抗大肠癌相关抗原SC系列单抗的研究及其应用

为探讨大肠癌诊治的新途径,自1982年以来先后用新鲜大肠癌实体瘤细胞免疫并经细胞的杂交融合而研制出16种抗人大肠癌相关抗原的单克隆抗(体单抗),并命名为SC 系列单抗。这些杂交瘤细胞经连续多年的培养、冻存与复苏,至今仍能稳定分泌IgGl(13种)和 IgM(3种)型的单抗。     

肿瘤相关抗原CA—Hb3对大肠癌血清学诊断的价值

以抗大肠癌单抗Ｈｂ３进行免疫亲和层析从大肠癌细胞株ＨＲＴ－１８细胞提取溶液中纯化相应的大肠癌相关抗原ＣＡ－Ｈｂ３，用Ｓａｎｄｗｉｃｈ ＥＬＩＳＡ方法对大肠癌及消化道其他肿瘤患者血清中ＣＡ－Ｈｂ３水平进行定量检测。结果表明，大肠癌阳性率为６８．９％，胃癌阳性率为５６．７％，消化道良性疾病的阳性率为１１．９％。ＣＡ－Ｈｂ３对胃肠道肿瘤诊断的敏感性和特异性较高，是具有应用价值的新型大肠癌血清标志物。     

抗卵巢癌相关抗原单克隆抗体的制备及免疫分析方法的建立

目的　研究从卵巢浆液性乳头状囊腺癌患者的腹腔积液中 ,纯化获得携带CA 12 5抗原决定簇的卵巢癌相关抗原。以此为免疫原 ,制备单克隆抗体 ,并建立放射免疫分析方法并应用于临床中。方法　腹腔积液经高氯酸分离 ,凝胶层析及亲和胶蓝柱层析纯化CA 12 5相关抗原。应用细胞融合技术 ,抗体采用ELISA方法、免疫印记技术进行鉴定。单克隆抗体经碘原法标记放射性核素1 2 5I ,初步建立 1种碘标固相双位点夹心模式的免疫放射分析法。结果　腹腔积液中CA12 5相关抗原经纯化 ,其比活度达 1.1× 10 6 U mg。共获得 6株稳定分泌抗体的杂交瘤细胞株 ,采用 2株配对最佳的抗体 ,初步建立 1种碘标免疫放射分析法。该方法可测下限为 1.2U ml ,批内变异系数为 4.4% ,批间变异系数为 9.6% ,平均回收率为 97.0 %。初步临床应用的结果表明在正常人血清中CA 12 5相关抗原平均值为 (2 .14 7± 3 .0 92 )U ml。在 60例卵巢癌患者中 ,平均值为 (10 6.5± 187.5 )U ml ,两者比较有非常显著性差异 (P <0 .0 1)。结论　以患者腹腔积液中纯化的CA12 5相关抗原制备单克隆抗体 ,并建立放射免疫分析方法 ,初步应用于临床血清学测定中 ,对卵巢癌的辅助诊断具有一定价值     

单抗导向超抗原对人大肠癌细胞的抑制作用研究

[目的]研究单抗导向超抗原对人大肠癌细胞的抑制作用。[方法]MTT法观察融合蛋白对Colo205细胞的体外抑制效应;DNA电泳法和电子显微镜法检测融合蛋白处理后肿瘤细胞的凋亡。[结果]在效应细胞介导下,肿瘤细胞的生长抑制率与随融合蛋白作用浓度的增加而逐渐增大;DNA电泳法和电子显微镜法显示被融合蛋白活化的免疫效应细胞攻击后的肿瘤细胞表现出典型的凋亡特征性变化。[结论]在效应细胞介导下,融合蛋白能对表达相应抗原的肿瘤细胞显示出有效的抑制作用;在融合蛋白介导的肿瘤细胞体外生长抑制过程中伴随有靶细胞的凋亡。     

酶免疫—醋酸纤维薄膜电泳法诊断大肠癌

以抗结肠癌及胰腺癌的单克隆抗体用酶免疫──醋酸纤维薄膜电泳法，检测大肠癌及非癌疾病患者粪便中的癌相关抗原。其中大肠癌６１例，慢性肠炎２０例，正常人１００例。结果表明，四株单克隆抗体ＣＬ－３、ＣＬ－４、ＰＳ－２、ＰＳ－９对应抗原在大肠癌患者粪便中的平均含量显著高于正常对照组及非癌肠道疾病组，其阳性率分别为７５．４％、５９．４％，７２．５％和５１．３％，如将三株单抗ＣＬ－３、ＣＬ－４、ＰＳ－２组合应用，则大肠癌组阳性率达８６．２％，正常组为１５．２％，肠炎组为１６．６％，６５例大肠癌中，５例早期诊断（ＤｕｋｅｓＡ期）患者均为阳性。本方法可能为早期诊断大肠癌提供简便省时，价廉，便于推广的新方法。     

