An avidin-biotin ELISA assay for the measurement of de novo human IgE synthesis in culture supernatants |
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| Authors: | Y G Alevy C M Blynn |
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| Affiliation: | 3. From the Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 and;4. the Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany;1. School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China;2. Innostar Haimen Site, Shanghai Innostar Bio-tech Co. Ltd., 100 Dongtinghu Road, Haimen, Jiangsu Province, 226133, China;3. Shanghai Innostar Bio-tech Co. Ltd., 199 Guoshoujing Road, Shanghai, 201203, China;1. State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Key Laboratory of Magnetic Resonance in Biological Systems, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences – Wuhan National Laboratory for Optoelectronics, Hubei Optics Valley Laboratory, Wuhan, 430071, China;2. University of Chinese Academy of Sciences, Beijing, 100049, China;1. Cell Polarity, Migration and Cancer Unit, Institut Pasteur - CNRS UMR 3691, Université Paris-Cité, Équipe Labellisée Ligue Nationale Contre le Cancer 2023, 25 rue du Docteur Roux, F-75015, Paris, France;2. Sorbonne Université, Collège Doctoral, 4 place Jussieu, F-75005 Paris, France;1. Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, Texas 75390-9152, USA;2. Institute for Quantum Life Science, National Institutes for Quantum Science and Technology (QST), 4-9-1, Anagawa, Inage-ku, Chiba, 263-8555, Japan |
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| Abstract: | A very sensitive (100 pg/ml) solid-phase enzyme immunoassay (ELISA) for the determination of human IgE has been developed. This assay incorporates the avidin-biotin system to increase sensitivity and can detect as little as 100 pg/ml (10 pg/test) of human IgE. The assay is highly specific and allows quantitative determination of human IgE in supernatants of peripheral blood lymphocytes as well as in serum. The very high sensitivity of the assay was accomplished by optimizing concentrations of the following reagents: (1) affinity-purified rabbit anti-human IgE coating antibodies; (2) biotin-conjugated goat anti-human IgE; (3) avidin-horseradish peroxidase (HRP) conjugate. In summary, the assay described is rapid (6 h), reproducible, isotype specific, and has the sensitivity of radioimmunoassays usually employed for the quantification of IgE. This assay may be utilized in establishing concentrations of in vitro IgE levels synthesized by human peripheral blood lymphocytes (PBL). |
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