Development of novel markers for the characterization of chicken primordial germ cells

This study was undertaken to develop novel markers for chicken primordial germ cells (PGCs), which are of potentially enormous value in transgenic research. Gonadal cells collected from 5.5-day-old chicken embryos were cultured in a Dulbecco's minimal essential medium and the PGC colonies formed during the primary culture period were subcultured three times. Characterization of the PGCs with the candidate marker reagents was performed on the mixed cell population 2 hours after seeding, after the primary culture period (day 10), and after the third passage (day 40). Mouse embryonic stem (ES) cells were used as controls. The cytochemical reagents investigated included periodic acid-Schiff (PAS) stain, antibodies to stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), antibody to epithelial membrane antigen (EMA)-1, antibodies to integrins alpha6 and beta1, several lectins (Solanum tuberosum agglutinin [STA], Dolichos biflorus agglutinin [DBA], concanavalin A agglutinin [ConA], and wheat germ agglutinin [WGA]), and double staining with antibodies to SSEA-1, SSEA-3, SSEA-4, integrin alpha6, or integrin beta1 and then with the lectin STA. Densitometric quantification was used to identify PGC-specific markers. The results showed that chicken PGCs were stained selectively by PAS and by antibodies to SSEA-1, SSEA-3, SSEA-4, EMA-1, integrin alpha6, and integrin beta1. The control mouse ES cells reacted with PAS, anti-SSEA-1, and anti-EMA-1 antibodies, as well as with antibodies to integrins alpha6 and beta1, but not with antibodies to SSEA-3 and SSEA-4. Chicken PGCs reacted with the lectins STA and DBA, but mouse ES cells reacted with STA and WGA. The results of double staining of PGC colonies subcultured three times showed that the intensity of staining was not altered by concomitant use of the marker reagents. This study demonstrated that, in addition to PAS and antibodies to SSEA-1 and EMA-1, new specific markers of chicken PGCs are recognized by the lectins STA and DBA and by antibodies to SSEA-3 and SSEA-4 and integrins alpha6 and beta1. Double staining using these newly developed markers might be the method of choice for rapid characterization of chicken PGCs.     

A human thymocyte antigen defined by a hybrid myeloma monoclonal antibody.

Spleen cells from a BALB/c mouse that had been immunized with human thymocytes were fused with the myeloma line P3-NS 1/1 Ag 4.1. One of the resulting hybrid clones (NA 1/34) secreted an antibody that was highly specific for human thymocytes. Eighty-five % of thymocytes expressed the antigen designated HTA1. There were an estimated 15 x 10(4) molecules of HTA 1 per cell, and it is therefore a major surface molecule. The expression of this antigen on thymocytes appears to be reciprocal to HLA, as recognized by another monoclonal antibody W6/32. Immunoprecipitated material from [125I]-labeled thymocyte membranes was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate which disclosed a single component of 45,000 molecular weight.     

Production and characterization of monoclonal antibodies recognizing the alpha-chain subunits of human ia alloantigens

Two monoclonal antibodies, TAL-1B5 and TAL-3C3, specific for human Ia alpha-chain subunits have been produced by fusing P3/NSI/1-Ag4-1 mouse myeloma cells with spleen cells from a BALB/c mouse immunized with purified alpha-chains. Specificity for the alpha-chain subunits was initially established using a solid-phase radioimmunoassay. Indirect binding assays demonstrated that TAL-1B5 bound strongly to all human B lymphoblastoid lines tested and to CLLs, but only weakly to PBL-B cells and not to PBL-T cells or the T-cell lines Molt 4 and HSB-2. TAL-3C3 bound only weakly to B lymphoblastoid lines and not to CLLs or PBL-B cells. From 125I cell surface-labelled lysates TAL-1B5 immunoprecipitated a 33,000(alpha):28,000(beta) Ia dimer, but TAL-3C3 failed to immunoprecipitate cell surface molecules. Under denaturing conditions, however, both TAL-1B5 and TAL-3C3 immunoprecipitated the 33,000 alpha-chain subunit. Competitive inhibition studies demonstrated that both monoclonal antibodies recognize the same or spatially related alpha-chain antigenic determinants with some slight cross-reactivity against beta-chains. 2D-NEPHGE/SDS-PAGE analysis of TAL-1B5 immunoprecipitates from [35S]-methionine biosynthetically labelled cells revealed the presence of a number of alpha-chain spots in association with beta-chain products of three previously described loci (beta-1, beta-2, beta-3) suggesting that this antibody recognizes an antigenic site common to those human Ia alpha-chains so far identified.     

