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We have established a workflow allowing printing of allergens and control proteins into the wells of a MaxiSorp 96-well plate. Allergens are printed e.g., in a 4 × 4 pattern into each well of the plate. Prior to adding to the printed plates, a humanized rat basophil leukemia (RBL)-derived fluorescent reporter cell line (e.g., RBL NFAT DsRed) is sensitized overnight with the IgE-containing serum to be tested. Next, the humanized reporter cell line is added to the wells with the printed allergens and incubated for 24 h in a cell standard culture incubator. If the serum sample contains enough IgE specific for the printed allergens, NFAT-dependent signal transduction will occur, leading to synthesis of intracytosolic DsRed protein during an overnight incubation. Fluorescence can then be measured using a microplate reader using the well scan function.

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Use of Humanized NFAT-Dsred RBL Reporter for Detection of Allergen-Specific IgE Sensitization in Human Serum in 96-Well Microarray Format

  • 实验方案

  • 发表日期：

    2025年

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  • Daniel Wan,
  • Marcos J. C. Alcocer,
  • Franco H. Falcone

摘要

We have established a workflow allowing printing of allergens and control proteins into the wells of a MaxiSorp 96-well plate. Allergens are printed e.g., in a 4 × 4 pattern into each well of the plate. Prior to adding to the printed plates, a humanized rat basophil leukemia (RBL)-derived fluorescent reporter cell line (e.g., RBL NFAT DsRed) is sensitized overnight with the IgE-containing serum to be tested. Next, the humanized reporter cell line is added to the wells with the printed allergens and incubated for 24 h in a cell standard culture incubator. If the serum sample contains enough IgE specific for the printed allergens, NFAT-dependent signal transduction will occur, leading to synthesis of intracytosolic DsRed protein during an overnight incubation. Fluorescence can then be measured using a microplate reader using the well scan function.