Development of a monoclonal antibody to the aP2 protein to identify adipocyte precursors in tumours of adipose differentiation.

aP2 gene product (aP2 protein) expression has been shown to be a useful diagnostic marker for identification of lipoblasts and fetal fat cells in soft tissue tumours. A monoclonal antibody was developed by a mouse spleen cell-myeloma hybridoma technique to an 18 amino acid segment of the aP2 protein and was used to investigate the immunohistochemical expression of this protein in benign and malignant tumours of adipocytic differentiation and a wide variety of other soft tissue tumours. We found that aP2 protein was expressed by lipoblasts in liposarcomas and lipoblastomas and by brown fat cells in hibernomas and normal periadrenal fat. Other benign adipose tissue tumours and benign and malignant soft tissue tumours were distinguished from liposarcoma by absence of staining for aP2 protein. Immunohistochemical identification of the aP2 protein is likely to prove a useful means of distinguishing liposarcoma from other malignant mesenchymal and epithelial neoplasms, some of which contain cells that morphologically resemble lipoblasts.     

Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.     

Study of childhood renal tumours using a monoclonal antibody to Tamm-Horsfall protein.

A monoclonal antibody to Tamm-Horsfall glycoprotein was used for the immuno-localization of Tamm-Horsfall protein in formalin fixed, paraffin embedded tissue sections of childhood renal tumours, normal children's kidneys, and human fetal kidneys. The procedure was a dinitrophenyl hapten sandwich staining method. The antibody, diluted 1/100,000, gave a very strong and specific staining of the loop of Henle and distal tubules of normal and fetal kidneys. No staining was seen in Wilms' tumour, mesoblastic nephroma, and bone metastasizing renal tumour of childhood. In contrast, two of seven renal carcinomas and three of four rhabdoid renal tumours were positive for Tamm-Horsfall protein.     

Diagnostic utility of aP2/FABP4 expression in soft tissue tumours

Adipocyte P2 (aP2), also known as fatty acid-binding protein 4 (FABP4), is a fatty acid-binding protein found in the cytoplasm of cells of adipocyte differentiation. In this study, we examined a large number of soft tissue tumours with a commercial polyclonal anti-aP2/FABP4 antibody and a newly developed mouse monoclonal antibody raised against this protein to determine the diagnostic utility of aP2/FABP4 as a marker of tumours of adipose differentiation. A mouse monoclonal antibody, clone 175d, was raised against a mixture of synthetic peptides corresponding to the amino acid sequence of residues 10–28 and 121–132 of the human aP2/FABP4 protein. Antigen expression with polyclonal and monoclonal antibodies was immunohistochemically determined in paraffin sections of normal adipose tissue and a wide range of benign and malignant primary soft tissue tumours (n?=?200). aP2/FABP4 was expressed around the cytoplasmic lipid vacuole in white and brown fat cells in benign lipomas and hibernomas. Immature fat cells and lipoblasts in spindle cell/pleomorphic lipoma, atypical lipomatous tumour/well-differentiated liposarcoma, myxoid/round cell liposarcoma and pleomorphic liposarcoma also reacted strongly for aP2/FABP4. No specific staining was seen in non-adipose benign and malignant mesenchymal and non-mesenchymal tumours. aP2/FABP4 is expressed by mature and immature fat cells and is a marker of tumours of adipose differentiation. Immunophenotypic aP2/FABP4 expression is useful in identifying lipoblasts, and immunohistochemistry with polyclonal/monoclonal anti-aP2/FABP4 antibodies should be useful in distinguishing liposarcoma from other malignancies, particularly round cell, myxoid and pleomorphic soft tissue sarcomas.     

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A1 with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A1 scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the preferred selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv.     

