人绒毛滋养细胞单克隆抗体制备及初步鉴定

采集６～８周人工流产胎盘绒毛，经胶原酶处理及梯度离心后得到滋养细胞，再用ＮＰ－４０提取滋养细胞抗原。经常规免疫、细胞融合和克隆化后得到２０多株单克隆抗体（ＭｃＡｂ）细胞株。其中１０株ＭｃＡｂ进行了免疫学鉴定。免疫球蛋白分类，２株为ＩｇＭ，８株为ＩｇＧ，其中４株为ＩｇＧｌ。免疫斑点试验结果显示，１０株ＭｃＡｂ均为阳性，其滴度为１：８０～１：１２８０。免疫荧光定位，１０株中７株与滋养层细胞反应，２株与绒毛基底膜结合，１株为阴性。并对其进行了免疫转移印迹试验和交叉反应性研究，初步研究结果显示，单抗Ｔ６有着较好的特异性，值得进一步研究。     

腺苷脱氨酶在人精子膜上的定位及其单克隆抗体对人精子穿卵能力的影响

利用本实验室制备的抗人精子膜结合腺苷脱氨酶（ＡＤＡ）单克隆抗体（ＭｃＡｂ）和间接荧光免疫技术，分析了该酶在正常人精子膜上的位置。同时，利用该酶单抗分析了该酶活性对人精子穿透去透明带卵的影响。上述两个实验的结果显示：（１）ＡＤＡ结合于人精子中段、尾部膜的外侧；（２）利用抗ＡＤＡＭｃＡｂ抑制ＡＤＡ活性可明显抑制正常人精子穿透去透明带卵的能力。这一研究结果提示，ＡＤＡ作为精子的膜蛋白组分，可能参与对精子生理功能的调节过程。     

腺苷脱氨酶在人精子膜上的定位及单克隆抗体对人精子穿卵能力的影响

利用本实验室制备的抗人精子膜结合腺苷脱氨酶（ＡＤＡ）单克隆抗体和间接荧光免疫技术，分析了该酶在正常人精子膜上的位置。同时，利用该酶单抗分析了该酶活性对人精子穿透去透明带卵的影响。上述两个实验的结果显示：（１）ＡＤＡ结合于人精子中段，尾部膜的外侧；（２）利用抗ＡＤＡＭｃＡｂ抑制ＡＤＡ活性可明显抑制正常人精子穿透去透明带卵的能力。这一研究结果提示，ＡＤＡ作为精子的膜蛋白组分，可能参与精子生理功能的调节     

抗精浆免疫抑制物抗体的抗生育机理研究

为了探讨抗精浆免疫抑制物抗体（ＳＰＩＭ－Ａｂ）的抗生育机理，对１００例不育症患者和３０例正常对照进行了ＳＰＩＭ－Ａｂ的补体结合功能测定（ＣＦＡ）和ＳＰＩＭ－Ａｂ对精子凝集、制动、穿透力和杀精子影响的研究。结果：ＳＰＩＭ－Ａｂ阳性不育患者（ｎ＝２９）ＣＦＡ值为６．４７±１．５５ｋＵ／Ｌ，明显低于ＳＰＩＭ－Ａｂ阴性患者（ｎ＝７１，８．１１±１．６２ｋＵ／Ｌ）和对照组（ｎ＝３０，８．６０±１．８０ｋＵ／Ｌ），Ｐ＜０．０１。经ＳＰＩＭ－Ａｂ阳性血清和ＳＰＩＭ－Ａｂ阴性血清作用后，两组精子凝集，制动和死精子百分率分别为４１．４％、２１．１％，６９．０％、１６．９％，６２．１％、４．２％，精子穿透高度分别为３１．６±１３．０ｍｍ、３８．０±１２．９ｍｍ，有显著性差异（Ｐ＜０．０５～Ｐ＜０．０１）。结论：ＳＰＩＭ－Ａｂ能够以抗原抗体复合物形式激活补体，对精子凝集、制动、杀伤和穿透力的影响是干扰生育的重要原因之一。     

