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A three-dimensional numerical fluid dynamic model of antigen-antibody surface adsorption on piezoelectric immunosensors

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ANNALS OF BIOMEDICAL ENGINEERING 2002年第10期30卷 1313-1322页

作者： Barak-Shinar, D Rosenfeld, M Zisman, E Abboud, S Tel Aviv Univ Fac Engn Dept Biomed Engn IL-69978 Tel Aviv Israel Tel Aviv Univ Fac Engn Dept Fluid Mech & Heat Transfer IL-69978 Tel Aviv Israel Savyon Diagnost Ltd Ashdod Israel

A piezoelectric crystal is a unit that changes its frequency in parallel with a change in its mass. This characteristic is exploited in designing flow cell-based immunosensors for detecting the concentration of antibodies in liquid samples. In the present study, computational fluid dynamic techniques are used to optimize the antigen-antibody binding process on an electrode surface placed on the base of a conical flow cell. The geometry optimization of the flow cell was determined to minimize the test time. This time is needed for the electrode to be saturated by the antibody, a process that requires the maximization of the adsorption rate and be accomplished by increasing the shear rate in the vicinity of the electrode. To validate the numerical model and to determine its parameters, experiments were carried out using an identical flow cell. In the experiments, the system did not reach saturation within an acceptable time frame, therefore, the model parameters were determined based on the unsaturated state. The experimental results confirmed the applicability of numerical simulations in predicting the effect of changing the inlet section area of the flow cells, proving the computational model to be very valuable in designing immunosensors based on flow cells. (C) 2002 Biomedical Engineering Society.

关键词：吸附抗体/分析抗体/化学抗体细菌/分析抗体细菌/免疫学抗体单克隆/分析抗体单克隆/免疫学抗原抗体复合物/分析抗原抗体复合物/化学生物传感技术/仪器和设备计算机模拟计算机辅助设计结晶电化学/仪器和设备电化学/方法电极设备设计/方法大肠杆菌/免疫学流式细胞术/仪器和设备流式细胞术/方法免疫测定/仪器和设备免疫测定/方法模型生物学模型化学

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Monoclonal IgM:: difficulties with correct measurement

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ANNALS OF CLINICAL BIOCHEMISTRY 2001年第6期38卷 708-710页

作者： Bossuyt, X Vranken, G Mariën, G Blanckaert, N Univ Hosp Leuven Dept Lab Med B-3000 Louvain Belgium Analis Belgium B-5000 Namur Belgium

We describe three monoclonal IgM paraproteins for which nephelometric IgM quantification generated inaccurate results.

关键词：抗体单克隆/分析血液化学分析/方法电泳毛细管/方法免疫化学/方法免疫球蛋白M/分析散射测浊法和比浊法/方法副蛋白血症/免疫学人类

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Biodistribution and radioimmunotherapy of human breast cancer xenografts with radiometal-labeled DOTA conjugated anti-HER2/neu antibody 4D5

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BIOCONJUGATE CHEMISTRY 2000年第3期11卷 327-334页

作者： Tsai, SW Sun, YY Williams, LE Raubitschek, AA Wu, AM Shively, JE City Hope Natl Med Ctr Beckman Res Inst Div Immunol Duarte CA 91010 USA City Hope Natl Med Ctr Dept Anat Pathol Duarte CA 91010 USA City Hope Natl Med Ctr Div Diagnost Radiol Duarte CA 91010 USA City Hope Natl Med Ctr Dept Radioimmunotherapy Duarte CA 91010 USA City Hope Natl Med Ctr Beckman Res Inst Dept Mol Biol Duarte CA 91010 USA

