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1.
Summary IgM monoclonal antibodies directed against tumor cells which do not mediate antibody-dependent macrophage cytotoxicity (ADMC) even when they are cytotoxic in the presence of complement, have been shown to render macrophages tumoricidal when they carry an immunomodulating agent, i.e., muramyldipeptide (MDP).This statement is based on experiments using two IgM monoclonal antibodies selected for their ability to bind L1210 leukemia cells (F2-10-23-IgM) and 3LL Lewis lung carcinoma cells (6B6-IgM) specifically, as shown by flow cytofluorometry analysis.The MDP-IgM conjugates, containing 45 MDP molecules per IgM molecule, were prepared by allowing MDP-hydroxy-succinimide ester to react with IgM monoclonal antibodies.The MDP-IgM conjugates are shown to bind to relevant tumor cells and to induce the activation of thioglycolate-elicited peritoneal mouse macrophages leading to 80% growth inhibition of target cells at optimum concentrations of bound MDP. These concentrations of bound MDP were 10 times lower than the concentration of free MDP, giving a maximum activation that is limited to 20% growth inhibition.No macrophage activation was evidenced when tumor cells were incubated in the presence of irrelevant MDP-IgM conjugates and macrophages or when macrophages were preincubated in the presence of MDP-IgM conjugates and then incubated in the presence of relevant or irrelevant tumor cells but in the absence of the MDP-IgM conjugates.The reported results are discussed with reference to the mechanism of activation of macrophage by muramyldipeptide and to the usefulness of such MDP-IgM conjugates as potential antitumor agents in cancer therapy.Abbreviations ADMC antibody dependent macrophage mediated cytotoxicity - F-GAM fluoresceinylthiocarbamyl goat anti mouse antibody - -Man-BSA -mannopyranosyl-phenylthiocarbamyl bovine serum albumin containing some 25 mannose residues (neoglycoprotein) - MDP muramyldipeptide, 2-acetamido-3(2-0-d-lactyl-l-alanyl-d-glutamyl amine) glucopyranose - MDP-F2-10-23-IgM Murine monoclonal antibody specific of L1210 leukemia cells and substituted with 45 MDP molecules - MDP—6B6-IgM Murine monoclonal antibody specific of 3LL Lewis lung carcinoma cells and substituted with 45 MDP molecules - MEM minimal essential medium - TDM Trehalose-dimycolate  相似文献   

2.
Immunological activities of muramyl peptides   总被引:5,自引:0,他引:5  
Muramyl peptides are endowed with numerous modulatory effects on the immune and nervous systems. Studies with synthetic muramyl dipeptide (MDP), the smallest unit of bacterial cell walls that can replace Mycobacteria in Freund's complete adjuvant, revealed that this glycopeptide can regulate several functions of cells involved in the immune response. The adjuvanticity of MDP and the MDP-induced activation of macrophages against tumors were found to be potentiated in vitro and in vivo with monoclonal anti-MDP antibodies. When used on immunoadsorbent columns, the anti-MDP antibodies removed the somnogenic and pyrogenic activities contained in supernatants of stimulated rabbit peritoneal macrophages. Based on these data a hypothesis is put forward to explain the immuno- and neuro-modulatory effects of muramyl peptides.  相似文献   

3.
A rat IgG2a monoclonal antibody (mAb3A33) directed against the mouse Mac-1 antigen was conjugated with muramyl dipeptide (MDP) by using an intermediate polymer; under such conditions 75 MDP molecules were bound to one antibody molecule. A poly(L-lysine) polymer substituted with muramyl dipeptide and 3-(2-pyridyldithio)propionyl residues were prepared, the remaining lysine epsilon-amino groups were acylated with D-gluconolactone, leading to a neutral polymer; then a few polymer conjugates were coupled to mAb3A33 via a disulfide bridge. The binding capacity of the monoclonal antibody was preserved after conjugation with MDP-polymer molecules. Mouse peritoneal macrophages, incubated for 24 h with MDP-mAb3A33 conjugate became cytostatic against P815 mastocytoma cells, whereas unconjugated mAb3A33 and MDP-bound to a nonspecific rat IgG2a were ineffective. An enhancement of the cytostatic activity induced by MDP-mAb3A33 conjugate was obtained in the presence of gamma-IFN. These results show that several tens of MDP molecules can be linked to a macrophage-specific monoclonal antibody by using a neutral intermediate polymer without impairing the binding antibody capacity and that this type of MDP conjugate can efficiently activate macrophages and therefore could be the basis of the development of new antitumor therapy.  相似文献   