^Ⅰ—抗人成骨肉瘤单克隆抗体在荷瘤裸鼠体内的放射免疫显像

利用杂交瘤技术制备了抗人成骨肉瘤单克隆抗体（ＯＳ－ＭｃＡｂ），获得了ＯＳ－ＭｃＡｂ２两株特异性抗体。经＾１２５Ⅰ标记及荷瘤裸鼠体内放射免疫显像证明，ＯＳ－ＭｃＡｂ２明显优于ＯＳ－ＭｃＡｂ１，实验进一步证明ＯＳ－ＭｃＡｂ２对人肝细胞癌无交叉反应性。本研究为ＯＳ－ＭｃＡｂ２今后于临床显像诊断及导向治疗提供了前提条件。     

淋巴细胞CD28通路活化后Th1/Th2细胞因子mRNA表达及CsA的抑制作用

采用ＲＴＰＣＲ方法，观察ＣＤ２８通路活化后淋巴细胞Ｔｈ１／Ｔｈ２细胞因子ｍＲＮＡ表达水平及ＣｓＡ的抑制作用。取正常人外周血淋巴细胞，培养过程中分别给予抗ＣＤ３ｍＡｂ单独刺激或抗ＣＤ３ｍＡｂ＋抗ＣＤ２８ｍＡｂ共同刺激。提取细胞总ＲＮＡ并逆转录成ｃＤＮＡ，对Ｔｈ１细胞因子（ＩＦＮγ、ＩＬ２）和Ｔｈ２细胞因子（ＩＬ４、ＩＬ１０）ｃＤＮＡ进行扩增。结果显示：共刺激信号可使Ｔｈ１细胞因子基因ｍＲＮＡ转录增加，且对ＣｓＡ的抑制作用产生抵抗；而对Ｔｈ２细胞因子则不产生明显影响。研究表明：ＣＤ２８共刺激信号主要增强淋巴细胞Ｔｈ１细胞因子基因ｍＲＮＡ表达，并可能参与其分化调节；这一活化通路不被ＣｓＡ阻断。     

精索静脉曲张所致不育的免疫组化变化和临床研究（附编者按）

为了进一步探讨精索静脉曲张（ＶＣ）与不育症的关系，对２２例正常人，３５例左侧ＶＣ患者行远红外阴囊测温及血清抗精子抗体（ＡｓＡｂ）测定，及睾丸组织免疫组化染色检测三种ＡｓＡｂ（ＩｇＭ、ＩｇＧ、ＩｇＡ）含量。结果发现：精液异常ＶＣ患者睾丸温度显著升高（Ｐ＜０００１），睾丸温度与静脉曲张程度无关，而与精子总数、活动率呈显著负相关（Ｐ＜００１）；ＶＣ患者血清ＡｓＡｂ阳性率５７％，显著高于正常人（Ｐ＜００２５）；睾丸ＩｇＭ阳性率５３％，ＩｇＧ７３％，均显著高于正常人。ＩｇＭ、ＩｇＧ含量与睾丸温度呈显著正相关（（Ｐ＜００１，Ｐ＜００４）。其中ＩｇＭ含量与睾丸生精功能呈显著负相关（Ｐ＜００１）。病理检查发现Ｓｅｒｔｏｌｉ细胞变性。认为ＶＣ患者同时存在睾丸温度升高及抗精子免疫反应，共同损害生精功能，睾丸温度升高使Ｓｅｒｔｏｌｉ细胞变性，破坏血睾屏障导致自身免疫反应发生。     

人精子有效抗原基因表达克隆的研究

我们从人睾丸组织中提取ＲＮＡ，并进一步纯化出ｍＲＮＡ；以此为模板，在反转录酶和ＤＮＡ多聚酶的作用下，合成ｃＤＮＡ；将ｃＤＮＡ与λｇＩＩ载体重组后转染大肠杆菌，构建人睾丸ｃＤＮＡ表达文库。运用遗传显色法和噬菌斑原位杂交法鉴定表达文库后，用兔抗人精子抗体筛选人精子抗原基因表达克隆（ＨＳＧ）。对ＨＳＧ表达抗原（ＨＳＧＡｇ）和特异抗体（ＨＳＧＡｂ）进行纯化和抗生育效应的测定。结果显示：（１）人睾丸ｃＤＮＡ表达文库容量为１．８２Ｘｌ０６ｐｆｕ，遗传显色法示重组率为６７％，噬菌斑原位杂交法示重组率为５１％。（２）经兔抗人精子抗体筛选２Ｘｌ０４ｐｆｕ，得８株人精子抗原基因表达克隆。（３）人血清、兔血清ＨＳＧ２Ａｂ和兔血清ＨＳＧ８Ａｂ对人精子具有补体依赖细胞毒作用。（４）兔血清ＨＳＧ３Ａｂ能阻断人精子在顶体反应时顶体后区ＣｏｎＡ受体的暴露。结果提示，ＨＳＧ２、ＨＳＧ３和ＨＳＧ８克隆的表达抗原是精子有效抗原，能作为精子免疫避孕疫苗的候选成分。     

