Development and basic performance verification of a rapid homogeneous bioassay for agonistic antibodies against the thyroid-stimulating hormone receptor

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods Pub Date : 2024-03-05 DOI:10.1016/j.jim.2024.113655

Motoki Hoshina , Shiomi Ojima , Atsushi Kawasaki , Kosuke Doi , Satoshi Ohta , Asuka Inoue , Hiroshi Murayama

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引用次数: 0

Abstract

Graves' disease is a type of autoimmune hyperthyroidism caused by thyroid-stimulating antibodies (TSAb).¹ The combination of a porcine thyroid cell bioassay and cyclic adenosine monophosphate (cAMP) immunoassay (TSAb-enzyme immunoassay; EIA) is a clinically approved TSAb measurement method. Due to the requirement of multiple procedures and a long assay time of 6 h in the TSAb-EIA, a simplified and rapid assay is desired. Herein, we developed a rapid homogeneous TSAb bioassay (rapid-TSAb assay) using the human embryonic kidney cell line (HEK293), engineered to express the human thyroid-stimulating hormone receptor (TSHR), along with a cAMP-dependent luminescence biosensor. The measurement consists of three steps: thawing frozen cells, blood sample addition, and luminescence detection. The procedures can be conducted within 1 h. The World Health Organization International Standard TSAb (NIBSC 08/204) stimulated the cells co-expressing TSHR and cAMP biosensor. The intra- and inter-assay coefficients of variance were < 10%. Stimulation activity using wild-type TSHR and chimeric TSHR (Mc4) almost completely correlated with the tested Graves' disease and normal samples. In the rapid-TSAb assay, the evaluation of 39 samples, including TSHR antibody-positive sera, yielded a sensitivity of 100.0% and a specificity of 90.9%, compared to the TSAb-EIA control. The rapid-TSAb assay enables simple and rapid measurement of TSAb and is promising for improving the diagnosis of autoimmune thyroid diseases.

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针对促甲状腺激素受体激动抗体的快速均相生物分析法的开发和基本性能验证。

巴塞杜氏病是一种由甲状腺刺激抗体（TSAb）引起的自身免疫性甲状腺机能亢进症1。由于 TSAb-EIA 需要多个步骤，且检测时间长达 6 小时，因此需要一种简化且快速的检测方法。在此，我们利用表达人类促甲状腺激素受体（TSHR）的人类胚胎肾细胞系（HEK293）和 cAMP 依赖性发光生物传感器，开发了一种快速均相 TSAb 生物检测法（快速 TSAb 检测法）。测量包括三个步骤：解冻冷冻细胞、加入血液样本和发光检测。这些步骤可在 1 小时内完成。世界卫生组织国际标准 TSAb（NIBSC 08/204）刺激了共同表达 TSHR 和 cAMP 生物传感器的细胞。测定内和测定间的方差系数为

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来源期刊

Journal of immunological methods 医学-免疫学

CiteScore

4.10

自引率

0.00%

发文量

120

审稿时长

3 months

期刊介绍： The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.