肺癌单克隆抗体3C9及其相关抗原的研究

用肺腺癌细胞系Ａ５４９免疫ＢＡＬＢ／Ｃ小鼠，常规细胞融合，获得一株ＩｇＭ型单抗３Ｃ９。Ｗｅｓｔｅｒｎｂｌｏｔ研究发现该抗体识别５６．４ＫＤ蛋白带。免疫组化研究发现，３Ｃ９相关抗原（３Ｃ９Ａｇ）在肺癌组织中表达阳性率高达９２．５％。３Ｃ９Ａｇ血清学研究发现，其诊断肺癌的敏感性为６４．７％，特异性为９３．０％，准确性为８２．５％。其血清水平与组织学分布相关，在高、中分化的肺癌病人阳性率高于低分化者（Ｐ     

T抗原单克隆抗体法在大肠癌筛检中的应用初探

目的 探讨T抗原单克隆抗体法在大肠癌筛检中的临床价值.方法 同时采用T抗原单克隆抗体法和半乳糖氧化酶法检测207例直肠粘液中的T抗原.结果 T抗原单克隆抗体法和半乳糖氧化酶法的敏感性和特异性分别为69.2%、67.3%和64.2%、65.6%,两者的筛检价值无明显差别.结论T抗原单克隆抗体法对大肠癌筛检有较大临床价值,且操作简便.     

肿瘤相关抗原在人大肠癌中的表达

 目的 应用ND-1单抗和抗CEA单抗对大肠癌细胞的标记情况对比，探讨LEA在大肠癌中的表达及意义。方法 采用流式细胞仪和免疫细胞化学染色检测大肠癌细胞系LEA和CEA的表达。并采用间接ELISA法测定LEA和CEA单抗对大肠癌细胞系的特异性。应用免疫组化检测其在大肠癌组织中的表达。结果 流式细胞仪检测显示，LEA抗原在CCL-187、CX-1、CLone A和CCL-229细胞表达的阳性峰平均荧光强度呈递减趋势，并且强于CEA的表达量（P〈0．01）。LEA在高分化大肠癌细胞系CCL187和CX-1高度表达，并且低侵袭大肠癌细胞系CCL-187和CX-1 LEA表达量高于高侵袭大肠癌细胞系CLone A和CCL-229（P〈0．01）。ELISA检测表明，与抗CEA单抗相比，ND-1单抗对大肠癌细胞具有很强的特异性结合力（P〈0．01）。LEA的表达阳性率随大肠腺癌组织分化程度降低而下降（P〈0．01），而且对高分化腺癌表现出更高的选择性。抗CEA单抗对高、中、低分化癌选择性相似（P〉0.05）。结论 LEA是更加特异的高分化大肠癌相关抗原。LEA可能与癌细胞的分化程度、侵袭力、恶性程度有关，LEA可作为早期诊断和判断大肠癌恶性程度的有用指标。     

胃肠癌单抗在大肠癌和息肉的抗原表达

胃癌和肠癌单抗联合检测大肠癌和息肉抗原表达尚未见报道。本文报告这方面的检测结果。材料与方法经病理证实的大肠癌66例,大肠息肉68例(病理分类见附表),正常结肠粘膜10例,采用切除或活检标本、石蜡切片。鼠抗人结肠癌单抗MC_3及MC_5,胃癌单抗MG_7,均     

Characterization of mucin antigens recognized by monoclonal antibodies raised against human colon cancer cells

Two monoclonal antibodies, MLS 102, which recognizes cancer-associated mucin antigens, and MLS 103, which recognizes normal mucin, were used to isolate, by immunoaffinity chromatography, the corresponding antigens from cell lysates and spent medium of a human colorectal carcinoma cell line, LS 180. The MLS 102 antigen contained serine, threonine, and proline as major amino acids. The carbohydrate chains of the MLS 102 antigen were composed of O-linked NeuAc alpha 2----6GalNAc (56%), N-acetylgalactosamine (25%), and longer oligosaccharide chains. The MLS 103 antigen differed from the MLS 102 antigen in both amino acid and carbohydrate composition. Most O-linked oligosaccharides of the MLS 103 antigen were longer than the disaccharide found in the MLS 102 antigen. Immunostaining of LS 180 cells using MLS 102 and MLS 103 revealed that the cells are heterogeneous with respect to the expression of the antigens.     