抗精浆免疫抑制物抗体的抗生育机理研究

作者进行了抗精浆免疫抑制物抗体（ＳＰＩＭ－Ａｂ）的补体结合功能测定（ＣＦＡ）和ＳＰＩＭ－Ａｂ对精子凝集、制动、穿透力和杀精子影响的研究。结果表明，ＳＰＩＭ－Ａｂ阳性不育病人（ｎ＝２９）ＣＦＡ值为６．４７±１．５５ｋＵ／Ｌ，明显低于ＳＰＩＭ－Ａｂ阴性病人（ｎ＝７ｌ，８．１１±１．６２ｋＵ／Ｌ）和对照组（ｎ＝３０，８．６０±１．８０ｋＵ／Ｌ），Ｐ＜０．０１。经ＳＰＩＭ－Ａｂ阳性血清和ＳＰＩＭ－Ａｂ阴性血清作用后，两组精子凝集、制动和死精子百分率分别为４１．４％和２１．１％、６９．０％和１６．９％、６２．１％和４．２％，精子穿透高度分别为３１．６±１３．０ｍｍ和３８．Ｏ±１２．９ｍｍ，经统计学处理差异显著（Ｐ＜０．０５～ｐ＜０．０１）。提示ＳＰＩＭ－Ａｂ能够以抗原抗体复合物形式激活补体，其对精子凝集、制动、杀伤和穿透力的影响，可能是干扰生育的重要原因之一。     

人精子膜甘露糖结合蛋白的研究--蛋白的定位

作者运用生物素标记的山羊抗人巨噬细胞甘露糖受体（ＧｏａｔＡｎｔｉ－ｈｕｍａｎＭａｃｒｏｐｈａｇｅＭａｎｎｏｓｅＲｅｃｅｐｔｏｒ,ＡＭＭＲ）ＩｇＧ对人获得能精子膜上的甘露糖结合蛋白进行定位,经激光共聚焦显微镜分析结果显示：ＡＭＭＲＩｇＧ组阳性率显著高于运用正常山羊（ＮｏｒｍａｌＧｏａｔ,ＮＧ）ＩｇＧ的对照组：ＡＭＭＲＩｇＧ组的阳性率大约在７０％（６９．４１％）左右,而ＮＧ ＩｇＧ对照组中除少量精子显     

腺苷脱氨酶在人睾丸及附睾的分布

应用本实验室前期制备的特异性的抗人精子膜结合腺苷脱氨酶（ＡＤＡ）同功酶的单克隆抗体（ＭｃＡｂ）和免疫组化技术，我们分析了该同功酶在正常人睾丸和附睾中的分布。实验结果显示：（１）ＡＤＡ在正常人睾丸中无分布，而在附睾头部，附睾管腔上皮细胞中开始分布；（２）ＡＤＡ在正常人附睾管腔上皮细胞中的分布，由附睾头部至尾部，其密度递增。这一研究结果提示：（１）ＡＤＡ分布具有附睾特异性；（２）人精子膜结合ＡＤＡ可能来自附睾管腔上皮细胞。     

腺苷脱氨酶在大鼠睾丸,附睾中的分布

利用本实验室前期制备和鉴定了的抗人精子膜结合脱氨酶单克隆抗体和ＡＢＣ组分技术，我们分析了正常性成熟和性未成熟大鼠睾丸及附睾中的该酶分布情况。实验结果显示：（１）ＡＤＡ在正常性成熟和性未成熟大鼠睾丸和附睾中无分布；在性成熟大鼠中，ＡＤＡ的分布从际睾头部开始出现；（２）ＡＤＡ在正常性成熟大鼠附睾中的分布由附睾头部至尾部其密度未发现变化。     

