人类性激素结合球蛋白的单克隆抗体

1、. . . . Page 1第1页Monoclonal antibodies to human sex hormone-binding globulin 人类性激素结合球蛋白的单克隆抗体(SHBG): Characterization and use in a simple enzyme-linked （SHBG）：表征在一个简单的酶联 immunosorbent assay (ELISA) of SHBG in plasma 性激素结合球蛋白（ELISA）检测血浆中的免疫吸附试验 John G. Lewis*, Nicholas J. Longley, Peter A. Elder 约翰G

2、·易斯*，尼古拉斯J.朗利，彼得A. 爱尔德Steroid & Immunobiochemistry Laboratory, Clinical Biochemistry Unit, Canterbury Health Laboratories, Christchurch, New Zealand类固醇与免疫学生物化学实验室，临床生化组，坎特伯雷卫生实验室，新西兰基督城 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 antibod

3、ies to human sex hormone-binding globulin were raised and characterized. 提出了四个人类性激素结合球蛋白的单克隆抗体和特点 。 四分之三Three of the four antibodies四分之三个抗体 recognised different antigenic determinants on SHBG. 公认的SHBG不同的抗原决定簇 。 Two of the distinct antibodies were useful for Western blotting and recognized a 两个不同的抗体用

4、于免疫印迹，并确认 major 48 kDa band in human plasma as well as a 46 kDa minor component. 人体血浆中46 kDa的次要组成部分以与主要48 kDa区。 Carbohydrate residues do not form part of the antigenic 碳水化合物的残留物不会形成抗原的一部分 determinants of these two antibodies, although one of these showed increased signal following removal of N-linke

5、d oligosaccharides. 这两种抗体的决定因素，尽管这些发现增加的信号，去除N -连接低聚糖。 Some在同一天有些 of the antibodies were selected to form a basis of a same-day, non-competitive, enzyme-linked immunosorbent assay (ELISA) for SHBG 被选中的抗体在HSBG的基础上在血浆中，进行非竞争，酶联免疫吸附试验（ELISA） in plasma.。 The assay employs a purified IgG2a SHBG monoclona

6、l antibody adsorbed to the wells of a microtitre plate. 该试剂盒采用纯化IgG2a性激素结合球蛋白单克隆抗体吸附到一个微孔板井。 After blocking any 在阻止任何物质further adsorption to the plate, standards or diluted patient samples were added for a 5-h incubation at room temperature, after which the进一步吸附到板上，在室温下分别加入标准或稀释患者样本经过5 h的潜伏期后 plate

7、was washed and antibody-bound SHBG was detected with an anti-SHBG IgG1 monoclonal antibody followed by peroxidase-labeled 板被冲走，反SHBG过氧化物酶标记的IgG1单克隆抗体检测抗体绑定SHBG antimouse-IgG1 and o -phenylenediamine substrate. antimouse IgG1和 邻苯二胺底物。 The assay correlated well with an existing 2-day ELISA for SHBG in

8、 plasma using与现有的2天ELISA检测相关血浆SHBG使用 polyclonal antibodies and also correlated with a dihydrosterone (DHT) ligand-binding assay. 多克隆抗体，并与dihydrosterone（DHT）的配体结合法相关 。 The monoclonal antibody-based ELISA shows 单克隆抗体为基础的ELISA显示 excellent performance characteristics and is unaffected by added testoster

9、one or estradiol. 性能优良的特点，是增加睾丸激素或雌激素的影响 。 © 1999 Elsevier Science Inc. All rights reserved. © 1999爱思唯尔科技公司保留所有权利。 Keywords: Monoclonal antibodies; Sex hormone-binding globulin; SHBG, ELISA关键词：单克隆抗体;性激素结合球蛋白SHBG，ELISA Plasma sex hormone-binding globulin (SHBG) levels are 血浆性激素结合球蛋白（SHBG）的水

10、平 widely used together with sex steroid assays to determine 广泛使用性激素检测，以确定 the distribution of sex steroid hormones between protein- 蛋白质之间的分布性类固醇激素 bound and free fractions 1. 必然和自由的分数1 。 It is apparent that low levels of 很明显，水平低 SHBG may be associated with conditions of excessive an- SHBG条件可能与过度的一个