Six monoclonal antibodies to human pancreatic cancer antigens

Murine monoclonal antibodies were generated against FG, a human pancreatic carcinoma (HPC) cell line. Of the six monoclonal antibodies, five (S3-15, S3-23, S3-41, S3-60, and S3-110) reacted by indirect immunoperoxidase assays with HPC of the ductal type, and another (S3-53) reacted with both ductal and acinar HPC. Strong reactivity was also found with tumors of the stomach, colon, mouth, lung, and cervix, while a large panel of normal human tissues displayed little reactivity. Indirect immunofluorescence staining revealed that, except for S3-23, the antigens recognized by these antibodies are expressed at the cell surface. Immunoprecipitation of metabolically radiolabeled FG cells indicated that the epitopes recognized by these antibodies are carried by distinct proteins or glycoproteins, differentiated on the basis of the apparent molecular weight and/or subunit composition as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This panel of antibodies will be useful to study antigenic variations associated with malignant cell transformation in HPC as well as other tumors.     

Clinical application of carcinoembryonic antigen (CEA) monoclonal antibodies (McAbs)

CEA McAbs, recognizing three different epitopes on CEA molecules, were used to measure serum CEA level in cancer patients by enzyme-immunoassay (EIA). The results, as compared with those using polyclonal antibodies, indicated that the positive rate was higher while the false positivity was lower. Immunohistochemistry of tumour sections showed that the CEA McAbs are bonded to 80-90% of gastrointestinal cancers. Although the normal colon epithelium occasionally reacted with CEA McAbs, other normal tissues did not. After in vivo administration of radio-labeled CEA McAbs to nude mice xenograft with human colon cancer, the radio-isotope was found to be concentrated preferentially in the tumour. The ratio of tumour and normal tissue was 3.6-11.8 after 48 hours following administration. Thus, the CEA McAbs can be used clinically not only for serum CEA determination but also for diagnostic imaging.     

Characterization of human colon cancer antigens recognized by autologous antibodies

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) has proven to be a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. In the present study, 48 distinct antigens (NY-CO-1–NY-CO-48) reactive with autologous IgG were identified by SEREX analysis in 4 patients with colon cancer. Sequencing analysis showed that 17 of the cDNA clones were previously uncharacterized molecules and 31 represented known gene products. The individual cDNA clones were analyzed in the following manner: a search for mutations or other structural changes; an analysis of mRNA expression in a panel of normal tissues; and a frequency analysis of the antibody response to the expressed product in the sera of colon cancer patients and normal individuals. The initial analysis showed NY-CO-13 to be a mutated version of the p53 tumor suppressor gene. Three of the 48 antigens showed a differential pattern of mRNA expression, with NY-CO-27 (galectin-4) expressed primarily in gastrointestinal tract, and NY-CO-37 and -38 showing a pattern of tissue-specific isoforms. With regard to immunogenicity, 20 of the 48 antigens were detected by allogeneic sera; 14 of these were reactive with sera from both normal donors and cancer patients, and 6 other clones (NY-CO-8, -9, -13, -16, -20 and -38) reacted exclusively with sera from colon cancer patients (ranging from 14% to 27%). Our results on colon cancer illustrate both the complexity and the potential of the SEREX approach for analysis of the humoral immune response against human cancer. Int. J. Cancer76:652–658, 1998.© 1998 Wiley-Liss, Inc.     

A panel of monoclonal antibodies recognizing the Staphylococcus epidermidis fibrinogen-binding MSCRAMM SdrG

Staphylococcus epidermidis is an important opportunistic human pathogen that has recently emerged as a major cause of foreign-body infections. The most important stage contributing to the pathogenesis of this bacteria is the initial adherence to host tissue. SdrG is a cell-wall-anchored fibrinogen-binding adhesin of S. epidermidis that has been shown to be necessary for bacterial binding to fibrinogen-coated foreign bodies, such as catheters. Here we report the generation and characterization of a panel of monoclonal antibodies (MAbs) directed against this S. epidermidis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance, and flow cytometry, we have characterized a diverse array of MAbs that may prove to be beneficial in studies that address the precise biologic role of SdrG.     

Aberrant expression of intestinal mucin antigens associated with colorectal carcinoma defined by a panel of monoclonal antibodies