4.
Monoclonal antibodies were prepared against two different human tumour cell lines, the melanoma cell line SK-Mel-25 and the acute lymphoblastic leukemia T cell line CCRF-CEM. Presence of antibodies against human tumour cells in the supernatants of hybridoma cultures was tested by binding of 125I-F(ab′)2 anti-mouse IgG. On two occasions a hybridoma culture, initially selected for subsequent cloning as it seemingly produced antibodies against tumour cells, was later found to produce monoclonal antibodies specific for Mycoplasma hyorhinis. In immunofluorescent staining patchy structures were visible which seemed to be attached to the cell surface. By combined staining with FITC-conjugated anti-mouse immunoglobulin for monoclonal antibody, Evans blue for cytoplasm and Hoechst compound no. 33258 for DNA, the reaction against mycoplasma could be recognized. These results demonstrate that if cultured cells are used for preparation of monoclonal antibodies, there is a good chance that the selected hybridomas may produce antibodies against ‘culture artifacts’ such as mycoplasmas, in addition to the target antigens. Thus mycoplasma contamination of cell cultures poses a serious problem in the hybridoma research and the testing system for antibody specificity should be carefully monitored.  相似文献   

5.
Michael Sela 《Biopolymers》1983,22(1):415-424
The availability of synthetic antigens permitted a systematic elucidation of the molecular basis of antigenicity, as well as of other phenomena, and to establish the genetic control of immune response and its link to the major histocompatibility region of the species. Moreover, it allowed a conceptual approach to production of vaccines made up of synthetic fragments of relevant virus antigens. This was first demonstrated with a bacteriophage, MS-2, that can be efficiently inactivated with antibodies obtained with an immunogen in which a synthetic peptide corresponding to a sequence of 20 amino acid residues within its coat protein was covalently linked to a polymeric synthetic carrier. When the synthetic adjuvant N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) was chemically linked to the same molecule, the resulting immunogen was effective in aqueous solution. This approach has now been used for diphtheria: a synthetic peptide from the A-chain of the toxin, covalently attached together with MDP to the synthetic carrier multichain poly(DL-alanine) induced antibodies cross-reactive with the diphtheria toxin as well as protection against the dermonecrotic activity of the toxin. A similar approach has been described also by R. Arnon and her colleagues in our laboratory for influenza virus, making use of a peptide with sequence 91–108 of the hemagglutinin of type A H3N2 strains attached to tetanus toxoid. The results indicated that the conjugate led to partial protection against A/Texas mouse-adapted influenza virus.  相似文献   

6.
Mycobacterium tuberculosis infects not only host macrophages but also nonprofessional phagocytes, such as alveolar epithelial cells. Glycosaminoglycans (GAGs) are considered as the component of mycobacterial adherence to epithelial cells. Here we show that extracellularly occurring mycobacterial DNA-binding protein 1 (MDP1) promotes mycobacterial infection to A549 human lung epithelial cells through hyaluronic acid (HA). Both surface plasmon resonance analysis and enzyme-linked immunosorbent assay revealed that MDP1 bound to HA, heparin, and chondroitin sulfate. Utilizing synthetic peptides, we next defined heparin-binding site of 20 amino acids from 31 to 50 of MDP1, which is responsible for the specific DNA-binding site of MDP1. MDP1 bound to A549 cells, and exogenous DNA and HA interfered with the interaction. The binding was also abolished by treatment of A549 cells with hyaluronidase, suggesting that HA participates in the MDP1-A549 cell interaction. Adherence of bacillus Calmette-Guérin (BCG) and M. tuberculosis to A549 cells was inhibited by addition of HA, DNA, and anti-MDP1 antibody, showing that MDP1 participates in the interaction between mycobacteria-alveolar epithelial cells. Simultaneous treatment of intratracheal BCG-infected mice with HA reduced the growth of BCG in vivo. Taken together, theses results suggest that HA participates in Mycobacterium-lung epithelium interaction and has potential for therapeutic and prophylactic interventions in mycobacterial infection.  相似文献   

