The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.     

Monoclonal antibodies that inhibit the enzyme activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

Three hybridoma cell lines secreting antibodies against human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) were produced. Purified IgG2b from these cell lines recognized a distinct band of Mr 28,000 on SDS/PAGE from the purified enzyme as well as a band of Mr 56,000 from the crude enzyme preparation. These three monoclonal antibodies inhibited 15-OH-PGDH activity to different degrees. Inhibition of the enzyme activity could be prevented by prior incubation of the enzyme with NAD+ but not with prostaglandin E2 (PGE2) or NADP+. Inhibition by monoclonal antibodies appears to be non-competitive with respect to NAD+ and PGE2. An increased concentration of antibodies alters the apparent Km for NAD+ but not for PGE2, further supporting the notion that the antibodies bind to the coenzyme-binding site. The availability of these monoclonal antibodies should be valuable for probing the structure of the active site.     

Two monoclonal antibodies distinguish between human recombinant interferon-alpha 5s produced by Escherichia coli and by mouse cells

Two monoclonal antibodies against human IFN-alpha--one against natural leukocyte IFN-alpha and the other against recombinant human IFN-alpha 2 produced in E. coli--were prepared, and designated as HT-1, and 104-5-f, respectively. These monoclonal antibodies were used to examine the antigenicities of recombinant human IFN-alpha 5s produced by E. coli and by mouse cells. The HT-1 antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in mouse cells, but not recombinant human IFN-alpha 5 synthesized in E. coli. On the other hand, the 104-5-f antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in E. coli but not recombinant human IFN-alpha 5 synthesized in mouse cells. Then these monoclonal antibodies or sheep polyclonal antibody against human IFN-alpha were used to immunoprecipitate the radioactively labeled recombinant human IFN-alpha 5 synthesized either in E. coli or mouse cells, and analysed on polyacrylamide gel electrophoresis in the presence of NaDodSO4. The labeled recombinant human IFN-alpha 5 produced by mouse cells could be immunoprecipitated with the HT-1 monoclonal antibody or sheep anti-(human IFN-alpha) polyclonal antibody but not with the 104-5-f monoclonal antibody and showed a band of Mr. 17,500 on polyacrylamide gel electrophoresis in the presence of NaDodSO4. On the other hand, the labeled recombinant human IFN-alpha 5 produced by E. coli could be immunoprecipitated with the 104-5-f monoclonal antibody but not with the HT-1 monoclonal antibody and showed a band of similar Mr. on polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related

Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells. Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid. The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity. Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation. Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed. Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid. The reaction was inhibited by eicosatetraynoic acid but not by indomethacin. Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase. To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein. The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody. Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.     

Production and characterization of two monoclonal antibodies against human myeloperoxidase

Two monoclonal antibodies against human myeloperoxidase, designated 3-2H3 and 4-2C11, were produced and characterized. Both bound to the native enzyme, but neither bound to the denatured enzyme or to its two denatured subunits. 4-2C11 bound to the three types of leukocyte myeloperoxidase, I, II, and III, as well as to the four types of myeloid leukemia HL-60 cell myeloperoxidase, IA, IB, II, and III. 3-2H3 did not bind to enzyme IB, but bound to the other types of leukocyte and HL-60 cell enzymes. On incubation with myeloperoxidase III, 4-2C11 inhibited the enzyme activity, but 3-2H3 did not. Both antibodies belong to the IgG1 subclass.     

Monoclonal antibodies identify individual determinants on mouse mammary tumor virus glycoprotein gp52 with group, class, or type specificity