Regulation of tumor growth and antibody clone expression by antigen and anti-idiotype antibody ligands with specificity for receptor-binding sites

Dextran ligands, modified to increase epitope reactivity with receptors, were more effective in suppressing BALB/c mouse plasmacytomas MOPC 104 E and J-558, which bind alpha (1 leads to 3) dextran and have an idiotype (Id) in the common, than autoantibody (Ab) against the Id unique to each of the proteins secreted by the two tumors (the (IdI). BALC/c immunized with 104 E myeloma protein and expressing an antibody response to the 104 E IdI exhibited a specific, anti-104 E IdI transplantation resistance to lethal grafts of 104 E, but not J-558, tumors notwithstanding the shared common Id and similar ability to bind alpha(1 leads to 3) dextran. This autoantibody did not prevent modulation of the 104 E tumor to variant forms or the growth of the variants. On challenge with alpha (1 leads to 3) dextran, the immunized mice expressing the anti-104 C IdI responses failed to express the 104 D IdI-like antibody clone present in the normal, anti-alpha (1 leads to 3) dextran antibody repertoire. Passive, iso-anti-104 E IdI antibody had a transitory suppressive effect on the normal, 104 E IdI-like antibody clone but failed to circumvent 104 E tumor growth. It is apparent that the greater effectiveness of ligands strongly reactive in a nonphysiological manner with the tumor receptors lies in the stabilization of the tumor load without inducing variant escape or a disturbance of the immune network, and that receptor expression and malignancy state are not necessarily co-extensive functions.     

骨桥蛋白单克隆抗体的制备及其特异性分析

为了研制骨桥蛋白(OPN)特异性单克隆抗体(mAb),并鉴定其特异性。本研究在毕赤酵母中表达具有良好免疫原性的重组人OPN蛋白的基础上,用重组人骨桥蛋白(rhOPN)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,间接ELISA筛选杂交瘤细胞,并结合免疫印迹对抗体的特异性进行鉴定,通过竞争抑制试验对单克隆抗体识别的抗原位点进行分析。结果共获得4株能够识别OPN不同抗原位点的mAb,亚类测定显示,3株为IgG1,1株为IgG2a。这些mAb能与重组人OPN特异性结合。本研究的四株抗体中,只有4G2B5能够检出与肿瘤转移密切相关的OPN-c的条带,预示该抗体可用于判断肿瘤预后和高转移肿瘤的临床检测。本研究成功获得了针对骨桥蛋白的特异性mAb,同时为进一步研究OPN蛋白的结构和功能提供了重要工具。     

抗DESMOPLAKIN I单克隆抗体的制备

作者从水牛鼻表皮中分离桥粒,提取桥粒中的Desmoplakin I(DPI)。用DPI作免疫原,免疫BALB/C小鼠,建立了一株分泌抗DPI单抗的细胞AD-1。免疫组化染色证实,该单抗与上皮组织呈阳性反应,与非上皮性组织呈阴性反应。提示该单抗可作为区分上皮性肿瘤与非上皮性肿瘤的免疫组化探针。     

An indirect radioimmunoassay for mouse casein using 125I-labeled antigen.

A new indirect radioimmunoassay was developed for detection of casein in mouse milk and in mammary tissue extract. Preincubation of rabbit gamma globulin to mouse milk casein (Ca2+-rennin precipitate) with unlabeled casein, milk, extracts of mammary tissues of late pregnancy and lactation, virtually blocked subsequent binding of 125I-labeled mouse milk casein to the antibody. Preincubation with mouse serum, bovine serum albumin, rennin, extracts of liver or immature mammary tissue had little effect on [125I]casein binding to the antibody. The inability of [125I]casein to bind to the antibody after preincubation with protein samples, which are likely to contain casein, is indicative of a specific antigen-antibody reaction. The assay is capable of detecting 0.2 mug casein, 1 mug milk proteins and 10 mug lactating mammary tissue extract. The application of the assay was also demonstrated using organ culture of the entire mammary gland. The glands treated with the lactogenic hormones, insulin + prolactin + cortisol, showed a saturation level of antibody-antigen reaction, indicating hormonal induction of casein; whereas, no reaction was observed with the non-treated gland.     

