Hashimoto Y, Ikeda I, Ikeda M, Takahashi Y, Hosaka M, Uchida H, Kono N, Fukui H, Makino T, Honjo M
Institute of Biological Science, Mitsui Pharmaceuticals, Mobara, Chiba, Japan.
J Immunol Methods. 1998 Dec 1;221(1-2):77-85. doi: 10.1016/s0022-1759(98)00157-4.
Twenty kilodalton human growth hormone (20K-hGH) is a naturally occurring isoform lacking amino acid residues 32-46 of 22K-hGH. Due to this structural similarity to 22K-hGH, no one has constructed a specific and sensitive assay system for 20K-hGH, which can be used for measuring physiological concentration of this isoform in the circulation. To construct such a specific assay system, we have generated polyclonal antibodies (pAb) and monoclonal antibodies (mAbs) against recombinant 20K-hGH. Binding characteristics to 20K-, 22K-hGH and monkey GH (mGH) of these five mAbs, designated A23, B13, C02, D05, and D14, were analyzed by enzyme immunoassays (EIAs) and surface plasmon resonance analysis. Only one of them, the mAb D05, does not crossreact with 22K-hGH. It was also observed that mAb B13 does not crossreact with mGH, although the later is 96% homologous to hGH. Using these antibodies we have established several sandwich EIA systems for circulating 20K-hGH. The combination of A23 as a solid-phase antibody and B13 as a labeled antibody permitted both high sensitivity to 20K-hGH (< 0.1 ng/ml) and low cross-reactivities with 22K-hGH (< 2%), mGH (< 0.3%) and rat GH (< 0.1%). The clearances of administered 20K-hGH were determined by this combination in both rats and monkeys. In the assay of physiologically circulating 20K-hGH in humans, the combination of D05 and affinity-purified anti-20K-hGH pAb showed the highest sensitivity to 20K-hGH (< 10 pg/ml) and substantially no cross-reactivity with 22K-hGH (< 0.1%). The plasma 20K-hGH concentration in healthy female subjects was determined by this combination. The assay systems constructed here enables us to directly measure circulating 20K-hGH in physiological condition with no interference of 22K-hGH for the first time.
二十千道尔顿人生长激素（20K-hGH）是一种天然存在的同工型，缺少22K-hGH的氨基酸残基32 - 46。由于其与22K-hGH在结构上的相似性，尚未有人构建出用于20K-hGH的特异性且灵敏的检测系统，该系统可用于测量循环中这种同工型的生理浓度。为构建这样一个特异性检测系统，我们制备了针对重组20K-hGH的多克隆抗体（pAb）和单克隆抗体（mAb）。通过酶免疫测定（EIA）和表面等离子体共振分析，对这五种分别命名为A23、B13、C02、D05和D14的单克隆抗体与20K-、22K-hGH以及猴生长激素（mGH）的结合特性进行了分析。其中只有一种单克隆抗体D05不与22K-hGH发生交叉反应。还观察到单克隆抗体B13不与mGH发生交叉反应，尽管mGH与hGH有96%的同源性。利用这些抗体，我们建立了几种用于检测循环中[具体内容缺失，疑似“20K-hGH”]的夹心EIA系统。以A23作为固相抗体和B13作为标记抗体的组合，对20K-hGH具有高灵敏度（<0.1 ng/ml），与22K-hGH（<2%）、mGH（<0.3%）和大鼠生长激素（<0.1%）的交叉反应性较低。通过该组合在大鼠和猴子中测定了所给予的20K-hGH的清除率。在检测人体生理循环中的20K-hGH时，D05和亲和纯化的抗20K-hGH pAb的组合对20K-hGH表现出最高灵敏度（<10 pg/ml），并且与22K-hGH基本无交叉反应（<0.1%）。通过该组合测定了健康女性受试者血浆中20K-hGH的浓度。此处构建的检测系统首次使我们能够在无22K-hGH干扰的生理条件下直接测量循环中的20K-hGH。