Hybrid cell lines producing monoclonal antibodies against the C3H strain of mouse mammary tumor virus (C3H MMTV) were prepared by the fusion of mouse myeloma cells with the lymphocytes of BALB/c mice that were immunized with C3H MMTV. Approximately 10% of the hybrid cells initially plated after cell fusion produced immunoglobulins that reacted in antibody-binding assays with C3H MMTV; 40 of these cells were cloned, and 6 eventually yielded stable cell lines. High concentrations of monoclonal antibodies (5 to 20 mg/ml) were obtained from serum and ascites fluid of syngeneic mice inoculated with the hybrid cells. All of the monoclonal antibodies were directed against the envelope glycoprotein gp52. Three of the hybrid cell lines produced immunoglobulins of the immunoglobulin M subclass and three produced immunoglobulin G2a. The monoclonal antibodies showed limited charge heterogeneity in light and heavy chains when analyzed by high-resolution, two-dimensional gel electrophoresis. Three serologically distinct specificities were observed when these ascites fluids were tested against different strains of MMTV. The antigenic determinants detected were the following: (i) a type-specific determinant unique to the C3H strain of MMTV; (ii) class-specific determinants shared between C3H and GR MMTVs; and (iii) a group-specific determinant found on C3H, GR, RIII, and the endogenous C3H (C3Hf) MMTVs. Because monoclonal antibodies recognize single antigenic determinants, these results demonstrate for the first time that the three patterns of antigenic reactivity for MMTV are related to individual determinants on the gp52 molecule and also clearly show that one strain of MMTV can be distinguished from other strains.     

番木瓜环斑病毒单克隆抗体的制备及在病毒分型上的初步应用

本试验是用番木瓜环斑病毒(Papaya ringspot virus, PRV)提纯制剂免疫的BALB/c小白鼠脾细胞与Sp~2/o-Ag14骨髓瘤细胞融合,获得三个能稳定传代并分泌抗番木瓜环斑病毒的单克隆抗体的杂交瘤细胞系。其中23H1 McAb的效价较高,用ELISA检测,腹水抗体效价高达1:76800,能被PRV兔抗血清所阻断。这3个杂交瘤细胞系产生的单抗与TMV和CMV无血清交叉反应。它们可把PRV四个毒株初步区分为三个血清型。     

天然属性抗LDL及oxLDL IgM亚类抗体的制备与鉴定

目的：制备天然属性抗低密度脂蛋白（LDL）及抗氧化低密度脂蛋白（oxLDL）IgM亚类抗体。方法：给予Babl/c小鼠高胆固醇饮食,4周后取脾细胞直接与SP2/0细胞融合,以纯化的LDL及oxLDL为抗原,对阳性杂交瘤细胞生长孔进行间接ELISA筛选。鉴定杂交瘤上清的免疫球蛋白亚类,进而采用免疫沉淀和免疫印迹法对获得的抗体进行免疫学反应性鉴定。结果：杂交瘤细胞分泌的抗LDL及抗oxLDL的天然抗体通过ELISA法被筛选出来,可以与LDL或oxLDL发生高亲和力结合,经过4次克隆化,最终获得2株稳定分泌天然抗LDL的抗体,命名为5G8和2H7,及1株稳定抗oxLDL的抗体,命名为3A6,3株抗体均属于IgM亚类,无交叉反应,可以满足免疫印迹、免疫沉淀等实验要求。结论：成功制备了抗LDL及抗oxLDL IgM亚类抗体,为研究天然抗体在体内脂质代谢和相关心脑血管疾病如动脉粥样硬化等发生发展中的作用提供了重要的研究工具。     

Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line

HLA-A2 specific human cytotoxic T lymphocytes (CTL) cell lines have been developed using T cell growth factor and coculture of peripheral blood lymphocytes with selected allogeneic target cell lines. The CTL-8 line showed specificity for human leukocyte antigens (HLA)-A2 bearing target cells after 5 weeks in culture when tested against a panel of 14 lymphoblastoid cell lines in a 51Chromium (51Cr) release assay. Purified anti-human leukocyte antigens (HLA) monoclonal antibodies W6/32 and PA2.1 inhibited cytolysis by 85% and 60%, respectively. The CTL-8 line lysed non-HLA-A2 target cells in the presence of lectins concanavalin A (Con A) or phytohemagglutinin-P lectin (PHA-P) indicating the specificity of cytolysis was not due to nonspecific resistance of target cells to the CTL-lytic mechanism. The T5-1 HLA-A2 mutant cell series were tested as targets for the CTL-8 line. Cell clones 8.18.1, 8.21.1 and 8.6.1, which express altered HLA-A2 molecules as determined by their decreased reactivity with allospecific monoclonal antibodies, were lysed by the CTL-8 line as efficiently as the T5-1 wild type. These cell lines also acted as efficient cold target competitors for a normal HLA-A2 target cell. The 8.14.1 cell clone expressed a lower amount of HLA-A2 alloantigen and showed a corresponding decreased reactivity with CTL-8 in direct cytolytic and cold target competitive inhibition assays. In contrast, the M7 and DK1 HLA-A2 variant cell lines, which express normal HLA-A2 serological determinants, were inefficiently lysed by CTL-8 and did not act as competitive inhibitors of normal HLA-A2 target cells. These results support the concept that the alloantigenic determinant(s) recognized by T cells and antibodies occur at separate regions on the HLA-A2 molecule.     

