抗人VEGF受体Ⅱ胞外Ⅲ区单克隆抗体的生物学活性研究

目的研究抗KDR单体Ycom1D3抑制VEGF诱导脐静脉内皮细胞（HUVEC）增殖的体外生物学活性。方法采用FACS鉴定Ycom1D3与抗原结合特异性，采用免疫共沉淀测定Ycom1D3阻断VEGF165刺激KDR酪氨酸激酶受体磷酸化作用，并采用[^3H]-Thymidine掺入法、内皮细胞损伤愈合试验和内皮细胞血管腔形成实验进一步确定Ycom1D3抑制VEGF165诱导内皮细胞增殖的中和活性。结果Ycom1D3不但能与HUVEC结合，而且能阻断由VEGF165刺激HUVEC表面KDR酪氨酸激酶受体磷酸化，进而显著抑制VEGF165诱导HUVEC增殖、迁移及体外三维胶原模型上内皮细胞毛细血管样结构的形成。结论Ycom1D3可以通过封闭KDR而抑制VEGF活性，在肿瘤及其它血管新生疾病治疗中具有潜在应用前景。     

Immunohistochemical staining of normal, hyperplastic, and neoplastic adrenal cortex with a monoclonal antibody against alpha inhibin.

AIMS: To investigate the immunohisto-chemical staining of normal, hyperplastic, and neoplastic adrenal cortex with a monoclonal antibody against alpha inhibin. Also, to determine whether immunostaining with this antibody is useful in differentiating between adrenal cortical neoplasms and other tumours involving the adrenal gland that might mimic them. METHODS: Normal adrenal tissue (n = 20) and specimens from cases of adrenal hyperplasia (n = 13), adrenal cortical adenoma (n = 15), adrenal cortical carcinoma (n = 4), phaeochromocytoma (n = 8), and adrenal metastatic tumour (n = 7) were stained with a monoclonal antibody against the alpha subunit of human inhibin. RESULTS: Positive staining with the anti-alpha inhibin monoclonal antibody was seen in all normal adrenal glands. Immunoreactivity was largely confined to the inner cell layers of the adrenal cortex, with no staining of the adrenal medulla. All hyperplastic adrenal glands and adrenal cortical adenomas and carcinomas were also immunoreactive. The other tumours studied were negative. CONCLUSIONS: There is consistent immunoreactivity with the anti-alpha inhibin monoclonal antibody in normal adrenal cortex and in hyperplastic and neoplastic adrenal cortical lesions. In the normal adrenal cortex, positive staining is mainly confined to the zona reticularis. Other neoplasms involving the adrenal gland are negative. Immunohistochemical staining with anti-alpha inhibin monoclonal antibody, performed as part of a panel, may prove to be of value in the distinction between adrenal cortical carcinoma and phaeochromocytoma or metastatic tumour.     

Immunohistochemical characterization of Ki-M6 monoclonal antibody in Bouin-fixed,paraffin-embedded sections of normal and neoplastic human tissues

Summary A monoclonal antibody (Ki-M6) against the CD 68 antigen, which labels cells of the monocyte/macrophage system, was tested on Bouin-fixed, paraffin-embedded samples of normal, reactive and neoplastic tissues by an avidin-biotin-peroxidase complex method, with the aim of establishing its value in diagnostic pathology. In normal human tissues, Ki-M6 reactivity was confined to the so-called resident macrophages populating normal organs under physiological conditions. Moreover, restricted reactivity against cells of macrophage lineage was observed in reactive and inflammatory lesions. Granulocytes, monocyte/macrophage-related immune accessory cells, and other analysed normal tissue structures did not reveal any reactivity. Ki-M6 was strongly reactive with the cases of benign (4/4) and malignant (15/15) fibrous histiocytomas, in addition to the true histiocytic lymphomas (3/3). Cases of granular cell tumour (2/3) showed strong reactivity with Ki-M6, whereas only few immunoreactive cells, with weak staining, were seen in the other Ki-M6-positive neoplasms [neurofibroma (3/3), benign schwannoma (1/2), ganglioneuroma (1/1), malignant schwannoma (5/9), melanoma (9/28), dermatofibrosarcoma protuberans (1/1), myelomonocytic leukaemia (3/3)]. Among the epithelial malignancies tested (47 cases), Ki-M6 was positive only in renal cell carcinoma (11/14). Malignant lymphomas of the Hodgkin (56 cases) and non-Hodgkin type (67 cases) were uniformly non-reactive. From these data, Ki-M6 appears to be an excellent marker of monocyte/macrophage-related cells and appears to be a reliable indicator for fibrous histiocytomas and true histiocytic malignancies. The availability of this additional antibody capable of staining routinely processed tissue is of practical interest.     