7.
Our research on pathogenesis of disseminated candidiasis led to the discovery that antibodies specific for Candida albicans cell surface β-1, 2–mannotriose [β-(Man)3] protect mice. A 14 mer peptide Fba, which derived from the N-terminal portion of the C. albicans cytosolic/cell surface protein fructose-bisphosphate aldolase, was used as the glycan carrier and resulted in a novel synthetic glycopeptide vaccine β-(Man)3-Fba. By a dendritic cell-based immunization approach, this conjugate induced protective antibody responses against both the glycan and peptide parts of the vaccine. In this report, we modified the β-(Man)3-Fba conjugate by coupling it to tetanus toxoid (TT) in order to improve immunogenicity and allow for use of an adjuvant suitable for human use. By new immunization procedures entirely compatible with human use, the modified β-(Man)3-Fba-TT was administered either alone or as a mixture made with alum or monophosphoryl lipid A (MPL) adjuvants and given to mice by a subcutaneous (s.c.) route. Mice vaccinated with or, surprisingly, without adjuvant responded well by making robust antibody responses. The immunized groups showed a high degree of protection against a lethal challenge with C. albicans as evidenced by increased survival times and reduced kidney fungal burden as compared to control groups that received only adjuvant or DPBS buffer prior to challenge. To confirm that induced antibodies were protective, sera from mice immunized against the β-(Man)3-Fba-TT conjugate transferred protection against disseminated candidiasis to naïve mice, whereas C. albicans-absorbed immune sera did not. Similar antibody responses and protection induced by the β-(Man)3-Fba-TT vaccine was observed in inbred BALB/c and outbred Swiss Webster mice. We conclude that addition of TT to the glycopeptide conjugate results in a self-adjuvanting vaccine that promotes robust antibody responses without the need for additional adjuvant, which is novel and represents a major step forward in vaccine design against disseminated candidiasis.  相似文献   

8.
Summary Monoclonal antibodies (IgG1) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG1 antibodies. Inhibition of 3H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID50) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG1-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID50 values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.  相似文献   

9.
《Cellular immunology》1985,92(2):226-234
A surprising unidirectional carrier effect has been observed in the antibody response to myoglobin-ferritin conjugate. This conjugate serves as a hapten-carrier complex for myoglobin-specific T cells to help ferritin-specific B cells make anti-ferritin antibodies, but it does not function for ferritin-specific T cells to help myoglobin-specific B cells to make anti-myoglobin. Therefore, myoglobin-ferritin does not bypass the Ir gene defect of low responders to myoglobin. In contrast, myoglobin-fowl γ-globulin does induce anti-myoglobin antibodies in low responder mice and thus bypasses the Ir gene defect. Both complexes are covalently coupled. Since the myoglobin-ferritin conjugate serves for myoglobin-specific T cells to help myoglobin-specific B cells, the myoglobin in the conjugate is not altered in a way that would prevent recognition by myoglobin-specific B cells. Similarly, the conjugate serves for ferritin-specific helper T cells to help ferritin-specific B cells, so it can be recognized functionally by ferritin-specific T helper cells. Explanations such as unidirectional induction of or sensitivity to bystander help, or T-cell suppression, have been excluded. While the explanation for this unexpected observation is not yet certain, several possibilities are discussed to explain this novel phenomenon, which is believed to be the first example of such a unidirectional carrier effect between two proteins.  相似文献   