Major histocompatibility complex-unrestricted cytolytic activity of human T cells. Analysis of precursor frequency and effector phenotype

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. These conditions were shown to expand a mean of 96% of cells cultured. All of the 198 clones generated by this method were T cells (CD2+, CD3+, CD4+ or CD2+, CD3+, CD8+) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. In addition, the activity was not inhibited by monoclonal antibodies directed against class I or class II nonpolymorphic MHC determinants. Killing, however, was inhibited by soluble monoclonal antibodies against the CD3 complex. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur or K562 was not mediated by a soluble factor secreted by the clones. Some of the clones retained their cytotoxic activity when grown in rIL-2 alone for 4 to 6 wk, whereas others exhibited markedly diminished cytotoxicity after maintenance in this manner. Clones that exhibited diminished or no cytotoxic activity after prolonged maintenance in rIL-2 could be induced to kill by stimulation with immobilized but not soluble monoclonal antibodies to CD3 in the absence of lectin. All of the clones examined expressed NKH1 and CD11b but none were CD16 positive. The degree of cytotoxicity of resting or activated clones could not be correlated with expression of these markers. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.     

L-656,224 (7-chloro-2-[(4-methoxyphenyl)methyl]-3- methyl-5-propyl-4-benzofuranol): a novel, selective, orally active 5-lipoxygenase inhibitor

L-656,224 (7-chloro-2-[(4-methoxyphenyl)methyl]-3-methyl-5-propyl-4-benzofuranol) was a potent inhibitor of leukotriene biosynthesis in intact rat and human leukocytes and CXBG mastocytoma cells (IC50 values, 18-240 nM) and of crude human leukocyte and highly purified porcine leukocyte 5-lipoxygenase (IC50 value, 4 X 10(-7) M). The selectivity of L-656,224 for 5-lipoxygenase was shown through the relative lack of activity of the compound on 12-lipoxygenase, 15-lipoxygenase, cyclooxygenase, catalase, and myeloperoxidase. The compound showed (i) oral activity against hyperalgesia induced in the rat paw by injection of yeast or platelet-activating factor, (ii) dyspnea in sensitized inbred rats induced by an aerosol of antigen, and (iii) bronchoconstriction induced by an aerosol of Ascaris in squirrel monkeys, suggesting a role for 5-lipoxygenase inhibitors in the treatment of asthma and peripheral pain.     

Identification of a novel arachidonate 12-lipoxygenase in bovine tracheal epithelial cells distinct from leukocyte and platelet forms of the enzyme

We examined the characteristics of an arachidonate 12-lipoxygenase in bovine tracheal epithelial cells in relation to the enzyme expressed in leukocytes and platelets. Homogenous preparations of intact or disrupted tracheal epithelial cells metabolized arachidonic acid predominantly to (12S)-hydroxyeicosatetraenoic acid, and subcellular fractionation by differential centrifugation demonstrated that the 12-lipoxygenase activity was localized predominantly to the 100,000 x g supernatant (cytosol fraction). Analysis of cytosolic enzymatic activity for pH dependence (maximum activity at pH 7.4-8.0), divalent cation effects (no dependence on cations), and kinetic characteristics (lag phase elimination by addition of hydroperoxide) exhibited similarity to leukocyte and platelet 12-lipoxygenases. Immunoprecipitation experiments demonstrated that the epithelial 12-lipoxygenase reacted with a monoclonal antibody (lox-2) directed against leukocyte 12-lipoxygenase but not with an antibody (HPLO-3) against the platelet enzyme. Immunoaffinity chromatography of the epithelial 100,000 x g supernatant fraction using lox-2 linked to Affi-Prep 10 yielded a single predominant protein band (Mr = 72,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identical in apparent mass to the bovine leukocyte lipoxygenase. Western blotting using a polyclonal antibody to leukocyte 12-lipoxygenase showed peroxidase staining of the same 72-kDa protein band. Activity assays of the purified enzymes demonstrated that substrate specificity for the epithelial 12-lipoxygenase was similar to that of the leukocyte enzyme, but the epithelial enzyme more efficiently converted 18-carbon fatty acids to the corresponding monohydroxylated conjugated dienes. We conclude that bovine tracheal epithelial cells express a 12-lipoxygenase that has immunological reactivity similar to leukocyte and distinct from platelet 12-lipoxygenase and possesses substrate specificity distinct from both enzymes. We further suggest that lipoxygenase heterogeneity may provide a basis for different functional roles for the enzyme in different cell types.     

