抗末端脱氧核苷酰转移酶单克隆抗体的制备及其生物学特性的初…

本文用提纯的小牛胸腺末端脱氧核苷酰转移酶为免疫原，经一次融合，建立了３株能稳定分泌抗小牛胸腺ＴｄＴ单克隆抗体的杂交瘤细胞株。３株单抗均为ＩｇＧ１抗体；抗体相加试验证明抗ＴｄＴ２和抗ＴｄＴ３识别ＴｄＴ的同一表位，而抗ＴｄＴ１则识别另一表位；３株单抗识别的抗原分子量均为５８．８ｋＤ；与人白血病细胞株及各型白血病细胞的反应谱３株单抗和进口抗ＴｄＴ单抗基本一致。提示这３株单抗有可能替代进口抗ＴｄＴ单抗，供     

抗慢性B淋巴细胞白血病同种型和独特型单克隆抗体的研究

本研究以慢性B淋巴细胞性白血病患者血清IgM免疫BALB／C／J‘鼠，取其脾细胞，采用细胞融合技术建立分泌抗慢性淋巴细胞白血病（BCLL）同种型和独特型单克隆抗体的杂交瘤细胞株。采用一系列免疫化学，细胞免疫学等技术对10株独特型单抗和5株同种型单抗的特异性，亲和力，识别抗     

用抗独特型抗体主动免疫增强鼻咽癌放疗病人的免疫功能

针对鼻咽癌细胞膜抗原制备了一株单克隆抗体（Ａｂ１），且又对该（Ａｂ１）可变区制备了一株单抗（Ａｂ２），此Ａｂ２能象鼻咽癌细胞膜抗原一样与Ａｂ１可变区的抗原结合位结合，因此在功能上Ａｂ２能够模拟原抗原，刺激机体的免疫系统。用该Ａｂ２对鼻咽癌放疗病人作主动免疫治疗，并设放疗加生理盐水注射对照组。检测了血清中人抗鼠抗体（ＨＡＭＡ）的产生情况，并检测了治疗前后各项体液免疫指标，包括抗Ａｂ２抗体（Ａｂ３）和抗鼻咽癌细胞抗体（Ａｂ１）水平以及各项细胞免疫指标，包括血清中细胞因子ＴＮＦ－α、ＩＬ－２、ＩＦＮ－γ的水平和外周血单个核细胞（ＰＢＭＣ）ＩＬ－２ｍＲＮＡ的表达。结果表明，与对照组相比，鼻咽癌抗独特型抗体能使鼻咽癌放疗病人的免疫指标上升，有增强免疫功能的作用，可能是一种有益的辅助疗法。     

双特异抗体介导的CD3AK对人小细胞肺癌细胞株的细胞毒作用

目的：研究在双特异性抗体的介导下ＣＤ３ＡＫ细胞（ＣＤ３单克隆栓激活的杀伤细胞）对人小细胞肺癌细胞株ＬＴＥＰ－ｓｍｌ的细胞毒作用。方法：用＾３１Ｃｒ－Ｎａ２ＣｒＯ４释放试验检测单抗（２Ｄ６、ＵＣＨＴ１）和双特异性抗体（ＷＳＴ－Ｈ７）与ＣＤ３ＡＫ共同对人小细胞肺癌细胞株的杀伤活性。持异性抗体体外能明显增强ＣＤ３ＡＫ细胞对人小细胞肺细胞株ＬＴＥＰ－ｓｍｌ的杀伤活性结论：与单纯ＣＤ３ＡＫ相比,加入双特异性     

抗表皮生长因子受体与抗CD_3双功能抗体的制备及细胞毒的研究

制备抗ＥＧＦ┐Ｒ与抗ＣＤ３双功能单克隆抗体及其细胞毒的研究方法通过杂交－杂交瘤技术制备双功能单克隆抗体细胞株Ｅｔ２２，并了解其与ＩＬ┐２联合体外激活ＰＢＬｓ细胞对Ａ４３１癌细胞的杀伤活性。结果ＩＬ┐２激活的ＰＢＬｓ和未经ＩＬ┐２刺激培养的ＰＢＬｓ对靶细胞Ａ４３１的杀伤活性，效靶比为２０∶１的条件下，杀伤率分别为４９％，１７％。Ｅｔ２２双功能抗体介导ＩＬ┐２激活的效应细胞杀伤靶细胞（Ａ４３１）为７５％。结论双功能单克隆抗体（ＥＱ７５×ＯＫＴ３）介导ＩＬ┐２激活的效应细胞杀伤靶细胞（Ａ４３１）活性高于两种亲本单抗（ＣＤ３或ＥＱ７５     

