The distribution of LH39 basement membrane epitope in the tumour stroma of oral squamous cell carcinomas.

LH39 monoclonal antibody detects a novel component of epithelial and endothelial basement membranes. The expression of LH39 antigen was investigated by immunohistochemistry in 55 oral squamous cell carcinomas and compared with 15 pyogenic granulomas of skin and oral mucosa, 20 non-specific oral ulcers, and 20 specimens of normal oral mucosa. The distribution of this basement membrane epitope was compared with that of other basement membrane components, type IV collagen, and laminin. LH39 monoclonal antibody labelled basement membrane surrounding small blood vessels in normal human organs. In oral squamous cell carcinomas, in contrast to the other basement membrane antigens, the LH39 epitope was not detectable in vessels within histologically recognizable tumour stroma. Neovascularization is known to attend malignant neoplasms and this finding was interpreted as absence of LH39 antigen within newly formed vessels. In support of this hypothesis, LH39 immunoreactivity was absent in newly formed blood vessels within pyogenic granulomas and the granulation tissue within ulcers. As increasing neovascularization is reported to correlate with a rising rate of metastasis, assessment of tumour angiogenesis may be of value in selecting patients for initial aggressive therapy.     

A monoclonal antibody stains blastemal but not tubular components of Wilms' tumour

The monoclonal antibody PAL-E is specific for endothelial cells in a wide variety of normal and tumour tissue. In normal kidney, PAL-E reacts exclusively with the endothelium of non-glomerular blood vessels. In Wilms' tumour, binding of PAL-E was not restricted to the endothelium; staining of blastemal cells was observed in seven out of eight cases examined. Mesenchymal and tubular components, if present in Wilms' tumour, were negative. In contrast, a monoclonal antibody to Factor VIII-related antigen (RFF-8-R-1) bound only to endothelial cells in these tumours. In fetal kidney, PAL-E binding showed a wider distribution than in adult kidney and both stromal and glomerular capillaries were stained. Tubules and non-endothelial stromal cells were negative. These results indicate that the reactivity of the monoclonal antibody, PAL-E, is not restricted to cells of endothelial origin in all tissues. The implications of these findings for the differentiation of Wilms' tumour are discussed.     

A rat cytotrophoblast antigen defined by a monoclonal antibody.

A monoclonal antibody that recognizes specifically a cytotrophoblast antigen was obtained. The monoclonal antibody 22H6 was tested on rat choriocarcinoma (in vivo, in vitro), normal placenta, ectoplacental cone, blastocysts, and several normal organs. The antigen was detected on frozen sections and on tissue culture by indirect immunofluorescence. The monoclonal antibody 22H6 reacts with the cytotrophoblasts of rat choriocarcinoma. The giant cells do not display a positive reaction. It is not expressed on other tumors than choriocarcinoma. In adult rats the only cells revealing a positive reaction are the hepatocytes and the epithelial cells lining the small intestine. In the pregnant rat, the antigen is expressed on the cytotrophoblasts of the junctional zone in the placenta, but not on the giant cells. The mab reacts only with the small trophoblast cells of the ectoplacental cone, but not with trophectoderm of blastocyst. The mab has an IgG2b isotype and is not cytotoxic for choriocarcinoma cells in a complement-dependent cytotoxicity test. The described monoclonal antibody is to our knowledge the only known marker of rat benign and malignant cytotrophoblast.     

Determination of class and subclass of mouse monoclonal antibody by ELISA

Summary Characterization of monoclonal antibody molecules is important to provide the information necessary to make appropriate choices in antibody purification and use. This report describes a frequent first step characterization of antibody molecules: determination of immunoglobulin class and subclass by enzyme linked immunosorbent assay.     

A novel HLA-A determinant recognized by a cytotoxic human hybridoma IgG1 monoclonal antibody (TrJ14)

Abstract: TrJ14 is a cytotoxic human IgG1Λ hybridoma mAb that recognized a novel HLA-A epitope expressed by lymphoblastoid B cells that are homo- or heterozygous for A2, A3, A11, A30, A31, A33, A68 and A69. Based on these results, the HLA type of cell line TEM (10w9057) was retyped as A66. When peripheral blood T cells isolated freshly from 265 HLA-typed normal individuals served as targets, TrJ14 killed cells expressing two TrJ14-positive HLA-A alleles, as well as the majority of cells having one TrJ14-positive and one TrJ14-negative HLA-A antigen. However, TrJ14 failed to recognize or reacted weakly with most HLA-A2 and -A3 heterozygous normal T cells when A2 or A3 was coexpressed together with a TrJ14-negative antigen. The serological reactivity of TrJ14 correlated with the amino acid valine and aspartic acid at positions 76 and 77 of the αl-domain helix. These amino acids were shared exclusively by all the identified TrJ14⁺ alleles.     