Immunohistochemical evaluation of AKT protein activation in canine mast cell tumours

The pathogenesis of canine mast cell tumour (MCT) remains unknown. Moreover, therapeutic options are limited and resistance to targeted drugs and recurrences are common, necessitating the identification of additional cellular targets for therapy. In this study we investigated the expression of phosphorylated AKT protein in 25 archival canine MCT samples by immunohistochemistry and examined the correlation between the immunohistochemical scores and histopathological tumour grades. AKT protein was detected in all of the samples and 24 of the 25 samples expressed the phosphorylated form of the protein, albeit with variable intensity. However, when the immunohistochemical scores of weak, intermediate and strong labelling were compared with the histopathological grades I to III, there was no strong correlation. This study suggests that canine MCT cells have activated AKT and indicates the need for further research on the role of the AKT protein and the possibility of targeting the AKT signalling pathway in MCTs.     

Inhibition of T-cell differentiation by an antibody to a soluble thymic factor.

An antiserum to chicken soluble thymic factor (STF) was prepared in rabbits and absorbed with chicken thymocytes until it reacted only with thymic reticuloepithelial cells. When injected into 6-day-old embryonated eggs, this antiserum caused a severe depletion of small lymphocytes in the cortical region of the thymus which was filled with large undifferentiated cells. In addition, reticuloepithelial cells displayed a marked disruption of cytoplasmic elements. The percentage of thymic lymphocytes bearing the T antigen was greatly reduced. It is believed that a factor responsible for differentiation of stem cells into T cells has been inhibited.     

人源抗戊型肝炎病毒单克隆抗体的获得与鉴定

目的　获得人源中和性抗戊型肝炎病毒 (HEV)单克隆基因工程抗体。方法　采用五轮筛选法 (逐轮降低抗原包被量 ,严格洗脱条件 ) ,以固相化的 4种含中和抗原表位的HEV代表株ORF2重组混合抗原淘筛本室构建的HEV患者外周血源的噬菌体抗体库。获得有特异结合活性的噬菌体抗体 ,酶切鉴定抗体基因插入。切去噬菌粒上衣壳蛋白gⅢ基因构建表达载体 ,在大肠杆菌中可溶性表达抗HEV抗体Fab段 ,ELISA、Westernblot检测抗体免疫学活性 ;测序分析抗体基因。结果　筛选出 4株与含中和抗原表位的HEV代表株ORF2重组混合抗原特异结合而与BSA、HBsAg无交叉结合反应的噬菌体抗体 ,可能为中和抗体。切去gⅢ基因 ,在大肠杆菌中表达可溶性抗体Fab段 ,表达量在细菌培养液上清和细菌裂解液中差异无显著性。可溶性抗体与含中和抗原表位的HEVORF2重组混合抗原有特异结合活性 ,与BSA、HBsAg无交叉结合反应。Westernblot显示在相对分子质量 (Mr)略大于 4 5× 10 3 处有一明显条带 ,与Mr4 8× 10 3 的Fab大小一致。DNA测序分析表明 ,Fab段VH 属于IgGVH3基因家族 ,VL 属于Vκ1基因家族。结论　利用噬菌体抗体库技术成功获得抗HEV的人源单克隆抗体。     

Leptin and leptin receptor mRNA are widely expressed in tumors of adipocytic differentiation.

Adipose tissue is the principal source of leptin, a cytokine-like peptide with many biologic functions. Leptin binds to the leptin receptor, present in the hypothalamus and in many other tissues, and modulates energy balance and maintenance of body weight. The expression of leptin and leptin receptor in tumors of adipocytic differentiation has not been previously examined. Because normal adipose tissue is the principal source of leptin and expresses leptin receptor, we hypothesized that tumors of adipose tissue differentiation may also express leptin and/or the long functional form of the leptin receptor (OB-Rb). Leptin and OB-Rb were analyzed by immunohistochemistry, in situ hybridization, RT-PCR, and western blotting in 21 lipomas, 2 hibernomas, and 16 liposarcomas. Immunostaining and in situ hybridization showed leptin and OB-Rb mRNA expression in all cases of lipomas, hibernomas, and liposarcomas, including dedifferentiated and pleomorphic liposarcomas. RT-PCR analysis showed leptin and OB-Rb mRNA in both lipomas (n = 5) and liposarcomas (n = 5). Western blotting identified the 16 kDa leptin protein in a lipoma and a liposarcoma. No important difference in the expression of leptin and OB-Rb mRNA was found between lipomas and liposarcomas, although the level of leptin protein was higher in a lipoma than a liposarcoma by western blotting. These results show for the first time that leptin and OB-Rb mRNA are expressed in lipomas, hibernomas, and liposarcomas. The presence of leptin and its receptor may provide new insights into the pathobiology of these tumors.     