抗人生长转化因子15单克隆抗体制备及鉴定

目的:制备抗人生长转化因子15(GDF15)单克隆抗体(mAb)并鉴定其特性。方法:构建GDF15原核表达载体pGEX-4T-2-gdf15,诱导表达并纯化GST-GDF15融合蛋白。以该融合蛋白免疫BALB/c小鼠,取小鼠的脾细胞与同系小鼠Sp2/0骨髓瘤细胞常规融合,依次进行阳性杂交瘤细胞株的筛选及亚克隆,最终获得稳定分泌抗GDF15 mAb杂交瘤细胞株。以间接ELISA法检测抗体效价;Western blot鉴定抗体特异性;免疫共沉淀法初步检测在肝癌患者血清中GDF15表达水平。结果:获得1株稳定分泌抗人GDF15 mAb杂交瘤细胞株,命名为9G3;抗体免疫球蛋白亚类为IgG1,轻链为κ型;免疫共沉淀及质谱分析证实抗GDF15 mAb9G3能与肝癌患者血清中GDF15蛋白特异性结合并且GDF15水平在肝癌患者血清中较正常人显著升高。结论:成功制备了抗人GDF15mAb,为后期肝癌早期诊断试剂盒的开发奠定了良好的基础。     

Immunohistochemical analysis for desmin in normal and neoplastic ovarian stromal tissue

Ovarian stroma contains cells with the ultrastructural features of smooth-muscle cells. The purpose of this study was to analyze normal ovaries, ovarian stromal tumors (fibroma/thecomata and granulosa cell tumors), and ovarian leiomyomata for desmin reactivity. Groups of ovarian stromal cells that expressed desmin were noted in six of six normal ovaries. Desmin was also detected in two of six fibroma/thecomata and two of two ovarian leiomyomata. The number of tumor cells with detectable desmin was much greater in the leiomyomata. Desmin was not detected in any of six granulosa cell tumors. We conclude that stromal cells with an immunohistochemical feature of smooth-muscle cells are routinely found in normal ovaries. This study demonstrates the usefulness of immunohistochemistry in corroborating the diagnosis of ovarian leiomyomata, although desmin positivity per se is not diagnostic of ovarian leiomyomata, and also raises the possibility that some ovarian leiomyomata may be derived from stromal cells.     

Immunohistochemical localization of the epidermal growth factor receptor in normal human tissues

A monoclonal antibody recognizing an epitope of the external domain of the human epidermal growth factor (EGF) receptor was used to localize this protein in selected normal human tissues. Two patterns of reactivity were recognized: strong linear or granular cell surface staining, and granular cytoplasmic staining. In one tissue, the endometrium, a change in the reaction pattern associated with changes in hormonal stimulation was observed. In some tissues such as epididymis and skin, the antibody showed surface reactivity with cells considered to represent part of the proliferating cell compartment, whereas in liver, pancreas, and prostate, all cells were reactive with the antibody, though the predominant reactivity was localized in the cytoplasm. The differential distribution of the epidermal growth factor receptor to specific cell types and cellular compartments may signify adaptations that permit growth factor responsiveness in a milieu of available ligand.     

抗重组人表皮生长因子单克隆抗体的制备及生物学特性鉴定

目的　制备出高效价的抗重组人表皮生长因子 (EGF)单克隆抗体。方法　以重组人表皮生长因子作为抗原 ,免疫Balb/c小鼠 ,以未免疫的Balb/c小鼠脾细胞为饲养细胞 ,运用细胞杂交瘤技术制备 ,间接ELISA法筛选产生针对人表皮生长因子的单克隆抗体细胞株 ;以体内诱生法产生腹水 ,并采用ProteinA Sepharose柱对其纯化 ,快速定性试纸鉴定McAb的Ig亚类 ,采用间接ELISA法相加实验鉴定抗原识别表位。结果　获得 3株产生针对人表皮生长因子的单克隆抗体细胞株EGF B2 、EGF C7、EGF A8,Ig亚分别为IgG1κ型 ,IgG1λ型 ,IgG3 κ型 ,亲和力常数分别为 2 .76× 10 10 、3.2× 10 9、1.4 5× 10 9。结论　成功制备 3株稳定分泌抗rhEGF的杂交瘤细胞株 ,产生的McAb特异性好 ,亲和力高 ,为探讨EGF的作用机制及临床应用奠定了基础 ,为肿瘤的诊断与治疗提供具有实用价值的研究工具 ,此外 ,为EGF的纯化提供实验材料     