人精子制动单抗的制备、鉴定及其腹水型单抗的纯化

用杂交瘤技术建立的一株分泌抗人精浆蛋白的使人精子制动单抗的细胞株，Ｉｇ亚类分析表明，此单抗为ＩｇＭ。间接ＥＬＩＳＡ试验、精子制动试验、精子凝集试验和间接免疫荧光等试验分别证明所得单抗能与人精浆和无精子症患者的精浆产生特异免疫反应，有制动人精子的作用，并能特异结合到人精子的颈部，从而证实此单抗是精子制动单抗，其相应的抗原系精浆和精子共有的精子膜抗原，用ＦＰＬＣ－羟基磷灰石柱层析成功地从小鼠腹水纯化了此单抗。     

Development and characterization of a monoclonal antibody which recognizes a new prostate-organ specific antigen]

PURPOSE: Development and characterization of monoclonal antibodies which recognizes a new prostate-organ specific antigen. METHOD: For development of monoclonal antibodies, hybrid cells were prepared by fusion of spleen cells of BALB/c mice immunized with the homogenates of surgically resected prostatic tissue and P 3 x Ag 8 U 1 (P 3 U 1) murine myeloma cells. Supernatants of hybrid clones were primarily screened using an ELISA on human prostatic cancer cell line PC-3 and human bladder cancer cell line T-24. In the secondary screening, they were tested on normal tissues by immunohistochemical staining. To characterize the antigens, biochemical analyses were performed using seminal plasma as an antigen by western blotting and gel filtration, and the reactivity of antibodies were compared with that of antibodies against prostatic acid phosphatase (PAP), prostate-specific antigen (PSA) and gamma-seminoprotein (gamma-Sm). RESULTS: A monoclonal antibody termed KP-9 was obtained and it only reacted with PC-3 and prostate tissues, but did not react with other cell lines and normal tissues. Immunohistochemical staining of prostate tissue revealed that KP-9 stained grandular epithelium and grandular exudate of normal and malignant prostatic tissues, and especially, strongly stained the apical site of grandular epithelium. Western blotting and gel filtration of seminal plasma suggested that the molecular weight of the KP-9 antigen was more than 300,000 and was different from PAP, PSA and gamma-Sm. CONCLUSION: We have developed a monoclonal antibody, KP-9 which specifically reacts with prostatic cancer as well as benign prostatic tissues. The antigen recognized by KP-9 appeared to be a new prostate-organ specific antigen and may be a useful marker for prostatic cancer such as PAP, PSA and gamma-Sm.     

抗人精子蛋白22单克隆抗体细胞株的建立与鉴定

目的:制备小鼠抗人精子蛋白SP22单克隆抗体并鉴定其特异性。方法:用BL/21菌表达的SP22作抗原免疫BALB/c小鼠,用有限稀释法筛选分泌SP22单克隆抗体(M cAb)的杂交瘤细胞株,制备单克隆抗体,通过ELISA技术,W estern印迹方法鉴定其敏感性及特异性。免疫组化法探讨SP22在人精子表面的分布与定位。结果:获得3株稳定分泌SP22单克隆抗体的杂交瘤细胞株,其细胞培养液浓缩上清和混合腹水的效价分别为1∶1000和1∶3 200,抗体亲和力为1.0×107L/mol,小鼠IgG亚类均为IgG1,W estern印迹结果显示该抗体能特异性识别人精子SP22,免疫组化结果显示SP22主要定位于精子的顶体部位。结论:用杂交瘤技术制备的抗人SP22单克隆抗体具有高的效价和特异性,并且能特异地与人精子表面的SP22蛋白结合。     

Hemizona assay (HZA) demonstrates effects of characterized mouse antihuman sperm antibodies on sperm zona binding.

Characterized antihuman sperm monoclonal antibodies from mice were evaluated using the hemizona assay (HZA) to determine whether sperm:zona binding was effected. The seven monoclonal antibodies were characterized using human sperm in agglutination, immobilization, and penetration assays. Semen was provided by four fertile men and used in the HZA to determine if the presence of a monoclonal antibody would affect tight binding of the sperm to the zona pellucida. Pre-incubation of MA-14 for 1 h with the sperm induced a 33-54% reduction of the number of tightly bound sperm. This antibody reacts to an antigen located on the acrosome and midpiece. Experiments in which there was no pre-incubation of the antibody with sperm, resulted in no significant reduction in the number of sperm bound in the HZA. These findings suggest that an anti-human sperm antibody produced in mice can modulate sperm:zona binding. Reduction in zona binding could indicate a cause of immune-related infertility and this test may be useful in selecting an antigen for contraceptive vaccine development.     

