【正文】 Page 1 第1頁(yè) Monoclonal antibodies to human sex hormonebinding globulin 人類(lèi)性激素結(jié)合球蛋白的單克隆抗體 (SHBG): Characterization and use in a simple enzymelinked （SHBG）：表征在一個(gè)簡(jiǎn)單的酶聯(lián) immunosorbent assay (ELISA) of SHBG in plasma 性激素結(jié)合球蛋白（ELISA）檢測(cè)血漿中的免疫吸附試驗(yàn) John G. Lewis*, Nicholas J. Longley, Peter A. Elder 約翰G 劉易斯*，彼得A. 愛(ài)爾德Steroid amp。 Immunobiochemistry Laboratory, Clinical Biochemistry Unit, Canterbury Health Laboratories, Christchurch, New Zealand 類(lèi)固醇及免疫學(xué)生物化學(xué)實(shí)驗(yàn)室，臨床生化組，坎特伯雷衛(wèi)生實(shí)驗(yàn)室，新西蘭基督城 Received 22 May 1998。 accepted 23 November 1998 1998年5月22日，1998年11月 23日 Abstract 摘要 Four monoclonal antibodies to human sex hormonebinding globulin were raised and characterized. 提出了四個(gè)人類(lèi)性激素結(jié)合球蛋白的單克隆抗體和特點(diǎn) 。 四分之三Three of the four antibodies四分之三個(gè)抗體 recognised different antigenic determinants on SHBG. 公認(rèn)的SHBG不同的抗原決定簇 。 Two of the distinct antibodies were useful for Western blotting and recognized a 兩個(gè)不同的抗體用于免疫印跡，并確認(rèn) major 48 kDa band in human plasma as well as a 46 kDa minor ponent. 人體血漿中46 kDa的次要組成部分以及主要48 kDa區(qū)。 Carbohydrate residues do not form part of the antigenic 碳水化合物的殘留物不會(huì)形成抗原的一部分 determinants of these two antibodies, although one of these showed increased signal following removal of Nlinked oligosaccharides. 這兩種抗體的決定因素，盡管這些發(fā)現(xiàn)增加的信號(hào)，去除N 連接低聚糖。 Some在同一天有些 of the antibodies were selected to form a basis of a sameday, nonpetitive, enzymelinked immunosorbent assay (ELISA) for SHBG 被選中的抗體在HSBG的基礎(chǔ)上在血漿中，進(jìn)行非競(jìng)爭(zhēng)，酶聯(lián)免疫吸附試驗(yàn)（ELISA） in plasma.。 The assay employs a purified IgG2a SHBG monoclonal antibody adsorbed to the wells of a microtitre plate. 該試劑盒采用純化IgG2a性激素結(jié)合球蛋白單克隆抗體吸附到一個(gè)微孔板井。 After blocking any 在阻止任何物質(zhì)further adsorption to the plate, standards or diluted patient samples were added for a 5h incubation at room temperature, after which the進(jìn)一步吸附到板上，在室溫下分別加入標(biāo)準(zhǔn)或稀釋患者樣本經(jīng)過(guò)5 h的潛伏期后 plate was washed and antibodybound SHBG was detected with an antiSHBG IgG1 monoclonal antibody followed by peroxidaselabeled 板被沖走，反SHBG過(guò)氧化物酶標(biāo)記的IgG1單克隆抗體檢測(cè)抗體綁定SHBG antimouseIgG1 and o phenylenediamine substrate. antimouse IgG1和 鄰苯二胺底物。 The assay correlated well with an existing 2day ELISA for SHBG in plasma using與現(xiàn)有的2天ELISA檢測(cè)相關(guān)血漿SHBG使用 polyclonal antibodies and also correlated with a dihydrosterone (DHT) ligandbinding assay. 多克隆抗體，并與dihydrosterone（DHT）的配體結(jié)合法相關(guān) 。 The monoclonal antibodybased ELISA shows 單克隆抗體為基礎(chǔ)的ELISA顯示 excellent performance characteristics and is unaffected by added testosterone or estradiol. 性能優(yōu)良的特點(diǎn)，是增加睪丸激素或雌激素的影響 。 169。 1999 Elsevier Science Inc. All rights reserved. 169。 1999愛(ài)思唯爾科技公司保留所有權(quán)利。 Keywords: Monoclonal antibodies。 Sex hormonebinding globulin。 SHBG, ELISA 關(guān)鍵詞：?jiǎn)慰寺】贵w。性激素結(jié)合球蛋白SHBG，ELISA Plasma sex hormonebinding globulin (SHBG) levels are 血漿性激素結(jié)合球蛋白（SHBG）的水平 widely used together with sex steroid assays to determine 廣泛使用性激素檢測(cè)，以確定 the distribution of sex steroid hormones between protein 蛋白質(zhì)之間的分布性類(lèi)固醇激素 bound and free fractions [1]. 必然和自由的分?jǐn)?shù)[1] 。 