Immunohistochemical demonstration of nonspecific cross-reacting antigen in normal and neoplastic human tissues using a monoclonal antibody. Comparison with carcinoembryonic antigen localization

Nonspecific cross-reacting antigen (NCA) immunoreactivity was localized in normal and neoplastic human tissues using a monoclonal antibody to 55, 90 and 95 kDa molecules of NCA. This was compared to the localization of immunoreactive carcinoembryonic antigen (CEA) as demonstrated by polyclonal and monoclonal antibodies. In frozen sections, CEA was localized in normal surface epithelium of the stomach and colon where NCA was only weakly detected. Type 1 and type 2-like pneumocytes were positive for NCA, while CEA was localized only in type 2-like pneumocytes. CEA and NCA were both demonstrated in ductal cells of frozen pancreatobiliary and mammary tissues. The antigenicity of CEA and NCA in normal tissues was significantly lost after paraffin embedding as compared to frozen sections. NCA was consistently demonstrated in eccrine sweat glands embedded in paraffin. In various tumor tissues, CEA and NCA were colocalized and expression increased sufficiently to be detected in paraffin sections. Adenocarcinomas of the stomach and colon and cystadenocarcinoma of the pancreas, as well as neuroendocrine carcinomas of the lung and thyroid, showed a CEA predominance over NCA. In ductal adenocarcinomas of the pancreas and breast and in cholangiocarcinoma, NCA reactivity was greater than CEA. Keratinizing foci of most squamous cell carcinomas of mucosal origin and some adenocarcinomas equally expressed both. Hepatocellular carcinoma, lobular mammary carcinoma and papillary thyroid carcinoma were positive only with unabsorbed polyclonal antibody which widely recognizes CEA-related substances. Renal cell carcinoma, prostatic adenocarcinoma, transitional cell carcinoma, anaplastic carcinomas, choriocarcinoma and basal cell carcinomas showed little or no immunoreactivity. Hence the relative ratio of CEA/NCA expression in tumors was dependent on the tissue of origin and histologic type. The cytoplasmic granular staining of NCA in cancer cells was a noteworthy difference from the plasma membrane-associated localization of CEA.     

Immunohistochemical reactivity of a monoclonal antibody prepared against human breast carcinoma

Summary The reactivity profile of an IgM monoclonal antibody, MBR1, raised against the human breast cancer cell line MCF7, was studied in a variety of human tumours and non-neoplastic tissues by light microscopic immunohistochemistry. The range of reactivity included specific types of non-neoplastic epithelial cells and a number of epithelial tumours. Most mammary carcinomas reacted with MBR1, but adenocarcinomas and squamous carcinomas from different sites were also strongly positive. Different patterns of immunoreactivity were apparent in microscopically normal tissues, in tissues with inflammatory changes and in carcinomas. Heterogeneous staining, despite morphological similarities, was documented in neoplastic and non-neoplastic epithelial cells. The reactivity of MBR1 was different from that reported for other monoclonal antibodies, but revealed similarities to that of monoclonal antibodies and polyclonal sera against human milk fat globule membrane.     

A Schwann cell antigen recognized by monoclonal antibody 217c is the rat low-affinity nerve growth factor receptor.

Monoclonal antibody 217c was generated against an antigen expressed on the rat glial cell line, C6 glioma, 217c has been shown to recognize Schwann cells in mixed cultures as well as in tissue sections and has been used to identify Schwann cells independent of other markers, such as monoclonal antibody 192-IgG, which recognizes the rat low affinity nerve growth factor (NGF) receptor. Here we show that the antigen recognized by 217c is the rat low-affinity NGF receptor. This indicates that monoclonal antibodies 192-IgG and 217c are not independent markers and therefore that additional criteria need to be used for the identification of Schwann cells early in development.     

Use of monoclonal antibody KP1 for identifying normal and neoplastic human mast cells.

The monoclonal antibody KP1 (CD68) was used to stain normal and neoplastic monocytes and macrophages in routinely processed, paraffin wax embedded tissue: mast cells also exhibited strong, consistent cytoplasmic immunoreactivity. Light microscopic findings were corroborated by electron microscopical and immunocytochemical findings. The predominant sites of immunoreactivity were the specific intracytoplasmic granules of the mast cells. All mast cell subtypes--that is, normal and reactive mast cells, such as those in lymph nodes exhibiting chronic non-specific lymphadenitis, and malignant or neoplastic mast cells in various types of mastocytosis--reacted with this antibody. This finding is of diagnostic importance, because mast cell proliferation could be mistaken for histiocyte proliferation. It also supports the hypothesis that mast cells derive from the bone marrow.     