11、drogen action 2, and there is recent interest in the rela- 氢行动2，且有在近期利益关系 tionship between SHBG and plasma lipoproteins 3. 性激素结合球蛋白和血浆脂蛋白之间tionship 3。 In 在 addition, the role of unliganded plasma steroid-binding pro- 此外，unliganded血浆中的作用的类固醇结合蛋白 teins bound to membrane receptors as a delivery mechani

12、sm 作为一个传递机制的约束膜受体蛋白 of steroids to target cells has attracted much attention 4,5 靶细胞的类固醇备受关注4,5 and established a functional link to steroid hormone recep- 并建立了一个功能到类固醇激素受体 tors 6. 职权围6。 The study of SHBG at the cellular level and in SHBG在细胞水平上，并在的研究 circulation can be enhanced by the use of monoclo

13、nal an- 流通可以提高使用的单克隆 tibodies to SHBG. SHBG tibodies。 Here we report the production and char- 在这里，我们报告的生产和char acterization of four monoclonal antibodies to human SHBG acterization四个单克隆抗体对人类SHBG that may prove useful tools for the study of SHBG. SHBG研究可能证明有用的工具。 In 在 addition, we report their use in

14、 a same-day, simple, and 此外，我们在同一天，简单报告其使用，并 direct enzyme-linked immunosorbent assay (ELISA) for 直接酶联免疫吸附试验（ELISA） human SHBG in plasma. 人类血浆中SHBG。 Although others have reported 虽然其他人已经报告 immunoradiometric and ELISA assays for SHBG 7,8, this SHBG 7,8，放射免疫和酶联免疫吸附检测 is also the first report of an ELI

15、SA using exclusively mono- 也是第一份报告，使用专门的单声道的 ELISA clonal antibodies. 单克隆抗体。 1.1。Experimental实验。Materials材料Human SHBG was obtained from Scripps Laboratories, 人类性激素结合球蛋白是从斯克里普斯实验室获得， San Diego, CA, USA and Antimouse IgG1-peroxidase and 加利福尼亚州圣迭戈，美国和Antimouse IgG1的过氧化物酶和 antimouse IgG2a-peroxid

16、ase from Nordic Immunology, Pil- antimouse IgG2a北欧免疫过氧化物酶，弼 berg, Netherlands. 伯格，荷兰。 Antimouse Ig-peroxidase and isotyping Antimouse免疫球蛋白过氧化物酶和isotyping kits were from Life Sciences, Amersham, UK. 从生命科学学院，英国Amersham公司，试剂盒。 Rabbit poly- 兔聚; clonal antibodies to human SHBG, both intact and peroxi- 对人

17、类SHBG，都完好无损，过氧化物单克隆抗体 dase-labeled, were from DAKO Corporation, Carpenteria, 案例分析标记，DAKO公司，卡奔塔利亚， CA, USA. 美国加利福尼亚州。 * Corresponding author. *通讯作者。 PO Box 151, Christchurch, New Zealand. 邮政信箱151，新西兰基督城。 Tel: ： 64-3-364-0888; fax: 64-3-364-0889. 64-3-364-0888 ：64-3-364-0889。 E-mail address: johnL2chh

18、t.nz (JG Lewis)E - mail地址：johnL2t.nz（JG易斯） Steroids 64 (1999) 259265 类固醇64（1999）259-265 0039-128X/99/$ see front matter © 1999 Elsevier Science Inc. All rights reserved. 0039-128X/99 / $ -见前面问题© 1999爱思唯尔科技公司保留所有权利。 PII: S0039-128X(98)00119-6 有价证券投资收益：S0039 - 128X（98）00119-

19、6 Page 2第2页。Immunization and cell fusion免疫和细胞融合Female RBF-DN mice were immunized with 10 g of 女RBF - DN小鼠免疫10克 SHBG in complete Freunds adjuvant at 4-week intervals. 在完成Freunds辅助SHBG每隔4周。 One week after the third injection, spleens were excised and 一个星期后的第三次注射，脾脏切除 spleen cells fused with FOX

20、-NY myeloma cells at a ratio of FOX - NY骨髓瘤细胞融合的比例脾细胞 5:1 as previously described 9. 5:1如前所述9。 。Screening of supernatants筛选的上清ELISA plates (Falcon 3912 microtest III; Beckton Dick- 酶标板（猎鹰3912微量三; Beckton迪克 inson, Oxnard, CA) were coated overnight at 4°C with 100 inson，奥克斯纳德，CA）分别涂在4 