HER2/neu oncogene encodes a 185 kDa trans-membrane protein which is overexpressed in 20-30% of breast and ovarian cancers and portends a poor prognosis. We have studied the targeting and therapy of this oncoprotein with 4D5, a murine monoclonal antibody which recognizes a distinct epitope on the extracelluar domain of HER2/neu. We conjugated the antibody with an active ester of the macrocyclic chelating agent DOTA, radiolabeled the conjugate with either In-111 or Y-90, and studied the antibody distribution and therapy, respectively, in athymic mice bearing xenografts of MCF7/HER2/neu, a human breast cancer cell line transfected with the HER2/neu oncogene. For the biodistribution of In-111-labeled DOTA-4D5, a high specificity of tumor localization (30% ID/g) was seen with a tumor-to-blood ratio of greater than 2 at 48 h postinjection. Compared to a previously published study with I-125-labeled 4D5 in beige nude mice bearing NIH3T3/HER2/neu xenografts [De Santes et al. (1992) Cancer Res. 52, 1916-1923], In-111-labeled 4D5 antibody gave superior antibody uptake in tumor (30% ID/g vs 17% ID/g at 48h). In the therapy study, treatment of the nude mice bearing MCF7/HER2/neu xenografts with 100 mu Ci (3 mu g) of Y-90-labeled DOTA-4D5 caused a 3-fold reduction of tumor growth compared to untreated controls (injected with human serum albumin) in 40 days. Treatment of animals with 100 mu Ci of nonspecific antibody Y-90-labeled DOTA-Leu16 (3 mu g) had no tumor growth inhibition. Treatment with unlabeled DOTA-4D5 (3 mu g) had a slight effect on tumor growth compared to untreated controls. When analyzed at the level of single animals, no effect was seen in seven of nine animals;however, in two of the animals, tumor growth inhibition was observed. Although a cold antibody therapeutic effect was unexpected at this dose level (3 mu g), it may be possible that in some animals that 3 mu g of antibody of Y-90-labeled DOTA-4D5 augmented tumor growth reduction. To further explore the eff

关键词：抗体单克隆/分析抗体单克隆/治疗应用乳腺肿瘤螯合剂铟放射性同位素乳房肿瘤实验性/病理学乳房肿瘤实验性/放射疗法小鼠裸肿瘤移植有机金属化合物/治疗应用放射免疫疗法受体 erbB-2/免疫学组织分布移植异种肿瘤细胞培养的钇放射性同位素动物女(雌)性人类小鼠

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Quantitative measurement of bitagged recombinant proteins using an immunometric assay:: Application to an anti-substance P recombinant antibody

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ANALYTICAL BIOCHEMISTRY 2000年第2期284卷 221-230页

作者： Boquet, D Créminon, C Clément, G Frobert, Y Nevers, MC Essono, S Grassi, J CEA Saclay Serv Pharmacol & Immunol DRM DSV F-91191 Gif Sur Yvette France CEA Saclay Lab Associe CEA INRA F-91191 Gif Sur Yvette France

We have developed two different immunometric assays to directly quantify both the total and the active fractions of a recombinant antibody (single chain fragment variable, or ScFv) as obtained in a crude extract from an Escherichia coli expression system. For total determination, the assay is based on the simultaneous recognition of two different peptide Tag sequences (Ha-Tag and Myc-Tag) at each of the N- and C-terminal extremities of the recombinant protein. A monoclonal antibody (mAb 12CA5, directed against Ha-Tag), coated on microtiter plates, is used for capture, and the mAb 9E10 (directed against Myc-Tag), labeled with acetylcholinesterase (AChE, EC 3.1.1.7), acts as tracer. In parallel, for the determination of the active fraction, the capture is performed using microtiter plates coated with the antigen, while solid-phase-immobilized ScFv is measured using the same 9E10 tracer mAb, A synthetic peptide in which the two Tag sequences were joined was used as a standard, thus avoiding the laborious purification of a recombinant protein as reference. The method was applied to the direct measurement, in periplasmic extracts, of the total and active;fractions of an ScFv produced at different induction temperatures. (C) 2000 Academic Press.

关键词：氨基酸序列抗体单克隆/分析抗体单克隆/遗传学抗体单克隆/免疫学抗体亲和力抗体特异性碱基序列克隆分子 DNA引物 DNA 互补免疫测定/方法免疫球蛋白可变区/分析免疫球蛋白可变区/遗传学免疫球蛋白可变区/免疫学分子序列数据神经肽类重组蛋白质类/分析重组蛋白质类/免疫学可重复性结果 P物质/免疫学动物小鼠

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Equine macrophage identification with an antibody (Ki-M6) to human CD68 and a new monoclonal antibody (JB10)

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JOURNAL OF COMPARATIVE PATHOLOGY 2000年第2-3期122卷 145-154页

作者： Siedek, EM Honnah-Symns, N Fincham, SC Mayall, S Hamblin, AS Univ London Royal Vet Coll Dept Pathol & Infect Dis London NW1 0TU England