人BRDT-NY多克隆抗体的制备及其在消化道肿瘤中的表达检测

目的:构建人BRDT-NY原核表达载体,表达人BRDT-NY原核蛋白,制备其多克隆抗体,检测其在消化道肿瘤中的表达。方法:以质粒pTriplEx2-BRDT-NY为模板,用PCR法扩增人的BRDT-NY基因。将其克隆到原核表达质粒pET28a+中,构建重组质粒pET28a+-BRDT-NY。将该重组质粒转化BL21菌,以IPTG诱导原核蛋白的表达。应用纯化的原核蛋白免疫小鼠,制备多克隆抗体,ELISA测定滴度,并用免疫组织化学法分析该蛋白在消化道肿瘤组织中的表达。结果:成功构建了用于原核蛋白表达的重组质粒,在BL21菌中表达BRDT-NY重组蛋白,用亲和层析Ni柱进行纯化得到人BRDT-NY蛋白。用纯化的BRDT-NY蛋白免疫小鼠2个月后,得到相应的多克隆血清抗体,经ELISA检测,滴度均能达到1/10万。免疫组织化学染色显示BRDT-NY蛋白在消化道肿瘤中有较高的表达率。结论:该抗体的制备为进一步研究BRDT-NY在消化道肿瘤中的发病机制奠定了基础。     

Immunohistochemical localization of murine stage-specific embryonic antigens in human testicular germ cell tumors.

Monoclonal antibodies raised against and/or recognizing stage-specific antigens on preimplantation mouse embryos and stem cells of murine teratocarcinoma were used to localize these antigens immunohistochemically on human testicular germ cell tumors. SSEA-1, the antigen found on mouse embryonal carcinoma (EC) cells and embryonic cells from the 8-cell stage embryo onward, including the fetal primordial germ cells, was detected on yolk sac carcinoma components of human tumors, but not on EC cells. SSEA-3, the antigen found on follicular ova, fertilized eggs, early cleavage stage embryonic cells, and visceral endodermal cells of the mouse embryo, but not on mouse EC cells, was detected on human EC cells. Both antigens were found on the cell surface of fetal testicular germ cells but not in the seminiferous tubules of adult human testes. These data point out differences between human and murine EC cells suggesting that human EC cells correspond developmentally to a less mature embryonic cell than the murine EC cells. The possible histogenesis of human germ cell tumors from primordial and/or fetal germ cells is briefly discussed.     

Down-regulation of caveolin-1, a candidate tumor suppressor gene, in sarcomas

Caveolae are plasma membrane microdomains that have been implicated in the regulation of several intracellular signaling pathways. Previous studies suggest that caveolin-1, the main structural protein of caveolae, could function as a tumor suppressor. Caveolin-1 is highly expressed in terminally differentiated mesenchymal cells including adipocytes, endothelial cells, and smooth muscle cells. To study whether caveolin-1 is a possible tumor suppressor in human mesenchymal tumors, we have analyzed the expression using immunohistochemistry in normal mesenchymal tissues, 22 benign and 79 malignant mesenchymal tumors. Caveolin-1 was found to be expressed in fibromatoses, leiomyomas, hemangiomas, and lipomas at high levels comparable to normal mesenchymal tissues. The expression of caveolin-1 was slightly reduced in four of six well-differentiated liposarcomas and strongly reduced or lost in three of three fibrosarcomas, 17 of 20 leiomyosarcomas, 16 of 16 myxoid/round cell/pleomorphic liposarcomas, five of eight angiosarcomas, 15 of 18 malignant fibrous histiocytomas, and eight of eight synovial sarcomas. The immunohistochemical findings were confirmed by Western blot analysis in a number of tumors. High levels of both the 24-kd [alpha]- and the 21-kd [beta]-isoform of caveolin-1 were detected in the nontumorigenic human fibroblast cell line IMR-90. In contrast, in HT-1080 human fibrosarcoma cells, caveolin-1 is strongly down-regulated. We show that the [alpha]-isoform of caveolin-1 is potently up-regulated in HT-1080 cells by inhibition of the mitogen-activated protein kinase-signaling pathway with the specific inhibitor PD 98059, whereas the specific inhibitor of DNA methylation 5-aza-2'-deoxycytidine only marginally up-regulates caveolin-1. In addition, re-expression of caveolin-1 in HT-1080 fibrosarcoma cells potently inhibited colony formation. From these we conclude that caveolin-1 is likely to act as a tumor suppressor gene in human sarcomas.     