21、6; C过夜100 l/well rabbit anti-SHBG serum, diluted in phosphate-buff- L /以与兔抗- SHBG血清，稀释的磷酸 BUFF ered saline (PBS), 29 l of antibody in 10 ml PBS. ERED等效液（PBS），29升的抗体在10毫升PBS。 Phos- 磷 phate-buffered saline was 0.05 M NaH phate，缓冲液为0.05 M的NaH 2 2 PO 宝 4 4 , 0.15 M NaCl, ，0.15 M氯化钠（NaCl）， adjusted to pH

22、 7.4 with 5 M NaOH. 5个M氢氧化钠调整pH值至7.4。 The following day, the 次日， plates were washed four times with PBS containing 0.1% 板洗涤用PBS含0.1的四倍 Tween-20 (v/v) and blocked with assay buffer PBS con- 吐温20（V / V）和阻止缓冲液PBS CON - taining 0.1% Tween-20 (v/v) and 0.1% gelatin (w/v) 150 含0.1的Tween - 20（V / V）和0.1明胶（

23、W / V） 150 l/well for 510 min at 20°C. L / 5-10分钟，以与在20 ° C After the plates were emptied, 后板被掏空， 100 l of diluted, pooled, human pregnancy plasma (1:1000 100升稀释，汇集，人类妊娠血浆（1:1000 in assay buffer) containing elevated levels of SHBG (400 在缓冲液中），其中包含的SHBG水平升高（ 400 nmol/l) was added to each we

24、ll for an overnight incubation nmol / L的），每孔加入孵育过夜 at 4°C. 在4 ° C The following day, the plates were again washed, 翌日，板块再次洗净， and 50 l of assay buffer was added to each well, followed 缓冲液和50升，每孔加入，其次 by 50 l of supernatant for a further 2-h incubation at 20°C. 上清液50 L的2 - H进一步的潜伏期在20

25、° C The plates were washed four times, and sheep antimouse 板洗四次，羊antimouse Ig-peroxidase was added (100 l/well at 1:1000 in assay 免疫球蛋白过氧化物酶（100升/以与在1:1000在检测 buffer) for 1 h at 20°C, after which the plates were washed, 缓冲区）在20小时1，分别洗净后，板， and substrate solution was added. 和底物溶液加入。 The sub

26、strate was freshly 基板是新鲜 prepared by the addition of 40 mg of o -phenylenediamine 编制由40毫克 邻苯二胺 and 60 l of 30% H 60升30的H 2 2 O Ø 2 2 to 100 ml of 0.05 M Na 100毫升0.05 M娜 2 2 PO 宝 4 4 and 和 0.025 M citric acid, pH 5.0. 0.025 M柠檬酸，pH值5.0。 Color development was termi- 颜色发展端子 nated by the addition o

27、f 100 l of 1.25 MH 提早终止100升1.25氢 2 2 SO SO 4 4 per well, 每口井， and absorbance was read at 492 nm. 在492 nm处读取吸光度。 Using of a Behring 使用一个贝林 ELISA Processor-II (Hoechst, Germany) allowed semi-au- ELISA处理器II（德国赫司特，）允许半AU - tomation of processes. 进程tomation的。 。SHBG Dako ELISASHBG Dako公司酶联免疫吸附The S

28、HBG ELISA using Dako polyclonal antibodies SHBG使用Dako公司多克隆抗体的酶联免疫吸附 was carried out as recommended by the manufacturer. 制造商的建议进行了 。 Briefly, microtitre plates were coated overnight at 4°C with 简而言之，包被酶标板4，过夜 intact rabbit anti-SHBG serum diluted in PBS (29 l in 10 完整的兔抗SHBG血清稀释在PBS（29日升 10 ml P

29、BS) 100 l per well. 毫升PBS）中，每孔100升。 The following day the plates were 翌日板 washed and blocked with assay buffer (150 l per well) for 洗涤缓冲液（150升，每孔）阻塞 1 h at 20°C. 1小时在20 ° C The plates were then emptied by inversion, and 板倒置，然后清空 Fig. 图 1. 1。 (a) SDS-PAGE Western blotting of human pregnancy

