Immunoenzymetric Assays Research Articles

Basophil Phenotypes in Chronic Idiopathic Urticaria in Relation to Disease Activity and Autoantibodies

Potentially pathogenic IgG autoantibodies to IgE or its receptor, Fc epsilonRIalpha, have been detected in approximately 40% of chronic idiopathic urticaria (CIU) patients. CIU patients' basophils display distinct altered Fc epsilonRIalpha-mediated degranulation. CIU patients with basophil histamine release in response to polyclonal goat anti-human IgE > or = 10% are classified as CIU responders (CIU-R) and < 10% are CIU non-responders (CIU-NR). We compared the presence of autoantibodies to basophil degranulation phenotypes and to disease status (active or inactive). Sera were collected from non-CIU subjects and CIU subjects who participated in a longitudinal study of disease severity and had defined basophil degranulation phenotypes. Immunoenzymetric assays (IEMA) quantified IgG anti-Fc epsilonRIalpha and anti-IgE. IgG anti-Fc epsilonRIalpha antibody was detected in 57% of CIU-R (n=35), 55% of CIU-NR (n=29), and 57% of non-CIU subjects (n=23), whereas IgG anti-IgE was present in 43% of CIU-R, 45% of CIU-NR, and 30% of non-CIU subjects. Both the autoantibody levels and the functional basophil phenotype remained stable in subjects with active disease (n=16), whereas there was an enhancement in basophil function as subjects evolved into a state of remission (n=6), which appears independent of the presence of autoantibody. IEMAs detected a similar frequency of autoantibodies in CIU-R, CIU-NR, and non-CIU subjects. Basophil function may be independent of IEMA-detected autoantibodies.

Allergen concentration in natural rubber latex

Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens. This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy. The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library. The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations. Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Assessment of indoor allergen exposure

Of the four modes of treating human allergic disease, avoidance or separation of the allergic patient from the allergen source is most effective and least expensive. The clinical immunology laboratory has established efficient and inexpensive "reservoir" dust sampling and processing procedures to obtain a surface dust specimen that reflects the allergen burden of the environment. Following extraction, allergens are quantified by reproducible, validated immunoenzymetric assays for the quantification of "indicator" aeroallergen levels in home, school, and work environments. In this paper, the strategies and methods for collecting and processing dust samples are discussed, and assays are reviewed for quantifying indoor aeroallergen exposure from dust mites (Der p 1 and 2, Der f 1 and 2), animals (cat: Fel d 1; dog: Can f 1; mouse: Mus m 1; rat: Rat n 1), and insects (cockroach: Bla g 1 and 2). Accurate quantification of the levels of allergen in indoor environments facilitates avoidance therapy by identifying environmental risk factors for asthma and allergy exacerbation and allowing the allergic patient to monitor the effectiveness of environmental remediation actions.

Hev b 5 and Hev b 13 as allergen markers to estimate the allergenic potency of latex gloves

Background Sensitization to natural rubber latex has been linked to proteins from medical latex gloves. Various assays to estimate the amount of residual allergenic proteins extractable from latex gloves to assess their potential exposure hazard have inherent weaknesses. Objective This investigation was aimed at developing 2-site immunoenzymetric assays and identifying appropriate protein markers to assess the allergenic potential of latex gloves. Methods The presence of 6 latex allergens—Hev b 1, 2, 3, 5, 6, and 13—was measured in a cross-section of commercial latex medical gloves by using monoclonal and polyclonal antibody-based 2-site immunoenzymetric assays. The overall allergenic potential of these gloves was assessed by IgE-inhibition assay. Stepwise multiple regression analyses were performed to identify marker allergens that best explained the variation in latex glove allergenicity. Results All 6 latex allergens were detected in at least some of the glove samples. Hev b 5 and Hev b 13 were identified as the marker allergens that combined best to explain the variation in the glove allergenicity. The significant multiple correlation ( R = 0.855) between these 2 markers and glove allergenic potency forms the basis of an assay to gauge latex glove allergenicity. Conclusion The overall allergenic potential of latex gloves can be estimated by using Hev b 5 and Hev b 13 as indicator allergens. The correlation between glove allergenicity and the level of these allergens was maintained for low-protein gloves (<200 μg/g). This estimation of glove allergenicity was superior to that obtained by using total protein readings.

