M.K. Agarwal

An immunochemical approach to indoor aeroallergen quantitation with a new volumetric air sampler : studies with mite, roach, cat, mouse, and guinea pig antigens

We describe a new high-volume air sampler for determining antigen concentrations in homes and illustrate its use for quantitating airborne house dust mite, cat, cockroach, mouse, and guinea pig antigens. The concentration of house dust-mite antigen was similar from houses in Rochester, Minn. and tenement apartments in Harlem, N. Y., but cockroach and mouse urinary proteins were present only in Harlem. The amount of cat or guinea pig antigen varied as expected with the number of pets in the home. In calm air the airborne concentration of mite and cat antigen was similar throughout the house but increased greatly in a bedroom when bedding was changed. In calm air most of the cat and mite antigens were associated with respirable particles less than 5 microns mean aerodynamic mass diameter, but in air sampled after the bedding was changed, more cat antigen was found in particles greater than 5 microns. The apparatus and technique described can provide objective data concerning the magnitude and the relative distribution and duration of suspended particles of defined sizes, which contain allergen activity.

Airborne ragweed allergens: Association with various particle sizes and short ragweed plant parts

These investigations were undertaken to size airborne particles by use of a high-volume sampler and to measure the short ragweed allergen activity of airborne particles of different sizes. We found both in vitro and in vivo short ragweed allergen activity in particles of all size ranges including greater than 6, 3 to 6, 1.8 to 3, 1 to 1.8, and 0.3 to 1.0 micron in diameter. Furthermore, we investigated various parts of the short ragweed plant as possible sources of allergen. Plant parts collected before, during, and after the pollination season demonstrated significant in vitro and in vivo allergen activities. We demonstrated allergen activity in various plant parts, especially the inflorescence, as late as November 30. Appreciable ragweed allergenic activity was also associated with particles less than 1 micron in diameter. Collectively, these observations suggest persistent ragweed plant debris in different sized particles as a source of allergen in the air before and after the ragweed pollination season. This may contribute to out-of-season symptoms observed in highly ragweed-sensitive individuals.

An immunochemical method to measure atmospheric allergens

Because particulate aeroallergens may exist in amorphous form as well as in pollen grains and fungal spores and because symptoms of allergic diseases presumably correlate with the total amount of allergen exposure, an immunochemical method of assay of aeroallergens would be useful. We report such a method based on (1) capture of airborne pollen, fungal spores, and amorphous particles 0.3 micrometer in diameter on fiberglass sheets with a high-volume air sampler; (2) elution of the sheets with buffered saline; and (3) analysis of eluate allergen content by radioallergosorbent test (RAST) inhibition assays. In preliminary indoor experiments we applied various quantities of short ragweed (SRW) pollen or dry Alternaria powder to the sheets while airflow was maintained at 1.19 m3/min. We compared techniques for extraction of allergen from the sheets, including homogenization, cutting and soaking, and descending elution of sheets. Although all three methods successfully extracted allergen from the sheets, an 8-hr descending elution procedure was optimal from the standpoint of yield and convenience. Eluates from filters exposed to as little as 4 mg of SRW pollen or Alternaria powder produced satisfactory RAST inhibition curves. When the sampler was operated outdoors continuously we could measure the atmospheric allergenic activity for both Alternaria and SRW from July to September. This allergenic activity was highly correlated with the traditional morphologic counts of airborne ragweed pollen and Alternaria spores.

Immunochemical quantitation of airborne short ragweed, Alternaria, antigen E, and Alt-I allergens: a two-year prospective study

We conducted a 2 yr prospective study to measure atmospheric short ragweed and Alternaria allergens by RAST inhibition analysis of eluates from filter sheets exposed in air samplers. In both years ragweed pollen and Alternaria spore counts, obtained with a rotoslide sampler, correlated significantly with immunochemically measured airborne ragweed and Alternaria allergenic activity. Airborne levels of the purified allergens AgE and Alt-I were successfully quantitated; these levels correlated closely with total airborne ragweed and Alternaria allergenic activities, respectively, and also with ragweed pollen and Alternaria spore counts. Eluates from filter sheets exposed during late summer and fall produced positive wheal-and-flare skin tests in patients with fall hay fever. In both years immunochemical measurements of allergenic activity due to airborne short ragweed correlated closely with mean symptom score indices in groups of short ragweed-sensitive individuals. Measurable levels of atmospheric ragweed allergenic activity were noted before and after the ragweed pollination season, and at these times we noted small increases in mean symptom score indices in the short ragweed-sensitive groups. Thus immunochemical analyses provide important information concerning levels of environmental allergens.

Immunochemical measurement of airborne mouse allergens in a laboratory animal facility

The major allergens from the mouse, including mouse serum albumin (MSA) and mouse urinary protein complex, are present in various concentrations in urine, serum, and pelts of mice. Subjects clinically sensitive to laboratory animals were screened by the RAST for specific IgE antibodies to mouse urine (MU), MSA, and mouse pelt extract (MPE). Twenty-four hour air samples from both a mouse-care room (2000 mice) and an immunology laboratory (five to 100 mice) were collected with a high-volume air sampler that retained particles greater than 0.3 micron with 95% efficiency. Eluates from the filter sheets were dialyzed, lyophilized, reconstituted in water, and used as fluid-phase inhibitors in the MU RAST and MPE RAST. MPE allergenic activity could be quantitated in filter eluates from both locations, but MU allergenic activity could be quantitated only in the mouse-care room. Airborne allergen content ranged from 1.8 to 825 ng/m3 and varied with both the number of mice and the degree of work activity in the rooms. The sampling and assay techniques described can be used to accurately quantitate amorphous airborne allergens.

