【正文】 兩個不同的抗體用于免疫印跡，并確認 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)。 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進一步吸附到板上，在室溫下分別加入標準或稀釋患者樣本經(jīng)過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過氧化物酶標記的IgG1單克隆抗體檢測抗體綁定SHBG antimouseIgG1 and o phenylenediamine substrate. antimouse IgG1和 鄰苯二胺底物。 1999 Elsevier Science Inc. All rights reserved. 169。 SHBG, ELISA 關(guān)鍵詞：單克隆抗體。 The study of SHBG at the cellular level and in SHBG在細胞水平上，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。 1. 1。 Rabbit poly 兔聚。 Tel: 電話： 6433640888。 1999愛思唯爾科技公司保留所有權(quán)利。 . 。C with 100 inson，奧克斯納德，CA）分別涂在4 176。C. L / 510分鐘，以及在20 176。C. 上清液50 L的2 H進一步的潛伏期在20 176。 2 2 to 100 ml of M Na M娜 2 2 PO 寶 4 4 and 和 M citric acid, pH . M檸檬酸。 SHBG Dako ELISA SHBG Dako公司酶聯(lián)免疫吸附 The SHBG ELISA using Dako polyclonal antibodies SHBG使用Dako公司多克隆抗體的酶聯(lián)免疫吸附 was carried out as remended by the manufacturer. 制造商的建議進行了 。C. 1小時在20 176。 Molecular weight markers are indicated. 分子量標記表示。 Fig. 圖 2. 2。 The binding on the monoclonal antibodies to 單克隆抗體的結(jié)合 SHBG was detected by antimouse IgG1peroxidase and antimouse IgG2a SHBG檢測antimouse IgG1的過氧化物酶和antimouse IgG2a peroxidase (IgG1 and IgG2a, respectively). 過氧化物酶（分別為IgG1的和IgG2a） 。 C。 Fig. 圖 3. 3。 261 261 JG Lewis et al. JG劉易斯等人。 C。C. 刻劃為5 h在20 176。 C and subsequent processing as described. 和后續(xù)處理說明。 Briefly, plasma or standards were diluted 簡言之，血漿或標準進行稀釋 8fold with cold PBS and 400 l duplicate aliquots incu 用冷PBS和400升的8倍重復等分培育 bated with either 100 l of a mixture of ng DHT and 20 000 dpm 20 000 DPM 3 3 HDHT or 100 ng DHT and 20 000 dpm H DHT或100吳DHT和20 000 DPM 3 3 HDHT in PBS for 10 min at 4176。C after which the G）在4 176。 ProteinA chromatography 蛋白層析 Monoclonal antibodies to SHBG were purified from cul 性激素結(jié)合球蛋白單克隆抗體純化培養(yǎng) ture supernatants by chromatography on ProteinA by using TURE上清蛋白 A通過使用色譜 the Affi Gel Protein A Maps II kit (BioRad, Hercules, CA, 阿菲凝膠蛋白一個地圖II試劑盒（Bio Rad公司，大力士，CA， USA). 美國）。C until re 20℃，直到重新 quired. quired。 For antibody staining, 抗體染色， nitrocellulose was initially blocked by incubation in Tris 硝化棉最初封鎖潛伏期在三 buffered saline (TBS) ( M Tris, M NaCl, pH ) 緩沖生理鹽水（TBS）（ M氯化鈉（NaCl），中號三，） containing % Tween20 (v/v) and 1% bovine serum ％吐溫 20（V / V）和1％牛血清 albumin (BSA) (w/v) for 1 h at 20176。 C。 Finally, the nitrocellulose was washed and immuno 最后，硝化棉是洗滌和免疫 conjugates visualized by enhanced chemiluminescence 增強化學發(fā)光法通過可視化的結(jié)合物 (Amersham). （Amersham公司）。 The “ Table 2 表2 Effect of deglycosylation on purified SHBG levels: Removal of N and 純化的SHBG水平的deglycosylation影響：N的去除和 Olinked oligosaccharides O型掛鉤低聚糖 Detection antibody 檢測抗體 2G11 2G11 7H9 7H9 16D5 16D5 Deglycosylated SHBG Deglycosylated SHBG 114 114 11 11 145 145 14 14 218 218 16 16 Control SHBG 控制SHBG 110 110 10 10 113 113 11 11 106 106 11 11 SHGB was assayed by ELISA using 11F11 as the coating antibody and SHGB測定涂層抗體酶聯(lián)免疫吸附使用11F11和 the detection antibody as indicated. 