Virus respiratoire syncytial et virus para-influenza : diagnostic virologique

The diagnosis of infection due to the respiratory syncytial virus (RSV) and parainfluenza virus (PIV) rests chiefly on detection of the virus or its antigens in a nasal specimen. The diagnostic yield depends on the number of respiratory cells collected in the specimen. RSV infection can be diagnosed rapidly using immunofluorescent assays and immunoenzymetric assays. Immunofluorescent assays are more difficult technically than immunoenzymetric assays but provide an assessment of the quality of the specimen. Virus isolation in cell cultures and detection of the viral RNA by reverse transcriptase-polymerase chain reaction techniques are possible for both the RSV and the PIV; however, these sophisticated, time-consuming, and costly tests now have a limited role as diagnostic tools.

Comparison of the Tandem-E Immunoenzymetric and Immuno-1 PSA Assays

Comparison of the Tandem-E Immunoenzymetric and Immuno-1 PSA Assays

Asthma severity, atopic status, allergen exposure and quality of life in elderly persons.

Although asthma can be associated with significant airflow obstruction in those over the age of 65, it is often underdiagnosed and undertreated. To describe severity of asthma, allergy skin test sensitivities, indoor allergen exposures, and the impact on quality of life (QOL) and health status in elderly persons with asthma. A cross-sectional data analysis with 80 elderly persons with asthma recruited from medical, geriatric, and allergy/immunology tertiary care centers. Asthma severity was determined by symptoms and measurements of lung function. House dust specimens were collected from mattresses and bedroom carpets and analyzed separately for the major allergens of house dust, using monoclonal antibody-based immunoenzymetric assays. QOL was measured using Juniper's Asthma Quality of Life Questionnaire. Health status was measured using the Short Form Health Survey Medical Outcome Questionnaire which included Ferrans and Powers' Quality of Life Index subscales. Two-thirds of participants had either moderate or severe persistent asthma. Skin tests to a battery of common airborne allergens were positive to at least one allergen in 56 of the 75 participants tested (74.7%). Reservoir dust allergen levels were often high enough to place participants at risk of symptoms or at risk of developing sensitization. Increased asthma severity was associated with significantly lower QOL and a trend toward decreased health status. Asthma is a significant chronic problem in the elderly. Atopy was common. Asthma severity impacts on these participants' QOL and health status. Results support interventions aimed at identifying allergens precipitating attacks and reducing them in the home.

248 Quantification of Hev-b 1/Hev-b 6 levels in prospective latex allergen reference preparations by immunoenzymetric assays (IEMAs)

248 Quantification of Hev-b 1/Hev-b 6 levels in prospective latex allergen reference preparations by immunoenzymetric assays (IEMAs)