Small intestine mucin antigen (SIMA) is an oncofoetal antigen for the colon and is distinct from the normal large intestinal mucin antigen (LIMA). In the present study, a panel of anti-SIMA and anti-LIMA monoclonal antibodies (MAb) was used to charaterise altered mucin expression in colorectal adenocarcinomas, by immunohistochemistry and quantitative immunoassays of tissue extracts. These results are compared with CEA expression and correlated with various clinicopathological indices. All mucin MAb reacted with a high proportion of the 100 colon cancers of every stage, histological type (including non-mucinous cancers), differentiation, site, or size. Inappropriate SIMA production was detected by either anti-SIMA MAb 4D3 or 4A1, even in 85% of early stage cancers. MAb 4D3 reacted with a higher proportion of cancers of smaller size and better differentiation. At the subcellular level, both anti-SIMA MAb showed reactivity typical of normal mucin, i.e., goblet cell and extracellular mucin. The normal colonic antigen, LIMA, was also detectable in the majority of cases, but quantitatively overproduced in some cases and reduced in others. However, in contrast to SIMA, LIMA was detected in predominantly undifferentiated cancer cells but not in goblet cells. Heterogeneity of MAb reactivity between cases and complementarity within each cancer was frequently observed. Mucin reactive with at least one of the MAb was detected in all of the CEA-negative cancers. A high rate of inappropriate SIMA expression was also detected in the perineoplastic transitional mucosa (88%, c.f. CEA, 35%) and adjacent, morphologically normal mucosa (80% c.f. CEA, 24%), indicating biochemical changes similar to the cancer. This panel of anti-mucin MAb demonstrated altered mucin glycoprotein metabolism associated with the development and progression of most colorectal cancers, which emphasises their utility as indicators of neoplastic change in the colon, and their superiority to CEA.     

Production of monoclonal antibodies recognizing human hair follicle keratinocytes

To raise new antibodies against the cells contained in human hair follicles, we immunized mice with a mixture of primary cultured human embryogenic keratinocytes thought to contain very immature keratinocytes, as antigen. Using a monoclonal antibody (MAb) producing technique, we obtained two MAbs, which recognize a specific portion of the hair follicle. MAb-8G2 was specific to the companion layer between the inner and outer root sheath of the follicle. This pattern of recognition was quite similar to the expression pattern of K6hf protein, a human hair follicle-specific type II cytokeratin. MAb-sDP showed unique recognition. Immunohistochemistry indicated that MAb-sDP specifically recognized the cells surrounding the dermal papilla (DP). These cells are possibly the germ line cells of hair matrix.     

Characterisation of breast cancer infiltrates using monoclonal antibodies to human leucocyte antigens

Serial frozen sections from eleven patients with malignant breast tumours and five patients with benign disease were studied by indirect immunoperoxidase using a panel of mouse monoclonal antibodies to human leucocyte antigens. More infiltrating leucocytes were seen in tumour sections than those of benign conditions. A considerable proportion of the infiltrating cells were T cells, and more of these were of the suppressor/cytotoxic subset than the helper/inducer subset. The T cells were apparently not all activated as indicated by lower levels of staining with anti HLA-DR than anti-leucocyte antibody. Diffuse staining was sometimes seen with HLA-DR and T cell subset antibodies. Tumour cells did not stain or were only very weakly positive with anti HLA-A, B, C.     

Characterization of monoclonal antibodies to human melanoma-associated antigens

The hybridomas No. 165.28T, No. 473.54S, and No. 653.25N derived from the fusion of myeloma cells with splenocytes from mice immunized with cultured human melanoma cells secreted monoclonal antibodies recognizing antigenic determinants maximally expressed on cultured human melanoma cells and freshly explanted melanoma cells. Monoclonal antibody No. 376.74T reacted also with carcinoma cell lines but with a significantly lower titer. Rosette inhibition assay showed that these antigenic determinants were expressed on antigenic structures, which are not associated with beta 2 microglobulin and histocompatibility antigens. Two monoclonal antibodies recognized the same or closely associated antigenic determinants, and the remaining two monoclonal antibodies reacted with distinct antigenic determinants. All four monoclonal antibodies could mediate antibody-dependent cellular cytotoxicity of cultured melanoma cells, but none could mediate complement-dependent cytotoxicity.     

Detailed characterization of reactivities of anti-gastric cancer monoclonal antibodies to carbohydrate antigen

Four murine monoclonal antibodies (MoAbs), KM191, KM206, KM230 and KM231, raised against gastric cancer exhibited very similar reactivities to human carcinoma cells, whereas their abilities in probing the antigen shed from the cancer cells were quite different. We found in this study that the four MoAbs reacted immunologically with the same monosialo gangliosides derived from a gastric cancer cell line by two-dimensional high performance thin-layer chromatography. When the reactivities of the MoAbs to a number of purified gangliosides or oligosaccharides were examined, the carbohydrate structure of the antigen was determined as sialyl Lea. KM231 exhibited the highest binding avidity to the oligosaccharide and the ganglioside among the four MoAbs. In a comparative study of KM231 and NS19-9, which is a widely used sialyl Lea-reactive MoAb, KM231 bound to the oligosaccharide with higher affinity and detected the antigen in tissue sections with higher sensitivity. In addition, KM231 could detect a small amount of the antigen ganglioside in human gastric normal and cancerous mucosa and in gastric cancer cell lines by HPTLC-immunostaining.