檢測抗體的指示 。 / Steroids 64 (1999) 259–265 /類固醇64（1999）259265 Page 5 第5頁 following day, petition experiments to determine 翌日，競爭實驗，以確定 whether the IgG1 SHBG antibody supernatants (2G11, 7H9, 無論是IgG1的性激素結(jié)合球蛋白抗體上清（2G11，7H9 ， and 16D5) could block the subsequent binding of 11F11 16D5）能阻斷11F11隨后約束力 supernatant (isotype IgG2a), and vice versa were carried out 上清（同型IgG2a），反之亦然進行了 together with appropriate controls. 加上適當?shù)目刂啤?Deglycosylation of SHBG Deglycosylation的SHBG SHBG from third trimester, human, pregnancy plasma SHBG從孕晚期，人力，懷孕血漿 was partially purified by affinity chromatography using Pro 通過親和層析純化使用Pro tein A purified 11F11 antibody covalently linked to CNBr 蛋白質(zhì)純化的11F11抗體共價連接到CNBR activated Sepharose4B. 活化瓊脂糖 4B。 Following N and O 接下來的n 和O linked oligosaccharide release, plasma samples were as 掛鉤低聚糖的釋放，血漿樣品 sayed at three different dilutions in quadruplicate, and 賽義德在三個不同稀釋度一式四份， partially purified SHBG was assayed at one dilution in 部分純化的SHBG一稀釋法測定 duplicate. 重復。 Normal range plasma 正常范圍等離子體 Plasma for normal ranges was accessed from the 從訪問正常范圍內(nèi)的血漿 Christchurch Endolab reference range collected from con 基督城Endolab參考范圍收集CON senting adults on the electoral roll. senting的選民登記冊上的成年人 。 Results and discussion 結(jié)果與討論 The fusion protocol, together with screening and subse 融合協(xié)議，連同篩選和subse quent recloning, identified four hybridoma cell lines secret quent recloning，確定了4個雜交瘤細胞株的秘密 ing antibodies to SHBG. ING以性激素結(jié)合球蛋白的抗體 。 Two of the antibodies (11F11 and 7H9) were useful for 兩個抗體（11F11和7H9）是有用的 SDSPAGEWestern blotting, identifying a major 48K spe SDS PAGE電泳，Western印跡，確定一個主要的48K SPE cies and a minor 46K ponent in human plasma [12] with CIES和未成年人血漿中的46K組件[12 ] enhanced signal in human pregnancy plasma pared to 在人類懷孕等離子體增強的信號相比， normal adult male plasma [Fig. 正常成年男性血漿[圖。 1b]. 1B]。 Comparisons in the upper panel of Fig. 在上游面板圖進行比較 。 / Steroids 64 (1999) 259–265 /類固醇64（1999）259265 Page 6 第6頁 by antimouse IgG2aperoxidase, suggesting no interference antimouse IgG2a過氧化物酶，提示無干擾 by antibody 2G11. 抗體2G11。 Comparison of the Dako polyclonal SHBG ELISA to the Dako公司多克隆SHBG ELISA相比， monoclonal antibodybased SHBG ELISA for the 11F11/ 單克隆抗體為基礎(chǔ)的SHBG ELISA 11F11 / 16D5 bination and the 11F11/2G11 bination are 16D5組合和11F11/2G11結(jié)合 shown in Fig. 如圖。 SHBG determinations on SHBG測定上 partially purified and deglycosylated SHBG pared to 部分純化和deglycosylated SHBG相比 controls are shown in Table 2. 控制表2所示。 This is also the case with antibody 16D5, where even higher 這也是與抗體16D5的情況下，甚至更高 apparent levels of SHBG were measured following carbo 明顯的SHBG水平測定碳 hydrate removal. 水合物去