An Immunohistochemical study of HER-2 expression in feline mammary tumours

The aim of the present study was to evaluate HER-2 expression in feline mammary tumours. Five different immunohistochemical protocols were tested with 73 feline mammary carcinomas (MCs), 10 mammary adenomas and 73 hyperplastic or dysplastic mammary lesions. The histological features of these lesions, clinical follow-up and expression of Ki-67 and p53 were also examined. With an optimized immunohistochemical protocol, HER-2 overexpression was detected in only four of the 73 (5.5%) MCs and did not correlate with histological classification or with the 1 year post-surgical clinical outcome. No correlation was found between the expression of Ki-67 or p53 and HER-2. Five of the 73 (6.8%) hyperplastic or dysplastic lesions and one of the 10 (10%) mammary adenomas were HER-2 positive. These results suggest that HER-2 may not play as significant role in mammary carcinogenesis and prognosis in cats as it does in human patients.     

Immunohistochemical analysis of Bcl-2 protein regulation in cutaneous melanoma.

Cutaneous melanoma is becoming increasingly common. Genetic and environmental factors are thought to play a role in its pathogenesis. We have previously shown that normal human melanocytes strongly express the oncoprotein, Bcl-2. To determine the role of Bcl-2 in melanocytic tumors, we studied human benign nevi and melanomas for expression of Bcl-2 protein using immunohistochemistry. Our results show that benign melanocytes from 3 of 4 normal skin biopsies and 5 of 7 common acquired nevi strongly express Bcl-2. Conversely, only 3 of 23 primary cutaneous melanomas and 3 of 9 metastatic melanomas showed strong staining in comparison with melanocytes from normal skin and common acquired nevi (chi 2, P = 0.0021). Interestingly, 0 of 6 dysplastic nevi, a precursor of melanoma, demonstrated strong staining as compared with melanocytes and nevi (8 of 11; chi 2, P = 0.02), but similar expression to that of melanoma (6 of 32; chi 2, P = 0.6). We conclude that Bcl-2 expression decreases in malignant melanoma and suggest that this may be related to the autonomous growth characteristics of malignant melanoma.     

Lack of C-erbB-2 protein expression in pulmonary carcinoid tumours.

To determine if amplification of the C-erb-B2 proto-oncogene could be correlated with prognosis in carcinoid tumours, 49 pulmonary carcinoid tumours (26 typical, 23 atypical) were examined using a polyclonal antibody to the C-terminal peptide of the C-erb-B2 protein sequence. No C-erb-B2 gene product could be shown: the demonstration of C-erb-B2 does not seem to help, therefore, in determining diagnosis or prognosis in pulmonary carcinoid tumours.     

pICln蛋白单克隆抗体的制备及初步鉴定

目的:制备特异性的抗plCln蛋白的单克隆抗体(McAb),为plCln的纯化及检测等提供必要的工具.方法:以pICln的合成肽为免疫原,对BALB/c小鼠注射免疫,将免疫鼠脾细胞和小鼠骨髓瘤细胞NS-1进行细胞融合,用显微镜下挑选单个克隆的克隆化方法克隆化,用间接ELISA检测法进行筛选.单抗的效价、亚型及特异性用ELISA、免疫双向扩散实验和Western blot等检测.结果:获得了稳定的分泌型杂交瘤细胞株大2B7和4A3,2B7培养上清液单抗效价为1:64,属IgGl亚型,其腹水抗体效价及纯化抗体的效价分别为1:1.28×104、1:6.4×103,Westem blot及免疫耗竭实验呈现一条约40 kD)的特异性带,免疫组织化学染色显示在细胞核和细胞质中呈现阳性反应.结论:本次制备的单抗具有特异性、高效价,可应用于plCln蛋白的纯化及检测等研究.     

