A double monoclonal antibody assay for the Alternaria allergen GP70.

A double monoclonal antibody-based assay for the 70-kD Alternaria allergen GP70 is described. The assay is sensitive to GP70 concentrations as low as 0.2 microgram/mL and is highly specific for this allergen. The glycoprotein detected by this assay is shown to bind to human IgE from Alternaria-sensitive patients, proving that it is an allergen. Two of three commercial Alternaria preparations tested contained no detectable GP70. Several related fungi were shown to contain detectable concentrations of GP70, but the most abundant source was a commercial preparation of house dust. Further measurement of GP70 in biologic materials should increase our knowledge of the distribution and importance of this Alternaria allergen in the environment and in the development of allergic diseases.

Enhanced sensitivity of immunoblotting with peroxidase-conjugated antibodies using an adsorbed substrate method.

Immunoblots probed with immunoglobulin E (IgE)-containing sera from allergic patients are frequently used in allergy research. Current techniques for detection of specific IgE include radiolabeled and enzyme-linked methods. Although radiolabeled methods are very sensitive, many research groups prefer non-radioactive procedures with equal or greater sensitivity. Alkaline phosphatase (AP) and horse radish peroxidase (HRP) are the most frequently used conjugating enzymes for immunoblotting with the former generally recognized as more sensitive. We describe a method of immunoblot detection using HRP-conjugated immunochemicals with sensitivity equal to and for some systems greater than that of AP conjugates. An adsorbed substrate method for developing immunoblots probed with HRP immunochemical conjugates is compared with traditional AP and HRP methods. The adsorbed substrate system, when used to detect IgE binding to allergic proteins, gives high resolution and delineates bands not otherwise seen. The system has advantages of high sensitivity, rapid development and conservation of immunochemicals. Problems of fading, sensitivity to heat and light, and high background can be solved with increased washing, prompt photography and computer scanning.

Selection of representative Alternaria strain groups on the basis of morphology, enzyme profile, and allergen content.

In an attempt to identify representative strains, 12 strains of Alternaria alternata were contributed by four different research groups. Each was grown on two types of solid media and characterized with descriptions of pigmentation and morphology. Enzyme profiles were determined on both the cellular antigen and the culture filtrate material with use of a commercial kit. Concentrations of a previously described Alt a 1 and a 70 kd allergen, GP70, were measured by a two-antibody "sandwich" ELISA. Allergenic proteins were visualized by IgE immunoblots. With use of these criteria, four separate strains were identified that might serve as representative of Alternaria for further research.

Bronchoalveolar lavage T-cell and Ia antigen quantitation by flow cytometry in acute and chronic experimental hypersensitivity pneumonitis.

Increasing evidence implicates effector T-cells in the pathogenesis of hypersensitivity pneumonitis. We utilized T-cell- and Ia-specific antibodies, flow cytometry, and computer analysis to quantitate T-cell numbers and Ia expression on lung cells of established rabbit models of acute and chronic hypersensitivity pneumonitis (HSP). We used analysis of variance (ANOVA) and Turkey's follow-up to evaluate group differences. In the acute HSP group, increased percentages of T-cells and greater Ia expression were present on bronchoalveolar lavage (BAL) cells at 24 h after inhalational allergen challenge. Increased BAL T-cell numbers were found in the chronic HSP group produced by repeated inhalation of antigen and muramyl dipeptide, compared to "desensitized" animals and control groups. Both chronic HSP and desensitized groups demonstrated increased Ia expression on BAL cells. Pathology scores for individual animals in both acute and chronic protocols correlated significantly (Pearson correlation coefficients) with total numbers of BAL cells, percentages of T-cells, and percentages of Ia-positive cells recovered. Findings are consistent with the hypothesis that cell-mediated hypersensitivity is a central mechanism in the pathogenesis of experimental hypersensitivity pneumonitis.

T cell localization in rabbit models of acute and chronic experimental hypersensitivity pneumonitis.

Cell-mediated hypersensitivity has been increasingly implicated in immunologic diseases of the lung, including hypersensitivity pneumonitis (HSP) (extrinsic allergic alveolitis). We used a T cell-specific monoclonal antibody (L11/135) to localize T cells in the parenchyma and bronchus-associated lymphoid tissue of ethanol-fixed, paraffin-embedded lung sections in rabbit models of experimental HSP to define further their possible role in pathogenesis. T cells appeared within 4 hours in early lesions of rabbit models of acute HSP and heavily infiltrated alveolitis lesions at 8 and 24 hours after aerosol challenge. T cells were also present in lesions of rabbit models with chronic alveolitis and occurred peripherally in granulomas. Variable aggregate and follicular forms of bronchus-associated lymphoid tissue rich in T cells occurred in both experimental and control animals. Our findings document early and continuing presence of T cells in lesions in rabbit models of experimental HSP.

Cyclosporine immunomodulation in a rabbit model of chronic hypersensitivity pneumonitis.

Rabbit models of chronic experimental hypersensitivity pneumonitis and desensitization were used to evaluate the effects of systemic cyclosporine. When administered 12 to 18 h before each inhalational challenge with aerosolized antigen and the adjuvant muramyl dipeptide, cyclosporine suppressed the development of disease as well as the anamnestic antibody response, particularly in bronchoalveolar lavage fluids. When administered at the time of sensitization only, cyclosporine suppressed the primary antibody response but not the anamnestic antibody response or the disease. Antigen- and mitogen-induced blastogenesis was inhibited by cyclosporine in vitro, but antigen-specific blastogenesis was not abrogated by cyclosporine previously administered in vivo. These results indicate that cyclosporine caused profound immunomodulation in this model, which can be at least partially explained by transient suppressive effects on T cells, particularly the helper/inducer and delayed hypersensitivity subset(s).