Purification of immunoglobulin E (IgE) antibodies from sera with high IgE titers

A three stage method for the ultrapurification of polyclonal IgE from human serum is reported using anion exchange chromatography followed by monoclonal antibody based positive and negative affinity chromatography. Following dialysis of 25–100 ml of serum (2.3–14 μg IgE/ml, n = 4) against 0.05 M Tris pH 8, each specimen was subjected to diethylaminoethyl (DEAE)-cellulose chromatography ( serum matrix = 1 4 ). IgE was eluted with 0.05 M Tris, 0.05 M NaCl pH 8, yielding an IgE recovery of 61–93%, with removal of ∼ 90% of other serum proteins and an IgE purity ( [IgE] [Igs] ) of 0.1-1.1%. After adjusting to 0.1 M NaCl and concentrating ∼ 30-fold, the eluted IgE was further purified by affinity chromatography using a panel of IUIS/WHO-documented mouse monoclonal anti-human immunoglobulin antibodies (αhIg-MAbs). First, the IgE-enriched DEAE-cellulose chromatography fraction was incubated in a batch mode with two αhIgE-Fc MAbs (HP6029, HP6061) coupled to CNBr-Sepharose, CL-4B. IgE was eluted with 0.05 M glycine pH 2.8 and immediately neutralized. The IgE recovery was 32–52% and IgE purity was 72–97%. Silver-stained SDS-PAGE and noncompetitive solid-phase two-site immunoenzymetric assays for total human IgA, IgE, IgG and IgM indicated that IgA, IgG and IgM were the only contaminants. Next, the IgE was concentrated 10–30-fold in the presence of 0.1% HSA. One IgE specimen was ultrapurified in a batch mode by negative selection chromatography using three pairs of αhIg-MAbs (αhIgA: HP6111 + HP6123; αhIgG: HP6017 + HP6046; αhIgM: HP6081 + HP6083) coupled to CNBr-Sepharose, CL-4B. IgE purity increased from 91% to > 99.9% with ∼ 70% recovery of IgE for this step. The ultrapurified IgE antibody was shown to be functionally reactive for allergen and Fc εRI receptors on human basophils. We conclude that αhIg-MAbs are powerful tools to facilitate the affinity purification of functionally active human IgE from serum; however, when the analyte is present in low concentration, a carrier protein needs to be added to minimize non-specific loss of the material during this process.

IgA, IgG and IgM quantification in bronchoalveolar lavage fluids from allergic rhinitics, allergic asthmatics, and normal subjects by monoclonal antibody-based immunoenzymetric assays

Recent reports have suggested that human secretory IgA (sIgA) may have a role in the mediation of atopic disease. We have studied the levels of sIgA, IgA, IgG and IgM in bronchoalveolar lavage (BAL) fluids collected from lungs of healthy non-allergic adults ( n = 14), allergic subjects with rhinitis ( n = 15), and allergic asthmatics ( n = 13), using a panel of monoclonal antibody-based immunoenzymetric assays (IEMAs). In contrast to commercially available immunodiffusion and nephelometric assays, these IEMAs employ highly specific monoclonal antibodies and demonstrate required precision (intra-assay CVs < 17%), parallelism (inter-dilutional CVs < 20%) at minimal detectable immunoglobulin levels in the ng/ml range, and excellent specificity with < 0.1% crossreactivity for heterologous immunoglobulin isotypes. Using these assays, we have observed a significant correlation between sIgA levels and total IgA levels in BAL fluids from all the study patients ( r = 0.94; p < 0.01). The percentage of sIgA to total IgA was 84.0 ± 2.2%. sIgA in BAL fluids from allergic rhinitics (18.0 ± 2.5 μg/ml) and allergic asthmatics (15.5 ± 2.5 μg/ml) were higher than those from nonallergic subjects (10.2 ± 1.9 μg/ml). The only statistically significant difference in sIgA levels was observed in BAL fluids from the rhinitics and nonallergic groups ( p = 0.03). Similar differences among the groups were found for levels of total IgA in BAL fluid. There were no significant differences in the levels of IgM and IgG in BAL fluids among the three groups of subjects. We conclude from these results that IgA is the predominant immunoglobulin on the airway surface and that it appears to be produced locally.

IL-1, IL-4, and IFN-gamma differentially regulate cytokine production and cell surface molecule expression in cultured human thymic epithelial cells.