Bovine monoclonal alloantibodies to blood group antigens prepared by murine x bovine or (murine x bovine) x bovine interspecies fusions.

In order to obtain monoclonal alloantibodies against bovine blood group antigens, lymph node cells from calves immunized with bovine red blood cells (RBC) were fused with either murine NSO/1 myeloma cells or a HAT sensitive murine x bovine heterohybridoma cell line. Both fusion partners resulted in heterohybridoma cell lines, producing monoclonal alloantibodies against bovine red blood cell antigens. Several clones produced antibodies against identical antigens and some of these clones have been further analysed. The antibodies produced by these selected cell lines have been compared with conventional polyclonal antisera used in bovine blood typing service. Thus extensive tests--including the ISAG Comparison Tests 1989/90 and 1991/92--have proved that monoclonal alloantibodies specific for the internationally recognized bovine red cell antigens A2, I1, O1, Q, A', B', Q', C1, R1, X1, S and Z have been produced. The Q, A', B', and C1 antibodies react weakly with certain phenogroups, whereas the A2, I1, O1, Q', R1, X1, S and Z antibodies have proved to be excellent blood typing reagents and have now substituted the polyclonal antisera in routine bovine blood typing in our laboratory.     

Human monoclonal antibodies to Pseudomonas aeruginosa produced by EBV-transformed cells

Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD50) values were 0.5, 2.6, and 3.1 micrograms immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.     

Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone human B lymphocytes and derived tumors

Two monoclonal antibodies (LN-1, LN-2) reactive with B lymphocytes in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by utilizing cell extracts from pokeweed mitogen-stimulated peripheral blood lymphocytes and diffuse histiocytic lymphoma SU-DHL-4 cells, respectively. Both monoclonal antibodies were initially identified by indirect immunofluorescence screening techniques on paraformaldehyde-acetone-fixed cell preparations. Specificity screens with 36 well-characterized human lymphoma and leukemia cell lines showed that both LN-1 and LN-2 stained cell lines of B cell lineage but were unreactive with those of T cell or, with one exception, myeloid derivation. Null cell acute lymphoblastic leukemia cell lines were found to be LN-2+ but LN-1-. The B cell specificity of these reagents was confirmed on 15 lymphoma and 17 leukemia biopsy specimens by using indirect immunofluorescence techniques. Immunoperoxidase staining of sections from B5-fixed, paraffin-embedded human lymphoid tissues showed that LN-1 bound to the cell membrane and cytoplasm of germinal center cells whereas LN-2 stained the nuclear membrane and cytoplasm of germinal center and mantle zone B lymphocytes as well as interfollicular histiocytes and thymic medullary dendritic cells. Both monoclonal antibodies failed to stain cortical thymocytes, lymph node T cells, and peripheral blood T and myeloid cells. Immunoperoxidase staining of 20 nonlymphoid human organs and tissues revealed that LN-1 reacted positively with red blood cell precursors of the bone marrow, ciliated epithelial cells of the bronchus, distal tubular cells of the kidney, and ductal cells from several organs including the breast and prostate. In contrast, LN-2 was unreactive with all human nonlymphoid organs and tissues including the bone marrow. Indirect immunofluorescence staining of a panel of 26 solid tumor cells lines showed that LN-1 was reactive with the majority of epithelium-derived cell lines, glioblastomas, and astrocytomas but was unreactive with neuroblastomas, small cell carcinoma of the lung, and sarcomas. LN-2 was unreactive with 25 of 26 of the solid tumor cell lines by these techniques. Immunobiochemical studies have shown that LN-1 recognizes a cell surface sialoantigen whereas LN-2 is directed against a 35,000 dalton nuclear membrane protein. Because of their high specificity for B cell tumors and their ability to stain B5-fixed, paraffin-embedded tissues, LN-1 and LN-2 are useful reagents for the diagnosis and classification of the human lymphomas and leukemias.     