Monoclonal antibodies for use in an immunoradiometric assay for alpha-foetoprotein

The advantages offered by a mouse IgG1 monoclonal antibody to human alpha-foetoprotein (AFP) for the preparation of [125I]antibody for use in an immunoradiometric assay (IRMA) have been investigated. The antibody was isolated from ascites fluid by sodium sulphate precipitation followed by gel filtration on Sephadex G-200. The freeze-dried powder and solutions thereof were stable and were used for iodination to 1 atom 125I/molecule antibody by the chloramine-T procedure. At high antigen concentrations 70-80% of the added [125I]Ab was present in the sandwich. Linear response curves in the range 1-100 micrograms antigen/l incubate were obtained when [125I]Ab was in slight excess. In this region an Ag : Ab ratio of 1.9 : 1 was obtained which is consistent with the saturation of a bifunctional antibody. Although non-specific binding (in the absence of antigen) was consistently less than 0.1% of added [125I]Ab, this was the main factor in determining assay detection limits. The serum AFP levels from both non-pregnant and pregnant subjects as measured by the IRMA using the [125I]monoclonal Ab and by radioimmunoassay (RIA) using a sheep antiserum to AFP were in excellent agreement. The IRMA was manipulatively simple, employed a shorter incubation time (2 h), required shorter counting times than the RIA and gave a much wider working range. The provision of a monoclonal antibody for labelling removes the one major practicability barrier which otherwise limits the development and use of the potentially superior IRMA system.     

pap-2-encoded fimbriae adhere to the P blood group-related glycosphingolipid stage-specific embryonic antigen 4 in the human kidney.

A subtype of P fimbriae, encoded by the pap-2 gene cluster, has been analyzed for agglutination of erythrocytes and for binding to cryostat sections of the human kidney. We have demonstrated that pap-2-encoded fimbriae are capable of binding to erythrocytes from some animal species and to human erythrocytes which express globoside and the LKE (stage-specific embryonic antigen 4 [SSEA-4]) antigen. The pap-2 fimbriae bind to Bowman's capsule in the human kidney. Monoclonal antibodies directed against glycosphingolipids were used for the detection of specific P blood group-related antigens in the human kidney and on erythrocytes. Preincubation of kidney sections with monoclonal antibody MC813-70, which binds to the SSEA-4 antigen, inhibited adherence of purified pap-2-encoded fimbriae to Bowman's capsule. We suggest that one receptor for pap-2-encoded fimbriae is the antigen known as LKE (Luke) on human erythrocytes or SSEA-4 in the tissues.     

Chemically induced transplantable malignant fibrous histiocytoma of the rat. Analyses with immunohistochemistry, immunoelectron microscopy and [3H]thymidine autoradiography

Malignant fibrous histiocytoma was produced in rats by injection of 9,10-dimethyl-1,2-benzanthracene into their knee joints. The original tumors consisted mainly of fibroblast-like cells and histiocyte-like cells, often intermixed with bizarre giant cells, and they frequently showed the storiform-pleomorphic pattern. By immunohistochemistry, anti-rat macrophage monoclonal antibodies, TRPM-3, RM-1, and Ki-M2R, and anti-rat leukocyte common antigen reacted to the histiocyte-like cells but not to the fibroblast-like cells. By the single cell cloning method, we established six tumor cell lines, none of which reacted with the anti-rat macrophage monoclonal antibodies, possessed any Fc receptors, or conducted immune phagocytosis and Latex particle phagocytosis. The ultrastructure of the cloned tumor cells resembled that of long-term cultured dermal fibroblasts. Collagen production by the tumor cells was demonstrated immunohistochemically with a monoclonal antibody for type I collagen. Inoculation of the cloned tumor cells into rats produced tumors with the histology of malignant fibrous histiocytoma and induced prominent macrophage infiltration. In the rat tumors produced by the inoculation of [3H]thymidine labeled cells, no reactivity of tumor cells with the anti-rat macrophage monoclonal antibodies was observed. Transplantation of the cultured rat tumor cells into nude mice produced tumors similar in histology to the original rat malignant fibrous histiocytoma. Tumor cells in nude mice induced marked macrophage infiltration as detected by immunohistochemistry with the anti-mouse macrophage monoclonal antibody F4/80. No differentiation of tumor cells into macrophages was detected, since no cells were stained with biotinylated anti-rat macrophage monoclonal antibody TRPM-3. By the flash labeling method with [3H]thymidine, infiltrating macrophages in the nude mouse tumors were proved to derive from the bone marrow of the host animals. These results indicate a possible experimental reproduction of malignant fibrous histiocytoma by proliferation of malignant fibroblasts or their related cells in combination with macrophage infiltration.     

