Doping Control Center, Korea Institute of Science and Technology, Seoul, Republic of Korea.
Department of Microbial Engineering, College of Engineering, Konkuk University, Seoul, Republic of Korea.
Drug Test Anal. 2023 Nov-Dec;15(11-12):1439-1448. doi: 10.1002/dta.3562. Epub 2023 Sep 4.
Due to athletes' misuse of recombinant human growth hormone (rhGH) for performance improvement, the World Anti-Doping Agency has designated rhGH as a prohibited substance. This study focuses on the development and improvement of a simple and fast rhGH detection method using a fluorescence-incorporated antibody sensor "Quenchbody (Q-body)" that activates upon antigen binding. Camelid-derived nanobodies were used to produce stable Q-bodies that withstand high temperatures and pH levels. Notably, pituitary human growth hormone (phGH) comprises two major isoforms, namely 22 and 20 kDa GH, which exist in a specific ratio, and the rhGH variant shares the same sequence as the 22 kDa GH isoform. Therefore, we aimed to discriminate rhGH abuse by analyzing its specific isoform ratio. Two nanobodies, NbPit (recognizing phGH) and NbRec (preferentially recognizing 22 kDa rhGH), were used to develop the Q-bodies. Nanobody production in Escherichia coli involved the utilization of a vector containing 6xHis-tag, and Q-bodies were obtained using a maleimide-thiol reaction between the N-terminal of the cysteine tag and a fluorescent dye. The addition of tryptophan residue through antibody engineering resulted in increased fluorescence intensity (FI) (from 2.58-fold to 3.04-fold). The limit of detection (LOD) was determined using a fluorescence response, with TAMRA-labeled NbRec successfully detecting 6.38 ng/ml of 22 kDa rhGH while unable to detect 20 kDa GH. However, ATTO520-labeled NbPit detected 7.00 ng/ml of 20 kDa GH and 2.20 ng/ml 22 kDa rhGH. Q-bodies successfully detected changes in the GH concentration ratio from 10 to 40 ng/ml in human serum within 10 min without requiring specialized equipment and kits. Overall, these findings have potential applications in the field of anti-doping measures and can contribute to improved monitoring and enforcement of rhGH misuse, ultimately enhancing fairness and integrity in competitive sports.
由于运动员滥用重组人生长激素（rhGH）来提高运动成绩，世界反兴奋剂机构已将 rhGH 列为禁用物质。本研究专注于开发和改进一种简单快速的 rhGH 检测方法，使用一种荧光标记抗体传感器“Quenchbody（Q-body）”，该传感器在抗原结合时被激活。骆驼科纳米抗体被用于产生能够耐受高温和 pH 值的稳定 Q-body。值得注意的是，垂体人生长激素（phGH）由两种主要的同工型组成，即 22 和 20 kDa GH，它们以特定比例存在，而 rhGH 变体与 22 kDa GH 同工型具有相同的序列。因此，我们旨在通过分析其特定同工型比例来区分 rhGH 滥用。两种纳米抗体，NbPit（识别 phGH）和 NbRec（优先识别 22 kDa rhGH），被用于开发 Q-body。Escherichia coli 中的纳米抗体生产涉及使用含有 6xHis 标签的载体，并且 Q-body 通过半胱氨酸标签的 N 端与荧光染料之间的马来酰亚胺-巯基反应获得。通过抗体工程添加色氨酸残基导致荧光强度（FI）增加（从 2.58 倍增加到 3.04 倍）。使用荧光响应确定检测限（LOD），TAMRA 标记的 NbRec 成功检测到 6.38 ng/ml 的 22 kDa rhGH，而无法检测到 20 kDa GH。然而，ATTO520 标记的 NbPit 检测到 7.00 ng/ml 的 20 kDa GH 和 2.20 ng/ml 的 22 kDa rhGH。Q-body 无需专门的设备和试剂盒，在 10 分钟内成功检测到人血清中 GH 浓度比从 10 到 40 ng/ml 的变化。总体而言，这些发现具有在反兴奋剂措施领域的应用潜力，可以有助于改善 rhGH 滥用的监测和执行，最终提高竞技体育的公平性和完整性。