100例急性白血病患者多药耐药性检测分析

作者应用抗Ｐ－糖蛋白（Ｐ－１７０）单抗ＪＳＢ－１对１００例急性白血病（ＡＬ）患者进行了检测，结果表明：（１）包括Ｍ３在内的几乎所有ＡＬ亚型均可有ＭＤＲ阳性表达，初诊组ＭＤＲ表达率以Ｍ５（６２．５％）为最高，ＡＮＬＬ（２０％）虽高于ＡＬＬ（８．３％），但差异无显著性意义。（２）初诊组部分患者（１８．２％）呈ＭＤＲ阳性表达，提示ＭＤＲ的内在性。（３）Ｐ－１７０阳性率与ＡＬ临床状况密切相关，难治组Ｐ－１     

三氧化二砷对结肠癌细胞抑制作用的实验研究

砷剂是治疗恶性肿瘤的化疗药物，１９世纪Ｆｏｗｌｅｒ′ｓ液曾被应用于白血病的治疗［１］。中国传统医学也有砒石（主要成分为Ａｓ２Ｏ３）治疗乳腺癌等肿瘤。以前对砷剂的研究主要局限于致癌、致畸、致突变上。近年来由于Ａｓ２Ｏ３在急性早幼粒细胞白血病（ＡＰＬ）治疗的临床和基础研究上取得很大进展［２，３］。但关于Ａｓ２Ｏ３对实体肿瘤作用的研究报道很少。作者采用结肠癌ＬＯＶＯ细胞株作为对象，来研究Ａｓ２Ｏ３对结肠癌的抑制效应，探讨应用于大肠癌临床治疗的可能性。材料与方法１“癌灵一号”，成分０１％Ａｓ２Ｏ３，…     

CLL、PLL、HCL临床表现和细胞形态学探讨

幼淋巴细胞性白血病（ＰＬＬ）、毛细胞性白血病（ＨＬＬ）是从慢性淋巴细胞性白血病（ＣＬＬ）中分出来的病种,本文探讨了三者的临床表现和细胞形态差异。１　临床资料１９８６年４月～１９９８年１２月我院共收治ＣＬＬ７例,男５例,女２例,年龄３６～６７岁;ＰＬＬ６例,男５例,女１例,年龄６８～７９岁;ＨＣＬ３例,男性,年龄４１～６６岁。ＣＬＬ均以头昏、乏力、贫血为就诊症状,ＰＬＬ２例以头昏,３例以腹部肿块及１例以查体血象不正常就诊,ＨＣＬ３例均因腹部肿块、消瘦就诊。临床检查结果见表１、２。表１ＣＬＬ、ＰＬＬ…     

急性白血病并骨髓坏死5例

急性白血病并骨髓坏死５例郭艳珍１吴桂芬２急性白血病（ＡＬ）并发骨髓坏死比较少见，现将我们在临床中所遇５例报告如下。１临床资料５例患者男３例，女２例。中位年龄３４岁。急性淋巴细胞白血病（ＡＬＬ）４例，急性非淋巴细胞白血病（ＡＮＬＬ）１例。均经骨穿及活检...     

平阳霉素（A5）与抗人食管癌非组蛋白抗体交联物对人食管癌Eca－109细胞株的体外抑制效应

我们以非组生白质（ＮＨＰ）抗体作为抗癌药物平阳霉素（Ａ５）的载体，制备ＮＨＰＩｇＧＡ５交联物。应用（１）细胞培养隔日计数示生长曲线；（２）以Ｆｅｕｌｇｅｎ法示细胞ＤＮＡ相对含量；（３）３Ｈ－ＴｄＲ掺入实验、比较了ＮＨＰＩＧ－Ａ５、ＮＨＰＩｇＧ及Ａ５对人食管癌细胞株ＥＣａ１０９的体外抑制效应，四者比较，ＮＨＰＩｇＧ－Ａ５人食管癌Ｅｃａ－ｌ０９细胞株有较强的抑制效应。     

The intracellular location of terminal transferase does not vary with cell cycle stage

