enzyme immunoassay.

An enzyme-immunoassay was developed to measure the concentration of serum antibody specific for the secretory antigens released by migrating toxocaral larvae. This technique was evaluated by testing sera from healthy UK adults, and from patients with and without toxocariasis. In 922 healthy adults, 2.6% were found to have elevated specific antibody levels. Elevated values were observed twice as frequently in males as in females but showed no significant regression with age between 20 and 65 years. Of 62 patients with non-toxocaral helminthic infections, all had antitoxocaral antibody levels within the range of values observed in healthy controls and had a mean level which was not significantly elevated. All of 13 patients with clinical toxocariasis had enzyme-linked immunosorbent assay (ELISA) antibody levels above the 100th percentiles of both the healthy population and the helminth-infected group and had a significantly high mean value (p less than 0.001) more than 12 times that of the healthy or infected controls. The high degree of sensitivity and specificity of the toxocariasis enzyme-immunoassay indicates that this new test should be useful in reference immunodiagnostic applications and in large-scale seroepidemiological surveys.