A novel blocking monoclonal antibody recognizing a distinct epitope of human CD40 molecule

CD40, a member of the tumor necrosis factor receptor superfamily, is an important costimulatory molecule during the immune response. Here, we report a blocking mouse antihuman CD40 monoclonal antibody, mAb 3G3, of which the specificity was verified by flow cytometry and Western blot. It was shown by competition test that 3G3 bound to a different site (epitope) of CD40 from the reported CD40 mAbs, including clone mAb89, 3B2, and 5C11. It was also found that mAb 3G3 could inhibit homotypic aggregation of Daudi cells induced by the agonistic anti-CD40 mAb 5C11. Furthermore, mAb 3G3 effectively inhibited the proliferation of peripheral blood mononuclear cells in mixed lymphocyte reaction assay. Finally, a sensitive and specific soluble CD40 (sCD40) ELISA kit was established by matching mAb 3G3 with 5C11, and it was found that the levels of sCD40 in sera from patients with immune disorders such as hyperthyroidism, chronic nephritis, and rheumatoid arthritis were obviously higher than those from normal individuals. Thus, this blocking anti-CD40 mAb provides a novel tool for the study of CD40.     

抗人红细胞M型抗原单克隆抗体的研制及初步应用

应用杂交瘤枝术,用人M型RBC免疫的BALB/c小鼠脾细胞,与小鼠骨髓瘤细胞系NS-1融合,筛选出抗M血型抗原的单克隆抗体杂交瘤细胞株13H_2;经过8个多月连续传代培养增殖良好,仍能稳定分泌效价高、特异性强的单克隆抗体。细胞培养上清效价1∶512,亲和力11s。通过将此株单抗用于血型检测、标准红细胞谱细胞的鉴定和在法医上检测血痕,均证明此株单抗识别的只是红细胞上的M血型抗原,与其它血型抗原无关。较多克隆抗M型抗原血清特异性强,效价高,亲和力好,是检测MN血型的良好诊断试剂。     

一株鼠抗人PD-1功能性单克隆抗体的研制及其生物学特性的鉴定

研制特异性鼠抗人PD-1功能性单克隆抗体,并对其生物学特性进行鉴定。以高表达人PD-1分子的基因转染细胞L929/PD-1作为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-1作为抗体筛选细胞,L929/mock为对照细胞,经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,筛选出分泌特异性鼠抗人PD-1单克隆抗体的杂交瘤细胞株;采用Western blot、Ig亚型快速定性试纸法、间接免疫荧光法、竞争结合抑制试验和MTT增殖实验对单抗进行生物学特性的分析。结果表明,成功获得1株特异、稳定分泌鼠抗人PD-1单克隆抗体的杂交瘤细胞株,命名为6E2。对6E2生物学功能的研究结果提示,该单抗能够识别活化T细胞表达的PD-1分子,且在体外能够显著抑制T细胞的增殖和细胞因子的分泌。获得的特异性鼠抗人PD-1功能性单克隆抗体,通过激发PD-1负性途径能够有效抑制T细胞的功能,为进一步研究PD-1信号奠定了物质基础。     

抗艰难梭菌A毒素单克隆抗体的制备及特性分析

目的 :制备抗艰难梭菌A毒素的单克隆抗体 (mAb)并鉴定其特性。方法 :用纯化的艰难梭菌A毒素免疫BALB/c小鼠 ,将免疫小鼠的脾细胞与骨髓瘤细胞Sp2 / 0融合 ,采用间接ELISA筛选杂交瘤细胞。用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析 ;用Westernblot检测mAb的特异性。结果 :得到 6株杂交瘤细胞株 ,5C10株细胞分泌的mAb为IgG2a ,4B5和 8A1株细胞分泌的mAb为IgG1,其他 3株细胞mAb (2H7、3E9和 6G8)均分泌IgM。中和试验表明 ,所有的mAb均无中和活性。腹水mAb的效价均在 10 -4以上 ,其中mAb 2H7、6G8、5C10、4B5和 8A1具有共同的表位 ,而mAb 3E9识别的位点与其他 5株不同。mAb 8A1和 4B5的相对亲和力>10 5,其他 4株mAb的相对亲和力 >10 4。在非变性条件下 ,PAGE后Westernblot的结果显示 ,6株mAb均可与相对分子质量 (Mr)为 5 5× 10 4的A毒素产生反应 ;而在变性条件下 ,还原与非还原SDS PAGE后Westernblot均显示 ,6株mAb均可与Mr 为 5× 10 4～ 2 4× 10 4的A毒素产生反应。结论 :6株杂交瘤细胞株均能分泌抗艰难梭菌A毒素的特异性mAb ,为艰难梭菌A毒素的研究提供了有利的工具     

