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Five monoclonal antibodies to human tissue plasminogen activator (t-PA) were obtained. Three of them recognized a determinant designated alpha, and other two, another determinant named beta. A t-PA molecule was found to bear one each of determinants alpha and beta arranged in such a manner that the binding of alpha with one of its corresponding antibodies did not interfere with the binding of beta with one of its corresponding antibodies and vice versa. This allowed the development of a one-step sandwich enzyme-linked immunosorbent assay (ELISA) in which the antibody to beta was immobilized on wells of a microtiter plate and the bound t-PA was detected with the enzyme-conjugated antibody to alpha. The ELISA detected both one- and two-chain t-PA with a high sensitivity. We also discuss the determinants, alpha and beta.

A mouse IgM monoclonal antibody, NUH2, was raised after immunization of mice with the disialoganglioside fraction of human colonic adenocarcinoma. This antibody reacts specifically with disialogangliosides having the Structure 1 shown below. (formula; see text) NUH2 does not react with structures lacking the sialic acid at either the beta 1----3 or beta 1----6 side chain, nor with a binary structure having unequal chain lengths, nor with a binary type 2 chain structure having a trimannosyl core as found in the side chain of N-linked complex type oligosaccharides. (formula; see text) In humans, the disialoganglioside antigens defined by antibody NUH2 are present in low quantity in normal cells (e.g., erythrocytes) and tissues, but are expressed highly in some colonic cancers, placenta, trophoblast, and sperm, and can be regarded as oncodevelopmentally regulated antigens.

Namalva (or Namalwa) interferon (IFN)-alpha was partially purified using a combination of conventional methods and modified acid-ethanol extraction. Four mouse monoclonal antibodies against Namalva IFN-alpha were prepared by hybridoma technology after immunization with Namalva IFN-alpha thus purified. Three of these monoclonal antibodies recognized the same or a similar epitope on Namalva IFN-alpha. One of these antibodies was paired with the fourth recognizing a different epitope and used respectively as enzyme-conjugated antibody and solid-phase antibody in our one step enzyme immunoassay (EIA) for IFN-alpha. This assay is simple and was able to detect as little as 5 pg of IFN-alpha in 100 microliters of sample in the short time of 5 hr. There was a good correlation between the EIA and bioassay. The use of one of the monoclonal antibodies as an immunoadsorbant to purify Namalva IFN-alpha is also described.

The changes of minor glycosphingolipids associated with human hepatocellular carcinomas were studied using monoclonal antibodies. Glycosphingolipids of twelve patients were isolated from the tumor tissues and their adjacent cirrhotic liver tissues. The immunoreactivities of glycosphingolipids with two monoclonal antibodies FH2 and IB9, that define x-structure (Gal beta 1-4[Fuc alpha 1-3]GlcNAc) and sialosyl alpha 2-6galactosyl residue, respectively, were studied by a solid phase enzyme-linked immunosorbent assay and a chromatogram immunobinding assay. X-structure was detected in the upper-phase neutral glycosphingolipids from both the cirrhotic liver tissues and the tumor tissues, but was generally expressed more strongly in the latter tissues. The thin-layer chromatography patterns of x-active glycosphingolipids were more complex in the tumor tissues. Sialosyl alpha 2-6galactosyl residue was detected in two ganglioside fractions. One ganglioside with faster migration on a thin-layer chromatogram was found in both the non-tumorous tissues and the tumor tissues. Another ganglioside with slower migration was more often detected in the tumor tissues. It was concluded that some of minor glycosphingolipids detected by monoclonal antibodies FH2 and IB9 could be the 'tumor-associated' antigens.