Monoclonal antibodies (mAbs) recognizing equine macrophages are scarce. The present study compared the immunocytochemical staining of various equine tissues (lymphoid tissue, lung, liver, small intestine, skin and blood leucocytes) by an antibody, Ki-M6, which detects CD68 in human macrophages and dendritic cells, and by a new anti-equine mAb, JB10, with staining produced by two previously described anti-equine macrophage mAbs, CZ2.2 and CZ3.3. Ki-M6 was shown to identify equine macrophages, which had a distribution different from those identified by CZ2.2 and CZ3.3. JB10 identified equine macrophages with a distribution similar to those identified by Ki-M6, but additionally bound to polymorphonuclear leucocytes. Flow cytometry of peripheral blood leucocyte subpopulations and tissue immunocytochemistry were used to compare staining by JB10 with that of CZ2.2 and CVS19;the latter identifies the myeloid antigen, EqCD13, found on polymorphonuclear leucocytes. The staining by JB10 differed from that of both CZ2.2 and CVS19, suggesting that JB10 detects a different molecule. These additional mAbs should prove useful for the future study of new, defined, populations of macrophages in equine immune responses and pathology, and, in the case of:Ki-M/6 antibody, may make possible an analysis of the structure, distribution and function of the CD68 molecule in the horse. (C) 2000 Harcourt Publishers Ltd.

关键词：抗体单克隆/分析抗体单克隆/代谢抗原 CD/分析抗原 CD/免疫学抗原 CD/代谢抗原分化髓单核细胞/分析抗原分化髓单核细胞/免疫学抗原分化髓单核细胞/代谢流式细胞术马免疫组织化学白细胞单核/化学肺/化学淋巴结/化学巨噬细胞/化学小鼠近交BALBC 组织分布动物女(雌)性人类小鼠

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Is MIB-1 proliferation index a predictor for response to neoadjuvant therapy in patients with esophageal cancer?

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AMERICAN JOURNAL OF SURGERY 2000年第6期179卷 514-520页

作者： Imdahl, A Jenkner, J Ihling, C Rückauer, KD Farthmann, EH Univ Freiburg Chirurg Klin Dept Surg D-79106 Freiburg Germany Univ Freiburg Dept Pathol D-79106 Freiburg Germany

BACKGROUND: The overall survival rate for patients with an esophageal cancer remains poor. As a consequence, preoperative chemoradiation was introduced for patients with tumor stage T >1 M0 regardless of tumor histology or localization. However, factors predicting response to this therapy pretherapeutically are largely unknown. METHODS: Clinical results of preoperative chemoradiation were investigated. The rates of proliferation and apoptosis were determined in pretherapeutic tumor samples and correlated with tumor response and long-term survival after surgery, RESULTS: A complete tumor response due to chemoradiation (n = 42;cervically localized tumors excluded) was achieved in 11 patients (26%) after resection. Five-year survival rate was significantly improved in these patients compared with those who did not respond to chemoradiation (48% versus 5.5%;P = 0.003). Chemoradiation was performed without benefit in 43%, Perioperative hospital mortality rate was 14.3% in all patients, No correlation of apoptosis with response to chemoradiation or postoperative long-term survival was observed. However, there was a clear correlation between the proliferation rate as determined by MIB-1 immunohistology. Five-year survival rate of patients with a proliferation index (PI) greater than or equal to 39% was 38% compared with 0% in tumors with a PI <39%. Tumors with a PI greater than or equal to 39% responded to chemoradiation in 71.4%, but 100% of tumors with a PI <39% did not. Mean survival time of these patients was 33 months and 11 months, respectively (P = 0.015). CONCLUSIONS: The results indicate that the PI may be used for stratification of patients treatment prior surgery. However, these results need further validation in larger patient numbers in the search for factors indicating response pretherapeutically to preoperative chemoradiation in esophageal cancer. (C) 2000 by Excerpta Medica, Inc.

关键词：腺癌/死亡率腺癌/病理学腺癌/治疗抗体单克隆/分析抗原核细胞凋亡癌鳞状细胞/死亡率癌鳞状细胞/病理学癌鳞状细胞/治疗细胞分裂化学疗法辅助综合疗法食管肿瘤/死亡率食管肿瘤/病理学食管肿瘤/治疗食管切除术/方法免疫组织化学 Ki-67抗原核蛋白质类/分析试验预期值手术前护理概率放射疗法辅助统计学非参数存活率肿瘤标记生物学/分析成年人老年人女(雌)性人类男(雄)性中年人

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Distribution of cells labelled by a monoclonal antibody (A3) against a cloned cell line derived from a rat malignant fibrous histiocytoma

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JOURNAL OF COMPARATIVE PATHOLOGY 2000年第2-3期123卷 77-87页