10.
Several components of mycobacteria including a water-soluble extract (WSA) and an interphase material (IPM) as well as the synthetic cell wall analog muramyl dipeptide (MDP) all stimulated human mononuclear cells (MNL) to produce a factor which was mitogenic for murine thymocytes. The mediator induced by MDP is probably lymphocyte-activating factor (LAF) because it was produced by adherent but not nonadherent MNL and yields two characteristic peaks of activity in the 16,000–22,000 and 60,000–70,000 molecular weight range when eluted from Bio-Gel P-100 columns. The 6-O-stearoyl derivative of MDP was an active inducer of MNL LAF production, whereas, the d-alanine analog of MDP was somewhat less potent. Unfractionated as well as adherent, but not nonadherent, mouse peritoneal cells also produced LAF in response to WSA, IPM, and MDP. P388D1 cell line macrophages, which are completely devoid of lymphocytes, could be stimulated by WSA, IPM, and MDP to produce LAF after prolonged incubation. These adjuvants did not stimulate nonadherent Balb/C or human blood cells to produce a mitogenic factor. However, when the P388D1 macrophages were stimulated with these adjuvants in the presence of nonadherent murine or human peripheral blood cells, a mitogenic activity was produced in a shorter period of incubation suggesting that activated lymphocytes can facilitate the production of LAF by macrophages.  相似文献   

11.
A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (Ka=1.5 × 109 L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.  相似文献   

12.
Abstract

A study was undertaken to determine if the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (C12MDP; Clodronat), a treatment known to deplete monocytes, as well as liver and spleen macrophages, would reduce the number of macrophages in the retina of animals with experimental autoimmune uveitis (EAU) and decrease the severity of the disease. EAU was induced in Lewis rats by immunization with S-antigen (S-Ag). Monocytes and macrophages were depleted via an intravenous injection of Cl2MDP encapsulated in liposomes. Control groups included rats that received no S-Ag (n= 18), S-Ag and no treatment (n=23), S-Ag and free drug (n = 20), or empty liposomes (n=14). Treated animals received injections of the Cl2MDP-liposomes, free drug, or empty liposomes. Animals were sacrificed at 14, 21 and 28 days post-S-Ag administration. Intravenous, Cl2MDP-liposomes produced a statistically significant reduction in the severity of the EAU when compared to controls at both days 14 and 21 following S-Ag injection. Immunohistochemical staining with the monoclonal antibody EDI demonstrated that the severity of the ocular inflammatory response correlated with the number of EDI-positive cells in the retina. Following the cessation of treatment, treated animals developed disease that was as severe at day 28 as that of untreated animals at day 21. These results confirm the importance of monocytes and macrophages in EAU by demonstrating the correlation between the presence of EDI-positive cells in the retina and the resultant damage to the retina. Although the dosing regimen employed here did not provide a cure, strategies designed to prevent the local recruitment and/or activation of mononuclear phagocytes may prove to be useful in the treatment of EAU.  相似文献   

13.
Summary Methotrexate (MTX) was conjugated to an anti-carcinoembryonic antigen monoclonal antibody (NP2) by using amino-dextran as an intermediate carrier. The drug was chemically linked to amino-dextran (averageM r = 40000), and the resulting MTX-dextran was then site-specifically attached to the carbohydrate moiety of the antibody. Athymic nude mice that carried human colonic GW-39 tumors (s. c.) were treated with the immunoconjugate. In this study, the specific conjugate caused a greater inhibition of the tumor growth than either free MTX or its conjugate with dextran and an irrelevant antibody. The intermediate MTX-dextran and the unlinked mixture of MTX-dextran with NP2 were both relatively ineffective in inhibiting tumor growth. The greatly reduced host toxicity permitted the use of the MTX-dextran-NP2 in a high-dose therapy of this tumor system.Supported in part by U.S.P.H.S. grant CA39 841 from the NIH  相似文献   

14.
Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam-equipped targeting modules. A model study was first performed with β-lactam conjugated to biotin. This conjugate efficiently and selectively modified the catalytic site lysine (LysH93) of mAb 38C2. We then conjugated a β-lactam to a cyclic-RGD peptide to chemically program mAb 38C2 to target integrin receptors αvβ3 and αvβ5. The chemically programmed antibody bound specifically to the isolated integrin receptor proteins as well as the integrins expressed on human melanoma cells. This approach provides an efficient and versatile solution to irreversible chemical programming of aldolase antibodies.  相似文献   