The biochemical activity of terminal transferase (TdT) in the thymocytes of leukemic AKR mice has no relationship to cell cycle stage, unlike the activity of replicative DNA polymerase which increases during the period of DNA synthesis. Moreover, such assays of DNA polymerase α reveal a shift in enzyme activity from cytoplasm to nucleus during S phase. In the present study, the role of TdT in DNA metabolism was explored further by examining the intracellular location of the enzyme during cytokinesis. Single cell suspensions of thymocytes from leukemic AKR mice were partially synchronized by velocity sedimentation in a sucrose gradient at unit gravity and harvested according to cell cycle stage. The content and location of TdT in individual cells was determined by indirect immunofluorescence using a rabbit antiserum to calf thymus TdT as the primary antibody. There was no relationship of fluorescence intensity or of the proportion of TdT-positive cells to cell cycle stage. In all samples examined (n = 6) the enzyme was located almost entirely in the nucleus throughout cytokinesis. These results do not support the hypothesis that the intracellular location of TdT may vary with cell cycle stage and a role for the enzyme in DNA synthesis remains to be defined.     

抗p16单克隆抗体的制备及鉴定

目的：制备抗Ｐ１６单克隆工进行鉴定。方法：以Ｐ１６合成肽为抗原免疫纯系小鼠。利用杂交瘤技术制备单克隆抗体,并利用亲和层析等法纯化抗体。通过酶免疫测定及免疫印迹等法对单克隆抗体进行分析及鉴定,并通过免疫组化方法与进我克隆抗体进行比较。结果：获得４株稳定分泌抗Ｐ１６单克隆抗体杂交瘤株,与进口多克隆坑体比较,检测阳性及阴性符合率达１００％,但使用单克隆抗体时,组织切片不需进行抗原修复,染色效果稳定。结论     

Prognostic significance of surface marker expression on blasts of patients with de novo acute myeloblastic leukemia

The prognostic significance of the expression of surface membrane antigens on the blasts of 123 consecutive patients with de novo acute myeloblastic leukemia (AML) was evaluated. For this purpose, reactivity of monoclonal antibodies (mAbs) CLB-ERY3 (antiblood-group H antigen), VIM-D5 (CD15), WT1 (CD7), MY7 (CD13), MY9 (CD33), VID-1 (antihuman leukocyte antigen locus DR [anti-HLA DR]), VIM-2 (CDw65L), VIM-13 (CD14), 63D3 (CD14) and anti-TdT with leukemic blast cell populations was prospectively analyzed with respect to the rates of complete remission (CR), continuous complete remission (CCR), and survival. The overall rate of CR was 65%, the 6-year rates of overall CCR and survival were 23% and 13%, respectively (median period of patient observation, 30 months). Of all Abs tested, four (CLB-ERY3, MY7, anti-TdT, and VIM-D5) were found to be of prognostic value. Reactivity of CLB-ERY3, MY7, and anti-TdT was predictive for CR (CLB-ERY3+, 43% v CLB-ERY3-, 73%, P less than .02; MY7+, 59% v MY7-, 91%, P less than .003; TdT+, 28% v TdT-, 71%, P less than .001, respectively) and probability of survival (significantly lower survival rates: CLB-ERY3+, P less than .02; MY7+, P less than .03; and TdT+ cases, P less than .001, respectively). Reactivity of VIM-D5 was significantly associated with a higher probability of CCR (P less than .01). Our results confirm earlier reports on the prognostic significance of expression of CD13 and TdT in AML and indicate CLB-ERY3 (antiblood-group H antibody) and VIM-D5 (CD15) as further markers predictive for the clinical outcome in patients with de novo AML.     

Classification of acute myeloid leukemias--a comparison of FAB and immunophenotyping

A large number of monoclonal antibodies (McAbs) directed against components on myeloid (granulocytic/monocytic) cells have been generated. Individual McAbs were identified which are selectively reactive with antigenic determinants expressed by myeloid cells at specific stages of differentiation in a lineage-restricted fashion. The composite phenotype obtained by a combination of antimyeloid McAbs allows for a precise definition of the normal or malignant cell type under investigation. Cell binding studies on normal and leukemic cells and the biochemical characterization of the antigens provided the basis for a grouping of those antimyeloid McAbs into clusters of differentiation (CD). The reactivity patterns of CD11, CD13, CD14, CD15, and CD33 McAbs and the characteristics of the respective antigens are reviewed. These CD McAbs distinguish leukemic cells of myeloid from those of lymphoid origin. The monocytic nature of AML cells can be recognized by CD14 McAbs, whereas the other CD McAbs react with both monocytic and nonmonocytic types of acute myeloid leukemia. The expression of these differentiation antigens is not concordant with the morphological-cytochemical French-American-British (FAB) classification of leukemia; nevertheless, tendencies for agreement are apparent. If used in combination, FAB typing and immunophenotyping could provide complementary information. Their potential use for mapping of myeloid differentiation and for cell type recognition in leukemia phenotyping demonstrates the utility of antimyeloid CD McAbs for biological or clinical investigations. The diagnostic value of antimyeloid McAbs is enhanced if the reagents are included in a panel of McAbs standardized for routine immunophenotyping.     