孕早期母血胎盘滋养层细胞的检测

为从孕妇血中检测和分离胎盘滋养层细胞，对５５例孕８～１４周母血中细胞滋养层Ｈ３１５和合体滋养层ＦＴ１－４１．１抗原阳性细胞分别进行了免疫细胞化学检测、流式细胞分析、荧光激活细胞分离（ＦＡＣＳ）及荧光原位杂交分析（ＦＩＳＨ）。结果在２８例和１４例孕妇外周血中分别检出了Ｈ３１５和ＦＴ１－４１．１抗原阳性细胞；而１３名怀男胎孕妇外周血Ｈ３１５阳性细胞经ＦＡＣＳ分选及Ｙ染色体探针的ＦＩＳＨ分析，其中杂交阳性细胞含量平均为１１．４±４．３％。认为：孕８～１４周母血循环中存在细胞滋养层和合体滋养层细胞，有可能利用这些细胞在孕早期对胎儿染色体非整倍性异常进行产前分子细胞遗传学诊断。     

Identification of fragments of proximal and distal tubular cells in the urine of patients under cytostatic treatment by immunoelectron microscopy with monoclonal antibodies

In an attempt to identify the origin of cellular fragments released in the urine of patients treated with potentially nephrotoxic drugs such as cytostatics, two monoclonal antibodies were applied: monoclonal antibody PM II 9 C2, directed against an antigen in distal tubular cells; and monoclonal antibody PM II 39 H11 specific for an antigen in proximal tubular cells. The specificities of both monoclonal antibodies were elaborated in the indirect as well as in the direct immunofluorescence technique. Both antibodies were then used to identify cellular fragments obtained from the urine of patients treated with cytostatic drugs by ultracentrifugation. By application of the indirect immunogold method, it was shown that material of proximal as well as distal tubular origin was shed by the damaged cells. Whereas the proximal tubular antigenic epitope recognized by PM II 39 H11 was always found in large irregular complexes of debris-containing vesicles, the distal tubular antigenic epitope recognized by PM II 9 C2 was always found associated with filament-like regular structures. This is the first report in which excretion of components of distal tubular cells is demonstrated as a consequence of the nephrotoxic side effects of cytostatic treatment. With the help of monoclonal antibodies, it has now become possible to identify and to investigate the damage inflicted on the distal part of the tubule system in addition to the well-documented proximal tubular damage.     

Differences in the antigen pattern recognized by antisperm antibodies in patients with infertility and vasectomy

PURPOSE: Antisperm antibodies may impair sperm fertilizing capacity. They are found in infertile patients and in men after vasectomy. Little is known to date of the biochemical nature of the antigens that induce the production of antisperm antibodies. MATERIALS AND METHODS: Sperm membrane proteins were prepared from donor spermatozoa, separated by 1-dimensional polyacrylamide gel electrophoresis and exposed to seminal plasma samples of 36 infertile men and 34 after vasectomy containing antisperm antibodies. RESULTS: Ten antigenic protein bands with different molecular weight were recognized by antisperm antibodies. Antisperm antibodies binding to the antigen band at 55 kDa. were significantly more common in infertile men, while those binding to the 72 kDa. band were more common after vasectomy. Significant differences also occurred in the incidence of detecting the 55 kDa. antigen band by the antisperm antibodies of patients with and without varicocele. Comparing antisperm antibodies from patients with or without a history of genital diseases or trauma did not reveal significant differences in the antigens detected. CONCLUSIONS: It seems likely that the development of antisperm antibody binding to different antigens is related to the mode of antibody induction. Since the antigenic properties of spermatozoa change during passage through the epididymis, the antigens detected by antisperm antibodies from men with vasectomy are mostly related to epididymal passage. The identification of human sperm antigens is essential for understanding the mechanism by which antisperm antibodies influence the fertilization capacity of spermatozoa. It is also necessary for the potential development of reliable diagnostic methods for antisperm antibodies that are relevant to infertility.