30、 plasma, lane （一）SDS - PAGE电泳免疫印迹人类妊娠血浆，车道 1, and human male plasma, lane 2, probed with antibody 11F11. 1，人类男性的血浆，2巷，探讨抗体11F11 。 Lanes 车道 contained identical sample loadings and the presence of mercaptoethanol. 载有一样的样品负荷和巯基乙醇存在。 Molecular weight markers are indicated. 分子量标记表示。 (b) SDS-PAGE Western

31、blotting （二）SDS - PAGE电泳免疫印迹 of partially purified human SHBG following deglycosylation, lanes 1 and 部分纯化的人类SHBG以下deglycosylation，泳道1和 3, probed with antibodies 11F11 and 7H9, respectively. 3，探测与抗体11F11和7H9，分别。 Partially purified 部分纯化 non-deglycosylated human SHBG, lanes 2 and 4, probed with antibod

32、ies 非deglycosylated人类SHBG，2和4车道，探讨与抗体 11F11 and 7H9 as indicated. 11F11和7H9表示。 Lanes contained identical sample loadings 车道包含一样的样品负荷 and the presence of mercaptoethanol. 巯基乙醇存在。 Fig. 图 2. 2。 Relative antigenic determinant mapping. 相对的抗原决定簇映射 。 ELISA plates coated with 酶标板涂有 polyclonal SHBG antibody

33、and the addition of standards 0 to 400 nmol/l. 性激素结合球蛋白多克隆抗体和0到400 nmol / L的标准除了 Polyclonal antibody-bound SHBG sequentially probed with monoclonal 多克隆抗体绑定SHBG顺序探讨用单克隆 antibodies, as indicated, with wash steps between each antibody. 抗体，如前所述，每个抗体之间的洗涤步骤 。 Mono- 单 clonal antibodies to SHBG are 2G11, 7

34、H9, 16D5 (isotypes IgG1), and 对性激素结合球蛋白的单克隆抗体2G11，7H9，16D5（亚型IgG1的），并 11F11 (isotype IgG2a). 11F11（亚型IgG2a）。 The binding on the monoclonal antibodies to 单克隆抗体的结合 SHBG was detected by antimouse IgG1-peroxidase and antimouse IgG2a- SHBG检测antimouse IgG1的过氧化物酶和antimouse IgG2a - peroxidase (IgG1 and IgG2

35、a, respectively). 过氧化物酶（分别为IgG1的和IgG2a） 。 260 260 JG Lewis et al.JG易斯等。/ Steroids 64 (1999) 259265/类固醇64（1999）259-265Page 3第3页100 l of SHBG standard plasma or patient plasma (1:1000 SHBG 100升的标准血浆或患者血浆（1:1000 dilution in assay buffer) was added to each well for over- 缓冲液稀释）以上，每孔加入 night incubation a

36、t 4°C. 晚上潜伏期在4 ° C。 The SHBG standard plasma was SHBG标准血浆 derived from pooled, female, third trimester, pregnancy 来自池，女，孕晚期，怀孕 plasma having an SHBG level of 400 nmol/l. 血浆中有400 nmol / L的SHBG水平 The standard 标准 plasma was initially diluted 1:1000 in assay buffer and then 等离子最初是在缓冲液稀释1:1000和

37、然后 serially to generate a series of standards from 0 to 400 连续生成了一系列的标准，从0到 400 nmol/l. nmol / L的 The plates were again washed, and peroxidase- 板块再次洗净，过氧化物酶 labeled rabbit anti-SHBG serum (1:3000 in assay buffer) 标记的兔抗血清性激素结合球蛋白（缓冲液1:3000） 100 l was added per well for 6 h incubation at 20°C. 在2

38、0 ° C孵育6小时，每孔100升 The “ plates were finally washed, and o -phenylenediamine sub- 板终于洗净， 邻苯二胺分 strate was added. strate上架。 Fig. 图 3. 3。 Comparison of plasma SHBG levels determined by the Dako ELISA using SHBG polyclonal antibodies and by ligand binding with plasma SHBG Dako公司酶联免疫吸附测定血浆SHBG水平，使用性

39、激素结合球蛋白多克隆抗体和血浆性激素结合球蛋白结合配体的比较 levels determined by ELISA using SHBG monoclonal antibodies of either the 11F11/16D5 (panels a and c) or 11F11/2G11 (panels b and d) combinations. ELISA法测定的11F11/16D5（面板A和C）或11F11/2G11（板B和D）的组合使用性激素结合球蛋白单克隆抗体的水平。 Table 1 表1 Effect of deglycosylation on serum SHBG level