Production and characterization of a monoclonal antibody to partially purified estrogen receptor from human breast tumor

A hybridoma cell line that secretes monoclonal antibody, MAb-ER-Br-1-15-4-18 is established. The MAb is highly specific for estrogen receptor (ER) from human breast tumor cells. In order to raise the antibody, the ER was first isolated from human breast tumor. Mice were immunized with the partially purified ER and the fusion of the spleen cells from the mouse, showing the highest serum titer, with the cells of the NS-1 mouse myeloma line, produced hybrid cells which continuously secreted antibodies specific for ER. Three of the hybridoma cultures which tested strongly positive were cloned using limiting dilution method and one of the cell lines was selected for further study. The recovery of the MAb from the cell culture was done by ammonium sulfate precipitation followed by dialysis and then hydroxylapatite liquid chromatography using linear gradients. The purity of the antibody was checked by polyacrylamide gel electrophoresis. The MAb was isotyped and found to be IgG1. When checked against other antigens the MAb showed a minimal cross-reactivity to ER from rabbit uterus and none to ovalbumin or rat liver ferritin. Further experiments showed that the MAb recognized the ER bound to the hormone and ER in the nucleus of breast tumor cells.     

Regulatory effects of isologous anti-idiotypic antibodies on the formation of different immunoglobulin classes in the immune response to phosphorylcholine in BALB/c mice

BALB/c mice were immunized with purified phosphorylcholine (PC)-specific myeloma protein of the TEPC-15 tumor, which bears the major idiotype of the anti-PC response (T15 Id) in this mouse strain. Four months after establishing the anti-idiotypic response, animals were immunized with PC-keyhole limpet hemocyanin under conditions which normally lead to IgE antibody formation. The expression of anti-PC antibodies of each immunoglobulin class was compared to a group of matched control mice not immunized with T15. In BALB/c mice producing anti-idiotypic antibodies the formation of anti-PC IgM, IgG1, IgG2, IgG3 and IgE was suppressed to various extents and for different lengths of time. An exception to this observation was the formation of anti-PC IgA antibodies, in that no significant difference was measured between the two groups of mice. BALB/c mice immunized in the same way with the PC-specific myeloma proteins of the MOPC-167, MOPC-511 and MOPC-603 tumors produced normal levels of anti-PC IgE and IgG antibodies. These results suggest that T15 idiotype-positive antibodies are probably formed within all classes of specific antibodies expressed during an immune response to PC. The possibility of extending the phenomena of anti-idiotype-induced suppression to the level of various classes of specific antibodies, including IgE, might be of interest in the treatment of some allergic diseases.     

The expression of BST2 in human and experimental mouse brain tumors

Glioblastoma multiforme (grade IV astrocytoma) is a highly malignant brain tumor with poor treatment options and an average lifespan of 15 months after diagnosis. Previous work has demonstrated that BST2 (bone marrow stromal cell antigen 2; also known as PDCA-1, CD137 and HM1.24) is expressed by multiple myeloma, endometrial cancer and primary lung cancer cells. BST2 is expressed on the plasma membrane, which makes it an ideal target for immunotherapy. Accordingly, several groups have shown BST2 mAb to be effective for targeting tumor cells. In this report, we hypothesized that BST2 is expressed in human and mouse brain tumors and plays a critical role in brain tumor progression. We show that BST2 expression is upregulated at both the mRNA and protein level in high grade when compared to low grade human astrocytoma (p < 0.05). To test the relevance of BST2, we utilized the intracranially (IC)-injected GL261 cell-based malignant brain tumor mouse model. We show that BST2 mRNA expression is increased in mouse brain IC-injected with GL261 cells, when compared to mouse brain IC-injected with saline at 3 weeks post-operative (p < 0.05). Furthermore, BST2 immunofluorescence predominantly localized to mouse brain tumor cells. Finally, mice IC-injected with GL261 cells transduced with shRNA for BST2 ± preincubated with BST2 mAb show no difference in overall lifespan when compared to mice IC-injected with GL261 cells transduced with a scrambled shRNA ± preincubated with BST2 mAb. Collectively, these data show that while BST2 expression increases during brain tumor progression in both human and mouse brain tumors, it has no apparent consequences to overall lifespan in an orthotopic mouse brain tumor model.     