Immunohistochemical studies of beta-hexosaminidase in neoplastic and normal tissues with monoclonal antibodies.

A total of 87 human specimens with 10 histological types of primary neoplasm were studied immunohistochemically with monoclonal antibodies specific for beta-hexosaminidase (Hex). High levels of Hex were found in malignant neoplasms of the skin, cervix, colorectum and in benign as well as neoplastic plasma cells, while no activity was detected in normal epidermis, normal colorectal epithelium or benign naevi. The strongest immunohistochemical reaction was revealed in tumor cells of malignant melanoma. Adenomas and adenocarcinomas of the colorectum showed high levels of Hex with a basal pattern of immunoreactivity more frequent in the tumor cells of adenocarcinomas than adenomas. Fibroblasts and macrophages in the tumors often disclosed immunoreactivity. In most of the sections (including those from plasma cell neoplasms), 7E4 antibody showed low immunoreactivity compared to 2E3, except for non-neoplastic plasma cells, which were as a rule positive with 7E4 and largely negative with 2E3 antibody. This result probably indicated different isoenzymes in benign and neoplastic plasma cells.     

Monoclonal antibody against human growth hormone receptor

GHR shows a high degree of homology with the prolactin receptor and with the other receptors that belong to the hemopoietic receptor superfamily. This paper describes a monoclonal antibody (MAb) (2B4B6) specific for both the extracellular domain of human GHR and human growth hormone (GH) binding protein. Mice were immunized against a seven-aminoacid peptide sequence screened by FASTA (sequence similarity search served by Genome-Net) from the European Bioinformatics Institute to exclude the existence of human membrane proteins with significant sequence homology. MAbs were screened against the peptide sequence and 2B4B6 was selected for its capability to recognize the full-length hGHBP. As evaluated by both enzyme-linked immunoadsorbent assay (ELISA) and FACS analysis, this MAb seems to recognize and bind to a hGHR positive cell line, IM-9, as well as a murine cell line, BaF3 (8/6), transfected with a chimeric construct, hGHR/hG-CSFR and expressing hGHR on the cell membrane. Studies investigating the biological effects of this MAb showed that anti-hGHR mediated inhibition of cell proliferation was not due to competition with GH binding but rather to prevention of receptor dimerization. Because of its specificity, this MAb may be usefully applied in situations in which GHR and receptors with a high degree of homology, such as PRLR (prolactin receptor), are expressed simultaneously, as occurs in the immune system.     

A new murine monoclonal antibody against human hepatoma.

A murine monoclonal antibody (MAb 336) reactive with human hepatocellular carcinoma has been raised after immunizing BALB/c mice with whole HepG2 cells. MAb 336 (IgG1) was reactive with HepG2 (whole cells and membrane fractions), but not normal liver or peripheral blood cells. Immunohistological studies indicated that 12/16 hepatocellular carcinoma and 6/11 cirrhotic livers expressed MAb 336-associated antigen, and most normal human tissues and tissues derived from other cancers were unstained. Direct and competitive binding assays ruled out the possibility that this MAb reacts with alpha-fetoprotein, carcinoembryonic antigen, or ferritin. Western blot analysis indicated that MAb 336 reacts with an antigen of approximately 30,000 daltons. This MAb may be potentially useful for studying antigenic expression in hepatocellular carcinoma and as a targeting agent for radioimmunodetection and immunoconjugate therapy.     

Nerve growth factor receptor in tumours of the human nervous system. Immunohistochemical analysis of receptor expression and tumour growth fraction

The expression of nerve growth factor receptor (NGFr) was investigated by means of immunohistochemistry in 135 tumours of the human central and peripheral nervous system. The results were compared to the proliferative activity of the tumours as determined by immunostaining for the proliferation-associated antigen Ki-67. Immunoreactivity for NGFr was most consistently observed in tumours derived from the neural crest such as neurinomas, neurofibromas and ganglioneuromas. In tumours of the central nervous system, NGFr-immunostaining was particularly strong in pilocytic astrocytomas while the majority of other gliomas were either NGFr-negative or contained only a minor fraction of NGFr-positive tumour cells. Among all other investigated tumours including medulloblastomas, pituitary adenomas and meningiomas only exceptional cases demonstrated a significant number of positive tumour cells. Choroid plexus papillomas and metastatic carcinomas were always NGFr-negative. Our results indicate that NGFr-expression in tumours of the human nervous system is heterogenous with respect to tumour type and appears to be unrelated to proliferative activity.     