Shared allergenic and antigenic determinants in Alternaria and Stemphylium extracts

Alternaria and Stemphylium extracts were compared by means of various immunochemical and biologic methods. Skin-test responses to Alternaria and Stemphylium in allergic patients were positively correlated, and RAST binding values with the sera of 30 patients utilizing solid-phase Alt-I, Alternaria (70% ammonium sulfate--precipitable fraction), and Stemphylium allergens showed significant correlation, giving evidence for the presence of common allergenic determinants among these three extracts. In RAST inhibition assays, Alternaria and Stemphylium extracts exhibited dose-related inhibiton with solid-phase Stemphylium, Alternaria, and Alt-I, confirming the presence of Alt-I as the major shared allergenic fraction in the two extracts. In repetitive absorption experiments, both Alt-I and Alternaria solid-phase allergens absorbed Stemphylium-specific IgE antibodies from a 12-person serum pool. Similarly, Stemphylium solid-phase allergen absorbed both Alternaria- and Alt-I--specific IgE antibodies. Double-antibody radioimmunoassay for Alt-I showed higher Alt-I activity in Stemphylium than in Alternaria extract. In double-immunodiffusion experiments with rabbit anti--Alt-I antibodies, both Stemphylium and Alternaria extracts produced precipitin lines of identity with Alt-I. The antigenic relationship of the two crude extracts was further confirmed by crossed and crossed-line immunoelectrophoresis experiments. Our results showed that Alternaria and Stemphylium extracts contain multiple shared allergenic and antigenic determinants, including Alt-I.

Allergen-controlled study of intranasal immunotherapy for ragweed hay fever

Previous studies of intranasal immunotherapy have not included control groups treated with an irrelevant allergen. In the present double-blind study, we tested the effectiveness of intranasal immunotherapy in 20 patients sensitive to both short ragweed (SRW) and orchard grass (OG). Patients sprayed increasing concentrations of either SRW (n = 11) or OG (n = 9) extract intranasally six times per day for 8 wk before the SRW pollination season. The effects of this treatment were determined by analysis of symptom score diaries and clinical examinations during the SRW pollination season. SRW-treated patients received cumulative AgE doses from 3 to 59 micrograms (mean 21); this mean dose was approximately sevenfold less than that used in a previous study from our laboratory. All patients reported immediate hay fever symptoms after use of the nasal spray. Five patients (four SRW- and one OG-treated) reported episodes of mild epistaxis during treatment; no other unexpected side effects were noted. During the treatment period, more SRW-treated patients showed signs of nasal obstruction and edematous nasal mucosa than OG-treated control patients (p less than 0.03). During the SRW pollination season, the SRW-treated patients reported lower mean weekly symptom scores than the OG-treated control patients, but the difference was not statistically significant. Supplemental antihistamine use was significantly higher (p less than 0.016) in the OG-treated control patients during the SRW pollination period. Subjective assessment of treatment efficacy by patients was similar in both treatment groups. We conclude that intranasal immunotherapy was of only marginal benefit in this study.

Immunochemical and physicochemical characterization of commercial Altemaria extracts: a model for standardization of mold allergen extracts

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p less than 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibiton using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibiton, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.

Occupational allergy to honeybee-body dust in a honey-processing plant

We studied a honey-plant employee who developed severe asthma coincident with the seasonal honey-packing process. Symptoms correlated with duration of exposure inside the plant during the honey pack and improved in other environments during that season. The patient was asymptomatic inside the plant at other times of the year. Skin tests to seasonal outdoor aeroallergens were negative, as were inhalation challenges with two insecticides used inside the building during the honey pack. Skin test, RAST, and bronchial provocation test with honeybee whole body extract were positive. We used high-volume air samplers to collect ambient airborne particles on filter sheets outside the patients home and inside the honey plant, both during and after the honey pack. Skin tests, RASTs, and bronchial provocation tests with eluates from these filters were positive only to eluate from the filter exposed inside the plant during the honey pack. The patients positive honeybee whole body RAST could be inhibited by preincubation of her serum with this filter eluate but not by preincubation with eluate from a filter exposed outside the patients home. Collectively, these data support an IgE-mediated, seasonal, occupational sensitivity to honeybee-body dust.

Occupational asthma in a florist caused by the dried plant, baby's breath☆

A 34-year-old gift shop owner with a history of virus-induced bronchospasm and seasonal allergic rhinitis developed daily asthma when he began working with dried plants. Extracts were made from these dried plants, including babys breath. German statice, leather leaf fern, and eucalyptus. Intradermal skin tests with these extracts were positive for the dried plant, babys breath (Gypsophilia panniculata). The patients serum contained elevated levels of IgE antibody to babys breath extract, his leukocytes released histamine after challenge in vitro with the babys breath extract, and bronchial challenge with babys breath extract produced greater than 20% fall in his FEV1 from baseline. He recovered fully after avoiding the plant. This is the first report of a case of IgE-mediated occupational asthma involving the dried plant, babys breath.