Detection of antibody to the PreS2 sequence of the hepatitis B virus envelope protein using an immuno-ligand assay with a silicon sensor detection system.

Sensitive immunoassays are essential for establishing the efficacy of recombinant vaccines to hepatitis B virus (HBV). These experimental vaccines include the PreS2 and S domains of the HBV envelope protein. To facilitate measurement of antibody against HBV PreS2, we employed the immuno-ligand assay with silicon sensor-based detection. Labeling of immune reagents with the haptens biotin and fluorescein allows adaptation to the immunofiltration light addressable potentiometric sensor (LAPS) system. A biotinylated monoclonal anti-PreS2 antibody and anti-PreS2 in clinical serum samples competitively bind in liquid phase to a fluorescein labeled PreS2 + S antigen. Streptavidin mediates the immobilization on biotinylated nitrocellulose membranes. Fluorescein mediates binding of an anti-fluorescein urease conjugate to the immune complex. Urease serves as the signal-generating component which subsequently is measured in the LAPS reader. In comparison to a competitive RIA, the immuno-ligand assay demonstrated a four-fold improved sensitivity using a smaller sample volume. The higher sensitivity resulted in earlier detection of seroconversion during a clinical vaccine study.     

Immunohistochemical detection of bcl-2 protein in thymoma

The protooncogene bcl -2 encodes a protein that inhibits apoptosis. The protein is expressed in most epithelial cells of the fetal thymic medulla but, to the best of our knowledge, no data are available on bcl-2 expression in thymoma. Expression of bcl-2 protein was analysed in 30 cases of thymoma by immunohistological staining of paraffin-embedded tissue. All cases were examined and classified according to the Salyer and Eggleston and the Müller-Hermelink classification. In four cases, the protooncogene bcl -2 was abnormally expressed in spindle cells of pure medullary thymoma, whereas the non-spindle cells in mixed and in cortical thymoma were negative. All the lymphocytes were also strongly positive in medullary thymoma while a few lymphocytes showed light staining in other thymomas.     

Immunohistochemical study of autoimmune pancreatitis using anti-IgG4 antibody and patients' sera

AIMS: Autoimmune pancreatitis (AIP), characterized by raised serum IgG4 levels, is frequently complicated by disorders of extrapancreatic organs. The aim of the present study was to examine immunohistochemically which extrapancreatic organs are affected, and whether an autoantibody to such organs is present in the serum of AIP patients. METHODS: Various tissues/organs obtained from AIP patients were studied immunohistochemically with an anti-IgG4 antibody. To examine the presence of an autoantibody in the serum of AIP patients, sera were incubated with various normal organs/tissues extracted for other diseases, followed by detection with an anti-IgG4 antibody. Sera were also examined before and after glucocorticoid therapy. RESULTS: Marked infiltration of IgG4+ plasma cells was observed in the pancreas, liver, bile duct and salivary gland of many of the AIP patients examined. The normal epithelia of the pancreatic ducts, bile ducts, gallbladder and salivary gland ducts reacting with the patients' sera were detectable by the anti-IgG4 antibody. Following glucocorticoid therapy the IgG4 antibody from the patients' sera showed decreased reactivity with these tissues. CONCLUSIONS: AIP may also affect extrapancreatic organs, the serum of AIP patients may contain an IgG4 autoantibody to various organs and glucocorticoid therapy may improve such disorders.