It is apparent that low levels of 很明顯，水平低 SHBG may be associated with conditions of excessive an SHBG條件可能與過(guò)度的一個(gè) drogen action [2], and there is recent interest in the rela 氫行動(dòng)2]，且有在近期利益關(guān)系 tionship between SHBG and plasma lipoproteins [3]. 性激素結(jié)合球蛋白和血漿脂蛋白之間tionship [3]。 In 在 addition, the role of unliganded plasma steroidbinding pro 此外，unliganded血漿中的作用的類(lèi)固醇結(jié)合蛋白 teins bound to membrane receptors as a delivery mechanism 作為一個(gè)傳遞機(jī)制的約束膜受體蛋白 of steroids to target cells has attracted much attention [4,5] 靶細(xì)胞的類(lèi)固醇備受關(guān)注[4,5] and established a functional link to steroid hormone recep 并建立了一個(gè)功能鏈接到類(lèi)固醇激素受體 tors [6]. 職權(quán)范圍[6]。 The study of SHBG at the cellular level and in SHBG在細(xì)胞水平上，并在的研究 circulation can be enhanced by the use of monoclonal an 流通可以提高使用的單克隆 tibodies to SHBG. SHBG tibodies。 Here we report the production and char 在這里，我們報(bào)告的生產(chǎn)和char acterization of four monoclonal antibodies to human SHBG acterization四個(gè)單克隆抗體對(duì)人類(lèi)SHBG that may prove useful tools for the study of SHBG. SHBG研究可能證明有用的工具。 In 在 addition, we report their use in a sameday, simple, and 此外，我們?cè)谕惶?，?jiǎn)單報(bào)告其使用，并 direct enzymelinked immunosorbent assay (ELISA) for 直接酶聯(lián)免疫吸附試驗(yàn)（ELISA） human SHBG in plasma. 人類(lèi)血漿中SHBG。 Although others have reported 雖然其他人已經(jīng)報(bào)告 immunoradiometric and ELISA assays for SHBG [7,8], this SHBG [7,8]，放射免疫和酶聯(lián)免疫吸附檢測(cè) is also the first report of an ELISA using exclusively mono 也是第一份報(bào)告，使用專(zhuān)門(mén)的單聲道的 ELISA clonal antibodies. 單克隆抗體。 1. 1。 Experimental 實(shí)驗(yàn) . 。 Materials 材料 Human SHBG was obtained from Scripps Laboratories, 人類(lèi)性激素結(jié)合球蛋白是從斯克里普斯實(shí)驗(yàn)室獲得， San Diego, CA, USA and Antimouse IgG1peroxidase and 加利福尼亞州圣迭戈，美國(guó)和Antimouse IgG1的過(guò)氧化物酶和 antimouse IgG2aperoxidase from Nordic Immunology, Pil antimouse IgG2a北歐免疫過(guò)氧化物酶，弼 berg, Netherlands. 伯格，荷蘭。 Antimouse Igperoxidase and isotyping Antimouse免疫球蛋白過(guò)氧化物酶和isotyping kits were from Life Sciences, Amersham, UK. 從生命科學(xué)學(xué)院，英國(guó)Amersham公司，試劑盒。 Rabbit poly 兔聚。 clonal antibodies to human SHBG, both intact and peroxi 對(duì)人類(lèi)SHBG，都完好無(wú)損，過(guò)氧化物單克隆抗體 daselabeled, were from DAKO Corporation, Carpenteria, 案例分析標(biāo)記，DAKO公司，卡奔塔利亞， CA, USA. 美國(guó)加利福尼亞州。 * Corresponding author. *通訊作者。 PO Box 151, Christchurch, New Zealand. 郵政信箱151，新西蘭基督城。 Tel: 電話(huà)： 6433640888。 fax: 6433640889. 6433640888傳真：6433640889。 Email address: johnL2@ (JG Lewis) E mail地址：johnL2@（JG劉易斯） Steroids 64 (1999) 259–265 類(lèi)固醇64（1999）259265 0039128X/99/$ – see front matter 169。 1999 Elsevier Science Inc. All rights reserved. 0039128X/99 / $ 見(jiàn)前面問(wèn)題169。 1999愛(ài)思唯爾科技公司保留所有權(quán)利。 PII: S0039128X(98)001196 有價(jià)證券投資收益：S0039 128X（98）001196 Page 2 第2頁(yè) . 。 Immunization and cell fusion 免疫和細(xì)胞融合 Female RBFDN mice were immunized with 10 g of 女RBF DN小鼠免疫10克 SHBG in plete Freunds adjuvant at 4week intervals. 在完成Freunds輔助SHBG每隔4周。 One week after the third injection, spleens were excised and 一個(gè)星期后的第三次注射，脾臟切除 spleen cells fused with FOXNY myeloma cells at a ratio of FOX NY骨髓瘤細(xì)胞融合的比例脾細(xì)胞 5:1 as previously described [9]. 5:1如前所述[9]。 . 。 Screening of supernatants 篩選的上清 ELISA plates (Falcon 3912 microtest III。 Beckton Dick 酶標(biāo)板（獵鷹3912微量三。 Beck