抗腺病毒载体单克隆抗体的制备及其初步鉴定

Objective:To prepare and identify monoclonal antibodies(McAbs)against the capsid proteins of adenovirus vector.Methods:BALB/c mice were immunized with a mixture of the purified adenovirus vector(Adv)and Al(OH)3.McAbs were produced using cell fusion technique in a conventional way.The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay(ELISA),immunocytochemical staining and Western blotting. Results:Six strains of hybridoma cells(A4H11,A8C7,F1H5,G1D2,G4E3 and H2G8)that can stably secrete the IgG1 McAb against Adv were obtained.After 3 months subculture and low concentration of serum adapting culture,six strains retained their stability to secrete McAb.The ascites titers were between 1:106 and 1:108.Western blot analysis demonstrated that all the McAbs reacted with one protein(about 114 kDa)which is present in wild type 3 adenovirus(wtAd3),wild type 5 adenovirus (wtAd5),wild type 7 adenovirus(wtAd7)and adenovirus vector.Conclusion:Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector,and provided the substantial foundation of preclinical research of adenovirus vectors.     

Detection of circulating ‘terminal transferase-positive’ cells does not predict relapse in acute lymphoblastic leukemia

Serial samples of peripheral blood were obtained from 35 children with ALL over a period of 18 months. The mononuclear cells were examined for TdT by indirect immunofluorescence using an unpurified anti-calf thymus TdT as the primary antibody. This analysis failed to distinguish those children who were destined to relapse (n = 9) from those who remained in continuous complete remission. Rather, the exhibition of fluorescence was linked to the co-existence of infection, with a negative predictive value of 0.91. Putative ‘TdT-positive’ cells were concentrated in the T-lymphocyte fraction and the very process of E-rosette formation seemed to contribute to this phenomenon. It appears as if the anti-TdT reagent recognizes not only TdT but also a variety of antigens which are expressed on or in immature and activated lymphocytes.     

Phorbol esters induce changes in adenosine deaminase, purine nucleoside phosphorylase, and terminal deoxynucleotidyl transferase messenger RNA levels in human leukemic cell lines

We have studied the expression of mRNA encoding adenosine deaminase (ADA; EC 3.5.4.4), purine nucleoside phosphorylase (PNP; EC 2.4.2.1), and terminal deoxynucleotidyltransferase (TdT; EC 2.7.7.31) in different leukemic cell lines of B- and T-cell lineage. Incubation of leukemic cells in the presence of the phorbol esters, 12-O-tetradecanoyl-phorbol-13-acetate or phorbol 12,13-dibutyrate, resulted in reduction of ADA and TdT mRNA levels, while PNP mRNA levels increased under the same treatment. The effect of TPA on the activity of these enzymes correlated well with its effects on their mRNA levels. TPA caused a 40% decrease in ADA and a 60% decrease in TdT enzyme activity, after 6 h of treatment. In contrast, PNP activity increased up to 200% after 12 h of incubation with the phorbol ester. The changes induced by the phorbol esters in the levels of mRNA of ADA, PNP, and TdT, and their enzyme activities in human leukemic cell lines mimic the changes in the activities of these enzymes in developing T-lymphocytes during differentiation in vivo, suggesting a role for protein kinase C in the regulation of ADA, PNP, and TdT gene expression during lymphoid cell differentiation.     