Monoclonal antibodies to small cell lung cancer (SCLC) were produced by fusion of P3X63/AgU1 mouse myeloma cells with spleen cells from BALB/c mice immunized against intact cells of SCLC tumors grown in BALB/c athymic nude mice. The antibody TFS-2 demonstrated "pancarcinoma" reactivity showing binding to non-small cell lung cancer (NSCLC) and carcinomas derived from other organs such as stomach, pancreas, and colon. This antibody failed to react with a variety of normal tissues including lung, bone marrow, spleen, stomach, colon, and pancreas. TFS-2 showed complement-mediated cytolysis of target cells. TFS-2 had no significant effects on hematopoietic stem cells. This antibody will serve as a powerful tool for the in vitro removal of tumor cells from bone marrow, thus facilitating high-dose chemotherapy of SCLC with autologous bone marrow transplantation. In contrast, TFS-4 monoclonal antibody reacted specifically with SCLC but not with NSCLS tumors. This antibody can distinguish SCLC from NSCLC tumors in histologic diagnosis at transbronchial biopsy, and in cytological identification of tumor cells in malignant effusion, and in infiltrated bone marrow. Radiolabeled TSF-4 specifically accumulated into SCLC tumors that were implanted subcutaneously into athymic nude mice. Thus, the antibody will be useful not only for diagnosis of lung cancer but also for targeting anti-tumor agents.

Immunization of mice with Ley-active trifucosylnonaosylceramide (III3FucV3FucVI2FucnLc6) isolated from human colonic adenocarcinoma (Nudelman, E., Levery, S. B., Kaizu, T., and Hakomori, S. (1986) J. Biol. Chem. 261, 11247-11253) followed by selection of hybridoma by positive reaction with this antigen and negative reaction with two other Ley antigens (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) resulted in successful isolation of the hybridoma producing IgM antibody, termed KH1, specific to Ley-active trifucosylnonaosylceramide, which does not cross-react with Ley-active hexaosyl- or octaosylceramides (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) without internal fucosyl substitution. The three-dimensional structure of the trifucosylnonaosylceramide was simulated based on previously published glycosidic torsion angles for fucosyl type 2 chain (Lex and Ley) and for GlcNAc beta 1----3Gal beta as predicted by hard sphere exo-anomeric calculations (Thøgersen, H., Lemieux, R. U., Bock, K., and Meyer, B. (1982) Can. J. Chem. 60, 44-57). The picture thus constructed showed a broad nonpolar area consisting of the hydrophobic surface of the pyranosyl ring and acetamido group of N-acetylglucosamine and three CH3 groups of L-fucose; this hydrophobic area is adjacent to a hydrophilic area. In analogy to the detailed structure of Leb or Ley involved in their interactions with antibodies and lectins (Spohr, U., Hindsgaul, O., and Lemieux, R. U. (1985) Can. J. Chem. 63, 2644-2652), such a wide hydrophobic area adjacent to a hydrophilic region could be recognized by the antibody KH1, as shown in the model illustrated in the text. Since the axis of ceramide, which is inserted in the lipid bilayer, is perpendicular to the plane of type 2 chain, the epitope recognized by the antibody KH1 is located at the external nonpolar surface of the carbohydrate chain that is overlaid on the lipid bilayer.

The epitope structure of the human sperm antigen reacting with antibodies present in sera of infertile women has been studied using mAb H6-3C4, which produces immobilization of human sperm in the presence of complement. Another antibody, NUH2, which also induces human sperm immobilization, was used to substantiate the presence of a receptor on sperm involved in susceptibility to immobilization. Both antibodies defined type 2 chain polylactosamine structure. H6-3C4 is directed to internally located repetitive N-acetyllactosamine, i.e., sialyl-i, i, or fucosyl-i. NUH2 defines binary alpha 2----3 sialyl type 2 chain, i.e., sialyl-I. Thus, the presence of antibodies in the sera of infertile women directed to sperm lactosaminoglycan or lactosaminolipid could be the basis for infertility in these cases.

We report here a useful method for elimination of small cell lung cancer cells in vitro from bone marrow. A monoclonal antibody, TFS-2, which mediates complement lysis and recognizes an antigen present on small cell lung cancer cells but not lymphoid cells or bone marrow cells, was used to clear infiltrated bone marrow. The antibody in the presence of complement effectively killed tumor cells, but it was not cytotoxic to bone marrow cells. When mixed populations consisting of tumor cells and bone marrow cells were treated with antibody and complement, the tumor cells were also effectively killed, except when large numbers of bone marrow cells were present, whereas TFS-2 had no significant effect on bone marrow stem cells, as judged by colony-forming unit assays.