Detection and inhibition of IgE antibodies reactive with cross-reactive carbohydrate determinants in an ELISA for allergen-specific IgE in horses.

BACKGROUND: It has been demonstrated that immunoglobulin (Ig)E specific for cross-reactive carbohydrate determinants (CCD) is present in the serum of sensitized humans, dogs and cats, and that these CCD-specific antibodies might confound serological testing. HYPOTHESIS/OBJECTIVE: The objective was to determine whether or not CCD-reactive antibodies occur in horses and to investigate the prevalence of CCD-reactive IgE antibodies in equine sera using a monoclonal cocktail-based enzyme-linked immunosorbent assay designed to detect allergen-specific IgE in horses, and to evaluate a means for successful inhibition of these CCD. METHODS AND MATERIALS: Sera from 28 horses suspected of clinical allergy were evaluated, with and without a proprietary inhibitor which contains carbohydrates derived from bromelain (BROM-CCD), using a panel of 72 allergens that include 15 grasses, 17 trees, nine weeds, five mites, 12 fungi, 12 insects and two environmental allergens. RESULTS: Twenty-five samples were shown to be reactive to at least one of the allergens, and 15 were reactive to 10 allergens or more. BROM-CCD had minimal effect on the mite reactivity in any of the positive samples; however, substantial inhibition for pollen allergens (trees, grasses and weeds) was demonstrable. Reduction in signal to pollens ranged from 20% to 100% for samples that were inhibited by CCD-BROM. CONCLUSIONS AND CLINICAL IMPORTANCE: These results demonstrate that CCD-reactive IgE antibodies are evident in horses and that BROM-CCD can be effective in reducing reactions with these irrelevant carbohydrates and will likely yield a more accurate in vitro allergen reactivity profile for selection of allergens included in an immunotherapeutic regime.

Detection and Inhibition of IgE for cross-reactive carbohydrate determinants evident in an enzyme-linked immunosorbent assay for detection of allergen-specific IgE in the sera of dogs and cats.

BACKGROUND: It has been demonstrated recently that immunoglobulin (Ig)E specific for cross-reactive carbohydrate determinants (CCD) is present in the serum of allergen-sensitized dogs and cats, and that these CCD-specific antibodies might confound serological testing. HYPOTHESIS/OBJECTIVE: The objective was to document the prevalence of CCD detectable in a monoclonal cocktail-based enzyme-linked immunosorbent assay designed for the detection of allergen-specific IgE in the sera of dogs and cats, and to define a means for successful inhibition of these CCD. METHODS AND MATERIALS: The incidence of reactivity to bromelain and a commercially available inhibitor of carbohydrate-specific antibodies (RIDA-CCD) was evaluated in 100 dog sera samples before and after inhibition with RIDA-CCD and a proprietary inhibitor containing carbohydrates derived from bromelain (BROM-CCD). Subsequently, sera from 600 dogs and 600 cats were evaluated using a serum diluent with and without BROM-CCD. RESULTS: Both the RIDA-CCD and BROM-CCD inhibitors demonstrated successful reduction of CCD reactivity, although a more efficient profile of inhibition was evident with BROM-CCD. Mite reactivity in dog and cat sera was largely unaffected; however, substantial inhibition for pollen allergens (trees, grasses and weeds) was shown. After BROM-CCD inhibition, 1% of canine samples and 13% of feline samples were rendered completely negative for allergen reactivity. CONCLUSIONS AND CLINICAL IMPORTANCE: The results demonstrate that BROM-CCD is effective in reducing reactions with irrelevant carbohydrates, and that inhibition of CCD reactivity might substantially alter the outcome of the in vitro reactivity profile used for selection of allergens to be included in an immunotherapeutic regime.

Performance characteristics of a monoclonal antibody cocktail-based ELISA for detection of allergen-specific IgE in dogs and comparison with a high affinity IgE receptor-based ELISA.

The purpose of this study was to define the operational and performance characteristics of a commercially available monoclonal antibody based (mac) ELISA for detection of allergen-specific IgE in dogs. The average intra-assay variance over 1 year was 9.7% (range 2.5-62.7%), while the interassay variance averaged 10.8% (range 8.1-13.8%). The average positive control responses observed for grass, weed, tree and mite allergens during each month remained relatively constant; the average monthly variance was 11.6% (range 8.3-19.2%) for grass pollens, 13.3% (range 9.1-20.4%) for weed pollens, 13.3% (range 9.8-18.2%) for tree pollens and 13.6% (range 8.9-18.7%) for mite allergens. The interlaboratory concordance of results for the macELISA was approximately 91%. The interlaboratory concordance of results comparing the macELISA and a high affinity IgE receptor-based ELISA was approximately 92%. The results demonstrate that the macELISA is reproducible and the results are comparable to the high affinity IgE receptor based ELISA within and between laboratories.

Intra and inter-laboratory reproducibility of a monoclonal antibody cocktail based ELISA for detection of allergen specific IgE in dogs: proficiency monitoring of macELISA in six laboratories.

The purpose of this study was to evaluate the reproducibility of results yielded using a monoclonal antibody based ELISA for detection of allergen specific IgE when run in six separate affiliated laboratories. On two separate occasions, duplicate samples of 15 different sera pools were independently evaluated by each laboratory in a single blinded fashion. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.2% (range 2.6-18.2%), while the average intra-laboratory inter-assay variance was 12.1% (range 8.0-17.1%). The overall inter-assay inter-laboratory variance was consistent among laboratories and averaged 15.6% (range 15.1-16.6%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose-response profiles observed in each of the laboratories were indistinguishable. Considering positive/negative results, inter-assay inter-laboratory concordance of results exceeded 95%. Correlation of OD values between and among all laboratories was strong (r>0.9, p<0.001). Correlation of OD values between the two separate evaluations was also high for all allergens except olive, which was attributed to lot-to-lot differences of allergen coated wells. Collectively, the results demonstrated that the monoclonal antibody based ELISA for measuring allergen specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory, but between laboratories using the same monoclonal-based ELISA.