Evidence for endocytosis of E-selectin in human endothelial cells.

E-selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)-activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule-1 (ICAM-1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E-selectin, was internalized at a rate of approximately 1.7% of the membrane-bound [125I]mAb per minute. In contrast, less than 0.1% of membrane-bound [125I]RR1/1, an mAb specific for ICAM-1, was internalized per minute. TNF-activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E-selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid-phase internalization, were detected in the same organelles, thus arguing against mAb interaction-induced E-selectin internalization. Furthermore, the amount of E-selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E-selectin kinetics. Taken together, these results indicate that TNF-activated HUVEC constitutively internalize E-selectin. Physiological significance of E-selectin internalization in the regulation of E-selectin membrane expression, and in clearing E-selectin ligands from the circulation, needs further investigation.     

Marker of peripheral blood granulocytes and monocytes of man recognized by two monoclonal antibodies VEP8 and VEP9 involves the trisaccharide 3-fucosyl-N-acetyllactosamine

Two hybridoma antibodies (VEP8 and VEP9) raised against the promyelomonocytic leukemia cell line HL60 have previously been shown to distinguish human granulocytes and monocytes from other cells of the peripheral blood. We report here that both antibodies recognize the carbohydrate structure 3-fucosyl-N-acetyllactosamine with the following sequence: (formula; see text) This structure is the same as that recognized by a hybridoma antibody against mouse teratocarcinoma cells (anti-SSEA-1) which recognizes an early embryonic antigen in the mouse. Until recently this carbohydrate structure was considered to be rare among glycoproteins and glycosphingolipids. However, there is a growing list of human and animal glycoproteins in which this sequence has been detected by chemical and immunochemical methods. In this article we survey this information and discuss how this and other carbohydrate structures behave as differentiation- or tumor-associated antigens.     

In vitro degradation of extracellular matrix by human ovarian carcinoma cells

Better in vitro models arc needed to elucidate the mechanisms underlying tissue destruction by human tumor cells. To address this matter recently isolated and characterized human ovarian carcinoma cell lines derived from either primary tumors, ascitic effusions or metastatic growths were plated in direct contact with extracellular matrix (ECM) previously deposited on culture dishes by bovine corneal endothelial cells. Light and electron microscopy of four of the five ovarian tumor cell lines demonstrated morphologic digestion with penetration of ECM by tumor cell microvilli, along with associated rarefaction. The ability of these same ovarian tumor cell lines to solubilize specific carbohydrate and protein moieties present in intact ECM was assessed with the use of metabolically prelabeled ECM employing tritiated fucose, galactose, glucosamine and proline. Results from these studies corroborated morphologic observations in which four of the five tumor cell lines tested extensively solubilized radiolabeled ECM. The kinetics of radiolabel release from ECM illustrated that three of the four invasive tumors released [³H]fucose, [³H]glucosamine and [³H]proline at high rates. Normal human ovarian fibroblasts and mesothelial cells were observed to be unable to digest ECM and this was consistent with their inability to release radiolabeled material from prelabeled ECM. The results from these studies suggest that some ovarian carcinomas have the ability to degrade basement membrane components. Knowledge regarding the mechanisms responsible for tissue degradation may eventually lead to the development of new chemotherapeutic modalities designed to restrict tumor cell invasion, growth and metastasis.     

Derivation and characterization of a monoclonal hybridoma antibody specific for human alpha-fetoprotein

A monoclonal antibody specific for human alpha-fetoprotein (HAFP) was derived by the hybridoma technique. Spleen cells from mice immunized with pure HAFP were fused with a mouse myeloma cell line (P3×63-Ag-8) and an anti-HAFP secreting hybridoma cell line was cloned in soft agarose. The HAFP specific antibody was shown to be a monoclonal IgG₁ subclass with extremely high avidity for HAFP.