抗重金属汞离子抗体的制备及鉴定

汞、镉、铅等重金属引起的环境污染已在世界范围内造成危害。快速、廉价地监测生境中重金属是减小其对人类及动物危害的先决条件。传统检测方法无法满足高通量的现场检测,建立更快速、更经济的免疫分析法检测汞离子是生产及经济发展的需要。本研究中,报道了汞特异性单克隆抗体的制备与筛选方法和结果。因Hg2+太小以至于不能引起免疫反应,所以用螯合剂(二乙烯三胺五乙酸,DTPA)将金属离子与载体蛋白(匙孔血蓝蛋白,KLH)连接起来。成功合成、鉴定汞复合物抗原后,免疫BALB/c小鼠,通过细胞融合获得了稳定分泌抗体的杂交瘤细胞。用极限稀释法亚克隆,通过ELISA筛选,获得了2株稳定分泌抗汞离子抗体的细胞株(H2H5,H1H8)。小鼠腹腔注射1×107H2H5、H1H8细胞株制备腹水,腹水抗体效价都在1∶51200以上。经鉴定两株杂交瘤均为IgG1亚类,轻链为kappa型且分泌抗体稳定性较好。实验结果为汞离子残留免疫学检测方法的建立提供了技术基础,对提高风险评估工作的效率和质量,保障食品安全有重要现实意义。     

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.     

Arachidonate 5-lipoxygenase of porcine leukocytes studied by enzyme immunoassay using monoclonal antibodies

The cytosol fraction of porcine leukocytes contained 5-lipoxygenase, the activity of which was masked by a predominant activity of 12-lipoxygenase. The 5-lipoxygenase was partially purified to a specific activity of about 10 nmol of arachidonic acid oxygenated/min/mg of protein and given to mice as an antigen to prepare monoclonal antibodies against the enzyme. Two species of antibodies recognized separate sites of the 5-lipoxygenase protein and did not cross-react with 12-lipoxygenase. They were utilized to develop a peroxidase-linked immunoassay of sandwich-type, which allowed a quantitative determination of the 5-lipoxygenase protein. The assay was applied to a screening of the 5-lipoxygenase content in various porcine tissues. By far the highest content of 5-lipoxygenase was found in leukocytes. About one-tenth the amount of the enzyme was found in lung, pancreas, ileum, and thymus, which could not be attributed to the contaminating leukocytes in these tissues.     

人凝血因子Ⅶ单克隆抗体的制备与鉴定

目的：制备抗人凝血因子Ⅶ单克隆抗体并鉴定其特性。方法：应用杂交瘤融合技术,以重组人凝血因子Ⅶ为抗原免疫BALB／c小鼠;取免疫小鼠的脾细胞与Sp2／0骨髓瘤细胞融合,经间接ELISA法筛选、融合细胞有限稀释法克隆、克隆化杂交瘤细胞株的亚类鉴定等方法筛选出单克隆抗体杂交瘤细胞株,并对单克隆抗体的特异性进行鉴定;用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行单抗的纯化。结果：获得了3株可稳定分泌单克隆抗体的杂交瘤细胞3E8、3D2和1C5,诱生的腹水效价分别为1：1×10^7、1：1×10^6和1：1×10^6;亚类鉴定表明388为IgG2a,其余2株均为IgGl;特异性鉴定显示它们与多种血浆蛋白均无交叉反应,表明单抗是特异的;经过亲和层析,获得了纯化的单抗。结论：获得了特异性的人凝血因子Ⅶ单克隆抗体,为建立人凝血因子Ⅶ检测及纯化方法奠定了基础。