Antigenic extracts obtained from Callistemon citrinis (bottlebrush) and Melaleuca quinquenervia (melaleuca) pollen were analyzed by crossed immunoelectrophoresis (CIE), tandem-crossed immunoelectrophoresis (TCIE), and trans-blot enzyme-linked crossed immunoelectrophoresis (TECIE). CIE analysis detected 14 and 12 antigenic components in bottlebrush and melaleuca, respectively. TCIE analysis of bottlebrush pollen, employing rabbit anti-melaleuca serum in the intermediate gel, demonstrated that all of the antigenic components detected were cross-reactive. Similar analysis of melaleuca using rabbit anti-bottlebrush serum in the intermediate gel also showed that all of the antigenic components were cross-reactive. At least three bottlebrush and two melaleuca cross-reactive pollen components were demonstrated to be allergenic by TECIE analysis of CIE and TCIE gels.

Pollen extracts of two trees, Callistemon citrinis (bottlebrush) and Melaleuca leucadendron (melaleuca), as well as the grass Paspalum notatum (bahia) were analyzed for antigenic and allergenic cross-reactivity using SDS-PAGE and Western blotting. SDS-PAGE analysis of all three pollen extracts revealed multiple antigenic components which were reactive with rabbit antisera made to each pollen extract. Comparison of reduced and nonreduced mobility patterns suggested the possible presence of multichain proteins linked by disulfide bonds. Clinical studies demonstrated that 81% of the patients skin test positive to at least one of the pollens were also positive to the other two. Sixty-three percent of allergic individuals studied showed a high correlation between skin test results and the number of IgE-binding components analyzed by immunoblotting. These IgE-reactive components were detected in the molecular weight range of 29–66 kD. Western blot analysis detected more IgE-binding components in bahia pollen extracts as compared with the tree pollens, consistent with other reports of grass pollens being more allergenic than those from trees and weeds. Each patient’s serum had a unique IgE-binding pattern, indicating heterogeneity of immune response; however, common major determinants were detected by a large percentage of the allergic patient’s sera.

Aqueous extracts of both Callistemon citrinis (bottlebrush) and Melaleuca quinquenervia (melaleuca) were analyzed for allergenic cross-reactivity. Inhibition analysis using the radioallergosorbent test (RAST) was performed on the ammonium bicarbonate extracts of bottlebrush (NH₄B) and melaleuca (NH₄M) pollens. RAST inhibition analysis demonstrated that the extracts contained allergenically cross-reactive components. Sephadex G-100 column chromatography of NH₄B and NH₄M extracts resulted in at least 4 distinct peaks for each extract analyzed. These fractions were designated NH₄B1-NH₄B4 and NH₄M1-NH₄M4. A modified dot-blot assay for detection of allergenic components was utilized to show that the first elution peaks of bottlebrush and melaleuca, NH₄B1 and NH₄M1, respectively, contained allergenic components. These allergenic components, NH₄B1 and NH₄M1, had estimated molecular weights of 50,000 and 35,000 daltons, respectively.

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