The number of radiolabeled antihuman IgE molecules that bind to the allergen is indicative of the amount of specific IgE in the patient's serum. If antibody is not present in excess in the serum, however, or if there is competing allergen-specific antibody of another isotype, interference can occur.
The enzyme-linked immunosorbent assay (ELISA) is analogous to RAST except for the detection system (Voller and Bidwell, 1986). In the case of ELISA, the antihuman IgE is conjugated to alkaline phosphatase, which catalyzes a colorimetric change proportional to the level of serum-specific IgE. The same interferences described above for RAST can occur with ELISA. In addition, false positives may occur in both assays if the serum has a high total level of IgE resulting in nonspecific binding of IgE with the allergen being tested. Several other immunoassays for specific IgE have been reported but are not widely used.
By using antihuman antibody directed to other immunoglobulin classes, such as antihuman IgG, ELISA can be used to demonstrate specific isotypes other than IgE. The clinical significance of such antibodies in asthma, however, is not clearly apparent. In cross-sectional studies of asthma and hypersensitivity pneumonitis, the presence of specific IgG has been interpreted as a biological marker of exposure (Biagini et al., 1990; Lushniak et al., 1990). There is also evidence, especially following immunotherapy with allergens, that antibody of a non-IgE isotype can be protective and prevent exposed individuals from manifesting IgE-mediated clinical sensitivity (Cooke et al., 1935).
In patients with hypersensitivity pneumonitis, precipitin assays can be used to demonstrate high titers of specific antibody. The most commonly employed assay of this type is the double-immuno-diffusion method of Ouchterlony (A. M. Johnson, 1986). In this assay, center and circumferential wells are cut in agar; sera are then placed in the circumferential wells, and the allergen is placed in the center. A precipitin band will form between the serum and the allergen wells if precipitating antibody, usually class IgG, is present in the serum. If a test serum is next to a positive control serum, researchers can determine whether the antibodies in the two sera recognize the same allergens, different allergens, or a combination; this judgment is based on the pattern of the precipitin lines, which show complete identity, nonidentity, or partial identity, respectively. Because commercial preparations of most hypersensitivity pneumonitis allergens are poorly standardized, the use of positive and negative control sera is critical. False-negative test results can also occur if a zone of equivalence (where precipitin lines form) is not obtained because of improper allergen concentration. Other techniques, including ELISA and the ammonium sulfate precipitation method of Lidd and Farr, have also been used as immunoassays for the evaluation of hypersensitivity pneumonitis (Lidd and Farr, 1962; Zeiss et al., 1977).