The Journal of allergy and clinical immunology · 2009
Mediator release assay for assessment of biological potency of German cockroach allergen extracts.
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Nowak-Wegrzyn Anna H, Bencharitiwong Ramon, Schwarz John, David Gloria, Eggleston Peyton, Gergen Peter J, Liu Andrew H, Pongracic Jacqueline A, Sarpong Sampson, Sampson Hugh A
Аннотация
BACKGROUND: Cockroach is an important allergen in inner-city asthma. The diagnosis and treatment of cockroach allergy has been impeded by the lack of standardized cockroach extracts.
OBJECTIVE: We investigated the utility of a mediator release assay based on rat basophil leukemia (RBL) cells for comparing the potency of German cockroach extracts.
METHODS: RBL cells (line 2H3) transfected with human FcepsilonRI were passively sensitized with sera from subjects with cockroach allergy and stimulated with serial dilutions of 3 commercial cockroach extracts (1:10 weight/volume). In addition, the in-house prepared extract was tested in separate experiments with pooled sera that produced optimal performance in the RBL assay. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation and was examined in relation to the intradermal skin test (ID(50)EAL) and serum cockroach-specific and total IgE levels.
RESULTS: The median cockroach-specific IgE concentration in 60 subjects was 0.72 kU(A)/L (interquartile range, 0.35-2.97 kU(A)/L); 19 sera (responders) produced a minimum 10% NHR to more than 1 extract. Responders had higher median cockroach-specific IgE (7.4 vs 1.0 kU(A)/L) and total IgE (429 vs 300 kU/L) levels than nonresponders. Ranking of extract potency was consistent between the mediator release assay and the ID(50)EAL. For the in-house prepared cockroach extract, the dose-response curves were shifted according to the concentration of the extract. NHR was reproducible between different experiments by using pooled sera.
CONCLUSION: The mediator release assay measures biologic potency and correlates with the ID(50)EAL. It should be further evaluated to determine whether it could be used to replace intradermal skin test titration for assessing the potency of cockroach extract.
OBJECTIVE: We investigated the utility of a mediator release assay based on rat basophil leukemia (RBL) cells for comparing the potency of German cockroach extracts.
METHODS: RBL cells (line 2H3) transfected with human FcepsilonRI were passively sensitized with sera from subjects with cockroach allergy and stimulated with serial dilutions of 3 commercial cockroach extracts (1:10 weight/volume). In addition, the in-house prepared extract was tested in separate experiments with pooled sera that produced optimal performance in the RBL assay. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation and was examined in relation to the intradermal skin test (ID(50)EAL) and serum cockroach-specific and total IgE levels.
RESULTS: The median cockroach-specific IgE concentration in 60 subjects was 0.72 kU(A)/L (interquartile range, 0.35-2.97 kU(A)/L); 19 sera (responders) produced a minimum 10% NHR to more than 1 extract. Responders had higher median cockroach-specific IgE (7.4 vs 1.0 kU(A)/L) and total IgE (429 vs 300 kU/L) levels than nonresponders. Ranking of extract potency was consistent between the mediator release assay and the ID(50)EAL. For the in-house prepared cockroach extract, the dose-response curves were shifted according to the concentration of the extract. NHR was reproducible between different experiments by using pooled sera.
CONCLUSION: The mediator release assay measures biologic potency and correlates with the ID(50)EAL. It should be further evaluated to determine whether it could be used to replace intradermal skin test titration for assessing the potency of cockroach extract.