40、s: Removal of N- and O-linked oligosaccharides deglycosylation血清SHBG水平的影响：去除N和O -连接寡糖 Detection antibody 检测抗体 2G11 2G11 7H9 7H9 16D5 16D5 Sample dilution 样品稀释 0.5 0.5 1.0 1.0 2.0 2.0 0.5 0.5 1.0 1.0 2.0 2.0 0.5 0.5 1.0 1.0 2.0 2.0 Deglycosylated Deglycosylated serum 血清 48 48 2 2 90 90 4 4 184 184 3

41、3 81 81 8 8 134 134 2 2 259 259 8 8 69 69 1 1 120 120 12 12 250 250 1 1 Control serum 对照血清 40 40 1 1 80 80 4 4 160 160 10 10 43 43 10 10 103 103 14 14 203 203 24 24 42 42 2 2 79 79 1 1 154 154 2 2 SHBG was assayed by ELISA using 11F11 as the coating antibody with the detection antibody as indicated.

42、 SHBG是通过检测抗体酶联免疫吸附使用11F11作为涂层抗体检测。 Values (nmol/l) are means of quadruplicates 值（nmol / L的）quadruplicates手段 SD. 政府统计处。 261 261 JG Lewis et al.JG易斯等人。/ Steroids 64 (1999) 259265/类固醇64（1999）259-265Page 4第4页。SHBG ELISA (monoclonal antibodies)SHBG ELISA法（单克隆抗体）Microtitre plates were coated with p

43、rotein A purified 酶标板包被蛋白的纯化 monoclonal antibody 11F11 diluted 1:1000 in 6 M aqueous 单克隆抗体11F11稀释1:1000 6米水 guanidine HCl (1.0 g/ml), 100 l per well, overnight at 盐酸胍（1.0克/毫升），100升，每孔过夜， 4°C. 4 ° C。 Plates were then washed and blocked in assay buffer 然后冲板和缓冲液受阻 for 1 h at 20°C. 在20 &#

44、176; C 1小时 The plates were emptied by inversion, and 板被掏空颠倒， either standards or samples were added as previously de- 无论是标准或样品分别加入先前 scribed for 5 h at 20°C. 刻划为5 h在20 ° C Plates were then washed four times, 板，然后洗4次， and 100 l monoclonal antibody supernatant 16D5 (1:20 in 和100升的单克隆抗体上清16D5

45、（1:20在 assay buffer) was added to each well for 30 min at 20°C. 缓冲液）20时为30分钟，每孔加入 The plates were washed and antimouse IgG1-peroxidase 板清洗和antimouse IgG1的过氧化物酶 added (1:1000 in assay buffer) for a further 30 min at 20°C 添加（1:1000缓冲液）为20分钟，另有30 ° C and subsequent processing as describe

46、d. 和后续处理说明。 A series of exper- 一个系列exper， iments measuring SHBG in male and female plasma that iments测量在男性和女性血浆的性激素结合球蛋白 contained increasing amounts of either added testosterone 或者补充睾酮所载越来越多 (0.7347 nmol/l) or estradiol (0.7368 nmol/l) to determine （0.7-347 nmol / L的）或雌二醇（0.7-368 nmol / L的），以确定 whe

47、ther the apparent SHBG level was affected by addition 有无明显的SHBG水平是受另外 of these steroids were carried out. 这些类固醇进行。 。SHBG ligand-binding assaySHBG配约束力的检测Plasma SHBG was also assayed by a ligand-binding 血浆SHBG是由配体结合检测 method 10. 法10。 Briefly, plasma or standards were diluted 简言之，血浆或标准进行稀释 8-fol

48、d with cold PBS and 400 l duplicate aliquots incu- 用冷PBS和400升的8倍重复等分培育 bated with either 100 l of a mixture of 0.75 ng DHT and bated同为100升0.75纳克的DHT的混合物和 20 000 dpm 20 000 DPM 3 3 H-DHT or 100 ng DHT and 20 000 dpm H - DHT或100吴DHT和20 000 DPM 3 3 H-DHT in PBS for 10 min at 4°C. PBS 10分钟的H - DHT在

49、4 ° C。 An equal volume of 等体积 cold, saturated ammonium sulfate was added, mixed, and 冷，饱和硫酸铵添加，混合，和 incubated for 10 min (1500 孵育10分钟（1500 g ) at 4°C after which theG）在4 ° C tubes were centrifuged and the supernatant counted. 管，离心，取上清液计算。 Radio- 无线电 active counts from the first set of