Development and characterization of a human monoclonal antibody probably detecting the leukocyte differentiation antigen CD39

A human monoclonal IgM antibody, referred to as TU223, has been produced. The reactivity of TU223 was tested in various cells and cell lines by complement-dependent microcytotoxicity test and fluorescence-activated cell sorter analysis. The antigen defined by TU223 was expressed on Epstein-Barr virus-transformed B-cell lines and on some Burkitt's lymphoma cell lines, but was not expressed on normal T cells, B cells or erythrocytes. In addition, expression of the antigen defined by TU223 was also induced on B cells activated by Epstein-Barr virus or pokeweed mitogen, and on T cells activated by phytohemagglutinin, concanavalin A, pokeweed mitogen or recombinant interleukin-2. However, no expression of the antigen detected by TU223 was induced at all on recombinant interleukin-4-treated B cells or macrophage-like cell line U937. When the ability of TU223 and various mouse monoclonal antibodies to bind to human differentiation antigens was compared, interestingly, the reactivity of TU223 was found to be very similar to that of mouse monoclonal antibody CD23 (H107), which reacts with Fc epsilon receptor II. Two-color analysis revealed that the antigen defined by TU223 is expressed on the cell surface of certain lymphoid cells expressing CD23 antigen. However, it can be concluded that the antigen defined by TU223 is clearly distinct from Fc epsilon receptor II, based on assay of cross-blocking between H107 and TU223. The surface antigen on B85 cells recognized by TU2232 had the molecular size of 80-82 kiloDaltons as determined by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody

Synuclein was initially named for its localization in both presynaptic nerve terminals and portions of nuclear envelope. However, subsequent studies only confirmed the presynaptic localization of this protein in the brain; its nuclear localization in the neurons remained elusive. Here, two new monoclonal antibodies against alpha-synuclein (alpha-SYN) were produced. Epitope mapping using phage peptide display showed that the epitopes of the two antibodies were localized in two distinct specific sequences of the C-terminal domain of alpha-SYN. One antibody named 3D5 recognized amino acids 115-121 of alpha-SYN and the other antibody named 2E3 identified the amino acids 134-138 of the protein. Western blot analysis demonstrated that both 2E3 and 3D5 detected a 19 kD protein from rat and human brain homogenates, which was identical to the molecular size of recombinant alpha-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of alpha-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic alpha-SYN but only 3D5 detected the nuclear alpha-SYN. The nuclear localization of alpha-SYN was further confirmed by Western blot analysis in isolated nuclear fraction where the same size of alpha-SYN was detected, and by immunoelectron microscopy using colloidal gold probes where gold particles were specifically localized in portions of peri- and intra-nucleus. The nuclear positive neurons were distributed extensively in almost all the brain regions. This is the first report well characterizing the extensive localization of alpha-SYN in the neuronal nuclei throughout the brain in normal conditions. This finding indicates an important physiological function of this molecule in the nuclei of brain neurons, which deserves further investigations.     

Association between epitopes detected by monoclonal antibody BIP-45 and the Xbal polymorphism of Apolipoprotein B

An epitope of Apolipoprotein B (ApoB), recognised by a monoclonal antibody BIP-45, is associated with the development of ischaemic heart disease (Duriez et al. 1988). We have examined the genetic relationships between this epitope and three Restriction Fragment Length Polymorphisms (RFLPs) of the gene for ApoB detected with the enzymes EcoRI, PvuII and XbaI in a sample of 53 unrelated individuals from France. There is an association between binding affinity to BIP-45 and the XbaI RFLP; the 8.6 kb XbaI allele (absence of cutting site) being associated with low-affinity binding to BIP-45. In this sample of individuals there is no significant association between serum cholesterol levels and BIP-45 binding affinity, but there is a significant correlation between serum cholesterol levels and XbaI genotype, with individuals of the genotype X1X1 having the highest and those with the genotype X2X2 having the lowest levels of serum cholesterol. This suggests that variation at the ApoB locus may be involved independently in the determination of serum lipid levels and in the development of ischaemic heart disease.     