15.
A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L2 component, and antibody 1C2 was specific for the L1 protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.  相似文献   

16.
Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody (mAb) at 1.85 Å resolution. Nicotine is almost completely (> 99%) buried in the interface between the variable domains of heavy and light chains. The high affinity of the mAb is the result of a charge–charge interaction, a hydrogen bond, and several hydrophobic contacts. Additionally, similarly to nicotinic acetylcholine receptors in the brain, two cation–π interactions are present between the pyrrolidine charge and nearby aromatic side chains. The selectivity of the mAb for nicotine versus cotinine, which is the major metabolite of nicotine and differs in only one oxygen atom, is caused by steric constraints in the binding site. The mAb was isolated from B cells of an individual immunized with a nicotine–carrier protein conjugate vaccine. Surprisingly, the nicotine was bound to the Fab fragment in an orientation that was not compatible with binding to the nicotine–carrier protein conjugate. The structure of the Fab fragment in complex with the nicotine–linker derivative that was used for the production of the conjugate vaccine revealed a similar position of the pyridine ring of the nicotine moiety, but the pyrrolidine ring was rotated by about 180°. This allowed the linker part to reach to the Fab surface while high-affinity interactions with the nicotine moiety were maintained.  相似文献   

17.
A procedure for production of monoclonal antibodies to testosterone is described. The method involves immunization of rats with a bovine serum albumin conjugate of testosterone 3-(0-carboxymethyl)oxime followed by polyethylene glycol induced hybridization of the immune lymphocytes with mouse myeloma cells. The resulting hybridomas were cloned and the antibodies produced by each clone were characterized. All the antibodies obtained showed high affinity for testosterone, (Ka = 1010 l/mol), but clones differed widely in the degree of cross-reaction of the antibodies with other steroids, such as 5α-dihydrotestosterone (range 2–100%) and androstenedione (< 0.1–4%). Large quantities of the selected specific antibodies can be obtained by mass growth of the hybridoma line in culture or as tumors in irradiated or nude mice. Monoclonal antibody preparations may improve standardization of immunoassay methods.  相似文献   

18.
Cloned and unselected bone marrow-derived macrophage cell lines were obtained from A/J, AKR/J, BIO.A(5R), CBA/J, DBA/2, HPC, NZW, and [NZB X NZW]F1 mice, and their interactions were studied in vitro with a lightly encapsulated natural serotype A isolate of Cryptococcus neoformans. Growth inhibition of C. neoformans was seen with all of the cell lines, as determined by enumeration of colony-forming units. Inhibition was enhanced by a high concentration (8%) of fresh mouse serum and was the same for serum obtained from AKR/J (C5 deficient) and BIO.A (C5 normal) mice. Macrophage incubation with fresh AKR/J serum which had been absorbed with heat-killed Cryptococcus cells also inhibited C. neoformans growth. Heat-inactivation, EDTA addition or anti-C3 antibody treatment of fresh serum abolished the opsonic activity for C. neoformans, while EGTA addition to fresh serum was without effect on opsonization. In addition, neither IgM nor IgG1 murine monoclonal antibodies specific for C. neoformans enhanced phagocytosis or killing of the yeast by macrophages. These findings are consistent with the interpretation that C3b is an important modulator of interactions between macrophages and C. neoformans.  相似文献   

19.
A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (Ka=1.5 × 109 L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.  相似文献   

20.
Karl Blau 《FEBS letters》1984,166(1):213-214
A competitive solid-phase immunoassay for the determination of testosterone in serum samples using time-resolved fluorescence is described. The solid phase is a testosterone-3-(O-carboxymethyl)-oxime-ovalbumin conjugate coated to polystyrene microtiter strips. Europium-labelled polyclonal and monoclonal antibodies against testosterone-3-(O-carboxymethyl)-oxime-bovine serum albumin were compared. Their behavior was quite similar although the polyclonal antibody was more sensitive, giving a detection limit of 15 fmol testosterone per assay. Correlation with RIA was very good (r = 0.982 and y = ?0.150 + 0.969x).  相似文献   

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