50、 tubes were corrected for 从第一套管积极计数更正 quenching by using the second set of tubes and results 所使用的管和结果第二组的淬火 interpolated on the standard curve. 插上的标准曲线。 。Protein-A chromatography蛋白层析Monoclonal antibodies to SHBG were purified from cul- 性激素结合球蛋白单克隆抗体纯化培养 ture supernatants by chromatography on

51、Protein-A by using TURE上清蛋白- A通过使用色谱 the Affi Gel Protein A Maps II kit (Bio-Rad, Hercules, CA, 阿菲凝胶蛋白一个地图II试剂盒（Bio - Rad公司，大力士，CA， USA). 美国）。 This system allowed the purification of up to 10 mg 这个系统允许的净化至10毫克 of mouse IgG from 200 ml of culture supernatant. 鼠标从200毫升培养上清抗体。 Follow- 后续 ing extensive

52、dialysis against running water, the purity of ING广泛透析对自来水的纯度 isolated mouse IgG was confirmed by agarose gel electro- 孤立鼠IgG证实了琼脂糖凝胶电 phoresis at pH 8.9. 电泳在pH值8.9。 The purified immunoglobulin was 纯化的免疫球蛋白 lyophilised in glass vials and stored at 冻干玻璃小瓶，并存储在 20°C until re- 20，直到重新 quired. quire

53、d。 。SDS-PAGE-Western blottingSDS - PAGE电泳，Western印迹Vertical sodium dodecyl sulfate-polyacrylamide gel 垂直十二烷基硫酸钠聚丙烯酰胺凝胶 electrophoresis (SDS-PAGE) was carried out in 10% poly- 电泳（SDS - PAGE）进行了10聚 acrylamide gels 11 with identical loadings of human 丙烯酰胺凝胶11与人类一样的负荷 plasma and using the Bio-Rad

54、 Mini Protean System. 血浆和使用Bio - Rad公司的迷你Protean的系统 。 Trans- 横贯 fer to nitrocellulose was verified by the use of Bio-Rad FER至硝酸纤维素核实使用Bio - Rad公司 Kaleidoscope Pre-stained markers. 万花筒预染色标记。 For antibody staining, 抗体染色， nitrocellulose was initially blocked by incubation in Tris- 硝化棉最初封锁潜伏期在三 buffered

55、 saline (TBS) (0.015 M Tris, 0.15 M NaCl, pH 7.4) 缓冲生理盐水（TBS）（0.015 0.15 M氯化钠（NaCl），中号三，pH值7.4） containing 0.1% Tween-20 (v/v) and 1% bovine serum 含0.1吐温- 20（V / V）和1牛血清 albumin (BSA) (w/v) for 1 h at 20°C. 白蛋白（BSA）（W / V）20 ° C时为1小时 The nitrocellulose 硝酸纤维素 was then washed (three times) i

56、n TBS containing 0.1% 当时含0.1的TBS洗（三次） Tween-20 and incubated with culture supernatant diluted Tween - 20与文化稀释上清孵育 1:20 in TBS containing 0.1% Tween-20 and 1% BSA over- 1点20含有超过0.1的Tween - 20和1BSA的TBS night at 20°C. 夜间在20 ° C。 Nitrocellulose was then washed (three times) 硝化棉是洗（三次） and incub

57、ated with antimouse Ig-peroxidase (1:10 000 in 孵育antimouse免疫球蛋白过氧化物酶（1:10 000 TBS containing 0.1% Tween-20 and 1% BSA) for 1 h at TBS的含0.1Tween - 20与1BSA）1 h后 20°C. 20 ° C。 Finally, the nitrocellulose was washed and immuno- 最后，硝化棉是洗涤和免疫 conjugates visualized by enhanced chemiluminescence 增

58、强化学发光法通过可视化的结合物 (Amersham). （Amersham公司）。 。Relative mapping of antigenic determinants相对的抗原决定簇的映射 Experiments were carried out to determine whether 进行了实验，以确定是否 SHBG monoclonal antibody 11F11 (isotype IgG2a) recog- 性激素结合球蛋白，单克隆抗体11F11（亚型IgG2a） recog nized a different SHBG determinant from those of su