新生血管特异抗体的制备及生物活性鉴定

目的 研制识别肿瘤新生血管的单克隆抗体及其活性。方法 用肝癌细胞条件培养基刺激人血管内皮细胞，以此作为抗原免疫小鼠制备抗体。用免疫组化、ELISA和流式细胞术筛选肿瘤血管特异抗体并鉴定其免疫特性。用MTT法研究抗体对血管内皮细胞增殖的影响。结果 抗体AA98选择性地识别肿瘤血管和人血管内皮细胞，抑制血管内皮细胞的增殖。结论 抗体AA98在肿瘤诊断和治疗方面具有潜在的应用价值。     

A Novel Anti-Human Syndecan-1（CD138） Monoclonal Antibody 4B3： Characterization and Application

Syndecan-1 （CD138）, a member of integral membrane heparin sulfate proteoglycans, is an essential matrix receptor for maintaining the normal morphological phenotypes. In this study, we generated a specific mouse anti-human syndecan-1 monoclonal antibody （mAb） 4B3 and identified it by competition assay with the available syndecan-1 mAb （BB4）. Stained by 4B3, the expression of syndecan-1 was detected on tumor cell lines, such as 8226, U266, XG-1, XG-2, Daudi and Jurkat. The expression was also found on neuron stem cells. It was established that 4B3 mAb could inhibit XG-1 and XG-2 proliferation. The data not only determined that 4B3 mAb was a functional anti-human syndecan-1 mAb, but also indicated that syndecan-1 might be a valuable surface antigen and play an important role in regulation of tumor pathology and differentiation of neural stem cells. This novel antibody 4B3 may be value of study of tumor proliferation/survival mechanism and contributes to diagnosis and treatment of diverse diseases. Cellular ＆ Molecular Immunology.     

A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type

Yasuda A, Uchida T, Nguyen LT, Kawazato H, Tanigawa M, Murakami K, Kishida T, Fujioka T, Moriyama M. A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type. APMIS 2009; 117: 893–9. Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme‐linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA‐positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver‐operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori‐infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up‐regulated by adhesion to epithelial cells.     

The effect of epitope density and antibody affinity on the ELISA as analysed by monoclonal antibodies

The antibody titre and the optical density values in an ELISA are influenced by the epitope density of the antigen and the affinity of the antibody tested. This has major implications in the interpretation of ELISA results.     

副流感病毒兰州株的分离与鉴定

目的 副流感病毒兰州株的分离与鉴定。方法 应用鸡胚和人胚肺成纤维突变细胞株分离病毒;应用血凝和血抑以及近年来多株流感国际和国际和国家株（Ｈ１Ｎ１亚型６株,Ｈ３Ｎ２亚型２株,Ｂ型４株）作抗原抗体交互试验,还用４类主要呼吸道病毒单克隆间接免疫荧光法与２株兰州株的病毒抗原基质片作特异性交叉反应。结果 ２株兰州株在鸡胚中培养血凝效价滴度高达１：５１２（＋＋）,它们与流感毒甲型（Ｈ１Ｎ１和Ｈ３Ｎ２）以及乙型     

A novel strategy for targeting CD4+ PPD-reactive T cells against tumour cells using PPD monoclonal antibody heteroconjugates.

We have constructed PPD monoclonal antibody heteroconjugates specific for a tumour-associated antigen of C57BL/6 melanomas or for human complement component C3d fixed de novo to murine fibrosarcoma cells (MC6A). The ability of our heteroconjugates to target CD4+ PPD-reactive T cells against the appropriate tumour targets was then determined in vitro. Heteroconjugate-treated B16-F10 and MC6A tumour targets were both able to present PPD to the specific T cells, resulting in activation and concomitant lymphokine secretion. Secreted lymphokines were then demonstrated to cause significant tumour cytolysis and cytostasis in vitro. Preliminary experiments in vivo suggest that this targeting system may provide the basis for a future immunotherapeutic strategy.     

Growth fraction in non-Hodgkin's lymphomas and reactive lymphadenitis determined by ki-67 monoclonal antibody in fine-needle aspirates

The fraction of proliferation cells was analysed in fine needle aspirates from a series of 448 non-Hodgkin's lymphomas and 199 reactive hyperplasias using an immunoperoxidase staining with monoclonal antibody Ki-67. There was a good correlation between proliferation fraction and cytologic assignment to high and low grade lymphomas. Thus high grade lymphomas had a high median percentage of Ki-67 positive cells with a figure of 82.1 for lymphoblastic, 60.0 for immunoblastic, and 59.7 for centroblastic lymphomas. For low grade lymphomas the figures were 17.1 and 11.1 percent for centroblastic/centrocytic and CLL/immunocytoma, respectively. the fraction of proliferation cells in reactive lymphadenitis varied between 1–50% with a median of 11.5%. Analysis of Ki-67 positivity can accordingly not be used to differentiate benign from neoplastic proliferations. Within all lymphoma subgroups but lymphoblastic lymphoma, there was a marked variation in fraction of Ki-67 positive cells, which resulted in a certain overlap between high and low grade lymphomas. the results show that cells procured through fine-needle aspiration can be used to analyse the fraction of proliferating cells which contributes information about the growth rate of the individual tumours that can not be obtained through cytologic classification. © Wiley-Liss, Inc.