A monoclonal antibody against neuron-specific enolase. Immunohistochemical comparison with a polyclonal antiserum

There is skepticism about the value of antisera to neuron-specific enolase (NSE) for immunohistochemical identification of neural and neuroendocrine differentiation in neoplasms, because of reports of detection of NSE, in a large percentage of nonneuroendocrine neoplasms. By immunohistochemical methods, the authors compared a monoclonal antibody to NSE (Mab NSE) with a heterologous antiserum to NSE (Het NSE) on 348 samples of tumors of diverse histogenesis. They studied 93 neural and neuroendocrine tumors and 255 nonneuroendocrine, nonneural tumors. The Mab NSE was slightly less sensitive but clearly more specific than the Het NSE in recognizing neural and neuroendocrine differentiation. Only 2% of the nonneuroendocrine, nonneural tumors reacted positively with Mab NSE; in contrast, 20% of the same tumors were positive with the Het NSE. Moreover, intense nonspecific staining was frequent with Het NSE, which often rendered interpretation difficult. Because of its superior specificity, the Mab NSE used in this study is more valuable than the heterologous antiserum as a diagnostic reagent in tumor diagnosis.     

A human monoclonal antibody against HLA-A25

We are reporting the production and characterization of a human monoclonal antibody recognizing antigen HLA-25. The antibody was developed by a line transformed in vitro by the Epstein-Barr virus. The immune B lymphocytes for transformation were generated by planned immunization of a volunteer with repeated doses of allogenic peripheral blood lymphocytes of one donor over the course of 7 years. The antibody showed correlation with A25 antigen on a panel of 244 individuals tested by microcytotoxicity. The antibody showed neither cytotoxic reactivity nor CYNAP phenomenon with antigens of HLA-10 CREG.     

抗人凋亡诱导因子单克隆抗体的制备与鉴定

背景：凋亡诱导因子不仅具有氧化还原和电子传递功能,还具有促细胞凋亡功能,从而在维持细胞正常生理活动中具有重要作用。 目的：制备抗人凋亡诱导因子单克隆抗体并进行生物学特性鉴定。 方法：利用Ensembl数据库及DNAstar软件包对凋亡诱导因子氨基酸序列进行分析,获得优势表位多肽,采用碳化二亚胺法将多肽与载体蛋白偶联制备完全抗原免疫动物,采用杂交瘤技术制备凋亡诱导因子单克隆抗体并纯化。 结果与结论：间接ELISA法检测显示,免疫鼠腹水中抗人凋亡诱导因子单克隆抗体效价达到1∶252 400。Western blot结果显示,存在与抗原带一致的相对分子质量67 000的特异条带。免疫组化检测结果显示,在结肠癌组织细胞中有棕色阳性颗粒表达,说明此抗体也可用于免疫组识化学染色。提示实验获得了高活性、高纯度、高特异性抗人凋亡诱导因子单克隆抗体。     

Expression of nerve growth factor receptor in paraffin-embedded soft tissue tumors.

Identification of growth factors and receptors in mesenchymal tumors may be crucial to understanding of growth regulation in sarcomas. During an immunohistochemical study of the expression of growth factors and receptors in human soft tissue tumors (STT), only 1 antisera capable of working in paraffin-embedded tissue was noted. A detailed study of 141 STT was undertaken to determine the frequency of expression of nerve growth factor receptor (NGF-R), its specificity and sensitivity for neural tumors, and the effect of fixation on detection. In normal mesenchymal tissue, only nerve sheath and perivascular staining was seen. No immunoreactivity was seen in many tumors including rhabdomyosarcoma, angiosarcoma, liposarcoma, Ewing's sarcoma, and alveolar soft part sarcoma. Less than 15% of tumors of smooth muscle, fibrous, or fibrohistiocytic origin showed immunoreactivity, usually focal. In contrast, a high frequency of immunoreactivity was noted in tumors of neural origin (74%). This included granular cell tumors (100%), Schwannoma/neurofibroma (91%), malignant Schwannoma (78%), neuroblastoma/neuroepithelioma (60%), and paraganglioma (57%). A high rate of reactivity was also seen in synovial sarcomas (80%), undifferentiated sarcomas (60%), and hemangiopericytomas (43%), suggesting a potential relationship to the neural phenotype. Among the neural tumors, Bouin's fixation was superior to formalin, suggesting that immunoreactivity for NGF-R is affected by fixation. This antibody may be a useful adjunct marker diagnostically.