The detection of residual acute lymphoblastic leukemia cells with immunologic methods and polymerase chain reaction: a comparative study

In this study we applied double color immunofluorescence analysis and polymerase chain reaction (PCR) amplification of rearranged TCR delta genes for detecting residual leukemia in the posttreatment bone marrow (BM) samples taken from four patients in morphological remission. In three of these patients (nos. 1-3; T-ALL) a combination of CD3 and anti-TdT antibodies (Abs) was used to identify residual blasts while in patient 4 (B lineage ALL) the combination CD13/TdT served to detect residual disease. Two rounds of PCR primed by nested amplimers were carried out to prepare clonospecific probes from presentation DNA and to investigate the follow-up samples. In patients 1 and 2 no cCD3+/TdT+ cells were seen posttreatment, but PCR amplification of the TCR V delta 1-D-J delta 1 region revealed residual disease in both patients. Patient 1 underwent allogeneic BM transplant (BMT) 8 months after diagnosis and is well 3 months post-BMT while patient 2 relapsed 12 months after presentation. In patient 3 the remission samples investigated 2 and 3 months after diagnosis did not contain cCD3+/TdT+ cells, but in the sample collected at 4 months a few such cells (0.0001-0.001%) could be detected. In the same sample, PCR amplification of the TCR V delta 2-D-J delta 1 region indicated the presence of 10(-4)-10(-3) residual leukemic cells. These findings predicted full morphological relapse which occurred 2 months later. In patient 4 CD13/TdT double positive cells were clearly seen 2 and 3 months after presentation. PCR amplification of the V delta 2-D delta 3 recombination also revealed residual blasts when applied to one of such "remission" samples. After further remission induction treatment, no immunologic evidence of residual disease was detected. This patient received an allogeneic BMT 8 months after diagnosis and is disease free 4 months after BMT. Our data indicate that both double color immunofluorescence and PCR analysis offer powerful tools to study residual leukemia and highlight the advantages as well as the potential limitations of each technique.     

The demonstration of terminal deoxynucleotidyl transferase on frozen tissue sections and smears by the avidin-biotin complex (ABC) method

A method using avidin-biotin complex (ABC) to detect the presence of the enzyme terminal deoxynucleotidyl transferase (TdT) is described and compared with a proven indirect immunofluorescence method. The material studied consisted of: (1) peripheral blood and bone marrow smears from 17 patients with leukemia (ALL, 8; CLL, 3; AML, 6), six normal controls, one T-ALL cell line, and (2) frozen tissue sections from four patients with lymphoblastic lymphoma (LL), two patients with nodular poorly differentiated lymphocytic lymphoma (NLPD), two patients with reactive follicular hyperplasia (RFH) and one calf thymus. The slides from patients with ALL had from 30 to 90% cells with nuclear positively by the ABC technique. Slides from patients with CLL were negative, as were the normal peripheral blood smears. Normal bone marrow smears contained less than 5% positive cells. The T-ALL cell line was 100% positive. The frozen tissue sections from the patients with LL and the calf thymus contained numerous positive cells, while all of the sections from the patients with NLPD and RFH were negative. A good correlation existed between the ABC and the indirect immunofluorescence methods. The ABC method described is both more specific and more sensitive than the previously described techniques in detecting TdT in tissue sections and smear preparations.     

Usefulness of a small scale indirect 125I-labelled protein A binding assay for detection of monoclonal antibodies against hematopoietic cell surface antigens

We describe a small-scale solid-phase indirect 125iodine protein A binding assay (IPA) and discuss its usefulness for detection of hematopoietic cell surface antigens and for screening hybridoma clones producing monoclonal antibodies (McAbs) against K562 cell surface antigens correlated with differentiation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA). This assay was performed in polystyrene Terasaki microtest plates instead of 96-well microtiter plates, using hematopoietic cells attached to the wells by air drying. This method facilitates washing procedures and reduces the radioactive waste. The specificity of this IPA method is as high as those of RIAs and ELISAs generally used. Titration curves of a McAb, My7, specific for CD13 on HL60 and TPA-treated HL60 (HL60-TPA) cells, were linear between the 10(-1) and 10(-4) dilutions. A difference in the CD13 expression between HL60 and HL60-TPA cells could be detected by both IPA and flow cytometry using My7 at 10(-1)-10(-3) dilutions. At the 10(-3) dilution, however, CD13 expressed on HL60 cells was detected by the IPA method but not by flow cytometry. These results show that the sensitivity of IPA is superior to that of flow cytometry. By screening hybridoma clones with this IPA method, we succeeded in producing three interesting and useful McAbs (21H73, 37G7 and 49C12) against K562 cell surface antigens whose expression was altered after the differentiation induced by TPA. We also demonstrated that the IPA method is suitable for cell surface marker analysis of leukemia cells freshly isolated from patients.