We investigated the response of purified and cloned human thymic epithelial cells (TEC) to IL-1, IL-4, and IFN-gamma stimulation in vitro. IL-1 alpha strongly up-regulated the production of granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), IL-6, and IL-8, as measured by specific immunoenzymetric assays and by increased steady state mRNA levels. IL-4 or IFN-gamma did not induce these cytokines in TEC but in a sustained and dose-dependent manner down-regulated the IL-1-induced GM-CSF protein and mRNA levels. Only IFN-gamma, and not IL-4, suppressed the IL-1-induced G-CSF and IL-8 production, as shown at both the protein and mRNA levels. The inhibition was dose dependent, sustained for at least 96 h, and more pronounced for G-CSF than for IL-8. In contrast, both IL-4 and IFN-gamma enhanced the IL-1-induced IL-6 production. IL-4 and IFN-gamma had additive effects to increase IL-6 secretion and to more completely suppress the IL-1-induced GM-CSF. Analyses of cell surface molecules showed that intercellular adhesion molecule 1 (ICAM-1) expression on TEC was increased by IL-1 or IFN-gamma. IL-4 slightly down-regulated constitutive ICAM-1 levels but did not significantly modify the levels of expression induced by either IL-1 or IFN-gamma. MHC class II expression was induced by IFN-gamma but not by IL-1 or IL-4. The combination of IL-1 and IL-4 with IFN-gamma did not alter the levels of class II MHC Ag induced by IFN-gamma. In conclusion, TEC cytokine production and cell surface molecule expression are differentially regulated via a complex cytokine network. Our data suggest that developing T cells provide, in part, the signals controlling the function of their supporting stroma.

Monoclonal antibody-based immunoenzymetric assays for quantification of human IgG and its four subclasses.

A panel of 5 immunoenzymetric assays (IEMAs) has been developed for quantification of the total and four subclasses of immunoglobulin G (IgG) in human serum using IUIS/WHO documented monoclonal antibodies (MoAb). Human IgG specific MoAb was adsorbed to microtiter plates and used to capture IgG from serum. Peroxidase conjugated forms of polyclonal mouse anti-human IgG Fc or a mixture of 4 MoAb (anti-kappa, anti-lambda, anti-IgG Fc PAN and anti-IgG Fd PAN) were used as detection antibodies. Use of monoclonal antibody in chromatographically purified form was required for acceptable assay sensitivity (S) and working ranges (WR). All 5 IEMAs displayed good precision (intra-assay %CV less than 5%, inter-assay %CV less than 12%) and parallelism (inter-dilutional CV less than 20%). Both HP6069 or HP6070 (anti-IgG1 Fc) worked well alone or together as capture antibodies in the IgG1 IEMA: WR = 20-1250 ng/ml, S = 15 ng/ml. HP6002 (anti-gG2 Fc) alone or in combination with HP6014 (anti-IgG2 Fd) produced an IgG2 IEMA with a WR of 5-200 ng/ml and S of 5 ng/ml. HP6047 (anti-IgG3 hinge) alone generated a sensitive IgG3 IEMA with a narrow working range: WR = 2-50 ng/ml, S = 1.6 ng/ml. Both HP6025 and HP6023 (anti-IgG4 Fc) worked equally well alone and together to produce a useful IgG4 IEMA: WR = 8-250 ng/ml, S = 7.8 ng/ml. HP6017 (anti-IgG PAN Fc) was combined in an equal molar ratio with HP6046 (anti-IgG PAN Fd) to produce a total IgG PAN IEMA with a WR of 5-530 ng/ml and a sensitivity of 5 ng/ml. All 5 IEMAs fulfilled requirements for robust clinical immunoassays that permit the quantitation of human IgG and its 4 subclasses.

False positive immunometric assays caused by anti-immunoglobulin antibodies: a case report

A serological phenomenon causing aberrant results with monoclonal immunoenzymetric assays (IEMA's) is reported. Two different commercial IEMA kits detected low levels of Choriogonadotropin (hCG) in the serum of a non-pregnant woman. These assays detected between 32 and 55 IU/1 of serum hCG over a 3-wk period; however, an RIA for β-subunit and two monoclonal immunoradiometric assays (IRMA's) detected no hCG (< 5 IU/1). An IEMA measurement of creatine kinase MB isozyme was also elevated. Antisera to either human immunoglobulin or specifically to human IgM, added to the serum prior to assay, substantially decreased these IEMA reactions. Addition of either mouse serum or purified mouse IgG totally abolished them. It is concluded that these spurious reactions were most likely caused by an IgM antibody which binds to native and enzyme-labelled mouse IgG, but not to iodinated IgG.