作者： Kumagai, D Yamate, J Tajima, T Tsukamoto, Y Yasui, H Kuwamura, M Kotani, T Sakuma, S Osaka Prefecture Univ Coll Agr Vet Teaching Hosp Osaka 5998531 Japan Osaka Prefecture Univ Coll Agr Dept Vet Pathol Osaka 5998531 Japan Osaka Prefecture Univ Coll Agr Dept Vet Microbiol Osaka 5998531 Japan Osaka Prefecture Univ Coll Agr Dept Vet Anat Osaka 5998531 Japan

To pursue the histogenesis of malignant fibrous histiocytoma (MFH), of which the cell of origin is still debated, 3 monoclonal antibody (A3) was produced against a rat MFH-derived cloned cell line (MT-8). Antigen recognized by A3 was around 80 kDa in molecular weight and was seen on the cytoplasmic membrane of MT-8 cells by immunoelectron microscopy. A3 reacted specifically with MT-8 cells, with another rat MFH-derived cell line (MT-9) and with their induced tumours in syngeneic rats, but not with ther rat tumours such as fibrosarcoma, histiocytic sarcoma, malignant meningioma, uterine leiomyosarcoma, endometrial stromal sarcoma, mononuclear cell leukaemia and malignant schwannoma. These findings indicate that A3 has a high specificity for rat MFH cells. In fetuses on gestation days 15, 18 and 20 and in postnatal rats aged 1, 4 and 8 days, A3 reacted with primitive mesenchymal cells in visceral organs and around arteries and bronchi, as well as in the lamina propria of intestinal mucosa, renal interstitium, meninges and perineurium. There were no A3-positive connective tissue cells in organs or other sites in adult rats more than 10 weeks old. It is therefore likely that MFH cells share antigens with primitive mesenchymal cells, which may be multipotent for mesenchymal differentiation. The present study suggests that MFH consists of 3 population of primitive. undifferentiated mesenchymal cells. A3 also immunolabelled endothelial cells of arteries, venules and pulmonary capillaries in fetal, postnatal and adult rats;vascular endothelial cells in chemically induced hepatic and renal lesions also reacted strongly with A3. However, the significance of endothelial immunoreactive with A3 remains to be elucidated. (C) 2000 Harcourt Publishers Ltd.

关键词：抗体单克隆/分析抗体单克隆/药代动力学主动脉/化学主动脉/胚胎学组织细胞瘤良性纤维性/免疫学组织细胞瘤良性纤维性/代谢组织细胞瘤良性纤维性/病理学免疫组织化学肾疾病/化学诱导肾疾病/代谢肾疾病/病理学肝/化学肝/胚胎学肝疾病/化学诱导肝疾病/代谢肝疾病/病理学肺/化学肺/胚胎学肺/超微结构小鼠近交BALBC 显微镜检查免疫电子大鼠近交F344 脊髓/化学脊髓/胚胎学肿瘤细胞培养的/免疫学肿瘤细胞培养的/代谢肿瘤细胞培养的/超微结构动物小鼠大鼠

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In vivo cytolysis and fusion of human immunodeficiency virus type 1-infected lymphocytes in lymphoid tissue

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JOURNAL OF INFECTIOUS DISEASES 2000年第1期182卷 338-342页

作者： Orenstein, JM George Washington Univ Med Ctr Dept Pathol Washington DC 20037 USA

Lymphoid tissue was examined to see whether in vivo cytopathic effects of human immunodeficiency virus (HIV) infection on lymphocytes could be detected. Transmission electron microscopy of mechanical suspensions prepared from lymph nodes showed both replication and phagocytosis of HIV particles by macrophages, Phagosomes contained cellular debris and virions, some of which were undergoing digestion. Neutrophils also contained HIV particles intermixed with cellular debris in phagosomes. Immunohistochemistry revealed whole Gag p24-positive lymphocytes and p24-positive cellular debris within the cytoplasm of paracortical macrophages. Lysing p24-positive lymphocytes were also seen. In the paracortex, p24-positive multinucleated lymphocytes with up to 5 nuclei were seen. In situ hybridization for HIV RNA in combination with immunohistochemistry for HAM56, a macrophage-specific marker, revealed colabeled cells. Thus, HIV-positive lymphocytes undergo lysis in lymphoid tissue. The cellular debris is phagocytized by macrophages, which themselves can replicate HIV. HIV-positive lymphocytes fuse in lymph nodes to form multinucleated cells.

关键词：抗体单克隆/分析细胞融合 HIV核心蛋白质p24/生物合成 HIV感染/免疫学 HIV感染/代谢 HIV感染/病理学 HIV-1/遗传学 HIV-1/代谢淋巴细胞/代谢淋巴细胞/病理学淋巴细胞/病毒学淋巴组织/免疫学淋巴组织/病理学淋巴组织/病毒学巨噬细胞/生理学巨噬细胞/超微结构巨噬细胞/病毒学显微镜检查电子中性白细胞/代谢中性白细胞/病毒学吞噬作用 RNA 病毒/分析人类

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Surface plasmon resonance (SPR) as a tool for antibody conjugate analysis

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BIOCONJUGATE CHEMISTRY 1999年第6期10卷 1032-1037页

作者： Adamczyk, M Mattingly, PG Shreder, K Yu, ZG Abbott Labs Div Diagnost Dept Chem D9NM Abbott Pk IL 60064 USA

Surface plasmon resonance (SPR) analysis was used to assess the immunoreactivity of anti-biotin (4) and anti-fluorescein (5) monoclonal antibody after conjugation with the N-hydroxysuccinimide ester of acridinium-9-carboxamide 1. Only minor changes in the apparent equilibrium dissociation constants of the antibody conjugates for their ligands resulted from the conjugation process. However, comparison of the initial binding rate of the conjugates with their ligands with those of the unmodified antibodies over a range of concentrations showed that the antibody conjugates were partially inactivated. The anti-fluorescein conjugates retained at least 90% of their immunoreactivity over the range of modification tested, while anti-biotin conjugates showed a progressive loss of reactivity with increased substitution by the label.

关键词：吖啶类/化学抗体单克隆/分析抗体单克隆/化学结合竞争性生物传感技术生物素/免疫学荧光素/化学溶液表面等离激元

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Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris - Evidence for a trophic paracrine relation

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AMERICAN JOURNAL OF PATHOLOGY 1999年第2期155卷 421-428页

作者： Blaauwgeers, HGT Hotkamp, BW Rutten, H Witmer, AN Koolwijk, P Partanen, TA Alitalo, K Kroon, ME Kijlstra, A van Hinsbergh, VWM Schlingemann, RO Univ Amsterdam Acad Med Ctr Dept Ophthalmol NL-1100 DD Amsterdam Netherlands TNO IVVO Gaubius Lab NL-2300 AK Leiden Netherlands Univ Helsinki Haartman Inst Dept Pathol Helsinki Finland Netherlands Ophthalm Res Inst Dept Ophthalmoimmunol NL-1100 AC Amsterdam Netherlands

The retinal pigment epithelium (RPE) maintains the choriocapillaris (CC) in the normal eye and is involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Vascular endothelial growth factor-A (VEGF) is produced by differentiated human RPE cells in vitro and in vitro and may be involved in paracrine signaling between the RPE and the CC. We investigated whether there is a polarized secretion of VEGF by RPE cells in vitro. Also, the localization of VEGF receptors in the human retina was investigated. we observed that highly differentiated human RPE cells, cultured on transwell filters in. normoxic conditions, produced two- to sevenfold more VEGF toward their basolateral side as compared to the apical side. In hypoxic conditions, VEGF-A secretion increased to the basal side only, resulting in a three- to 10-fold higher basolateral secretion. By immunohistochemistry in 50 human eyes and in two cynomolgus monkey eyes, KDR (VEGFR-2) and flt-4 (VEGFR-3) were preferentially localized at the side of the CC endothelium facing the RPE cell layer, whereas flt-1 (VEGFR-1) was found on the inner CC and on other choroidal vessels. Our results indicate that RPE secretes VEGF toward its basal side where its receptor KDR is located on the adjacent CC endothelium, suggesting a role of VEGF in a paracrine relation, possibly in cooperation with flt-4 and its ligand, This can explain the known trophic function of the RPE in the maintenance of the CC and its fenestrated permeable phenotype and points to a role for VEGF in normal eye functioning. Up-regulated basolateral VEGF secretion by RPE in hypoxia or loss of polarity of VEGF production may play a role in the pathogenesis of choroidal neovascularization.

关键词：抗体单克隆/分析细胞低氧细胞系细胞培养的脉络膜/解剖学和组织学脉络膜/血液供给脉络膜/代谢内皮生长因子/分泌上皮/解剖学和组织学免疫组织化学淋巴因子/分泌成束猴/解剖学和组织学旁分泌细胞细胞间通讯色素上皮眼/分泌原癌基因蛋白质类/分析受体蛋白质酪氨酸激酶类/分析受体细胞表面/分析受体生长因子/分析受体血管内皮生长因子视网膜/解剖学和组织学视网膜/代谢血管内皮生长因子A 血管内皮生长因子受体1 血管内皮生长因子受体3 血管内皮生长因子类青少年成年人动物儿童人类

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