Incorporation of monoclonal antibodies into cells by osmotic permeabilization. Effect on cellular metabolism

Incorporation of asparagine synthetase-specific monoclonal antibodies into L5178Y D10/R (L-asparaginase-resistant) murine lymphoma cells by osmotic lysis of pinocytic vesicles was used to evaluate the potential of the technique for macromolecular incorporation for metabolic studies. Nonspecific effects of the incorporation procedure included temporary inhibition of protein and DNA synthesis by 80-85% and a transitory loss of membrane integrity. Cells incorporated with an antibody inhibitory to tumor cell asparagine synthetase showed increased dependence upon an exogenous source of asparagine in the culture medium, while cells incorporated with a control antibody were not affected. These studies demonstrated that incorporation of inhibitory monoclonal antibodies into cells can be used to study the short term metabolic role of specific enzymes; however, the metabolic effects induced by the specific macromolecule must be evaluated within the context of the nonspecific effects caused by the osmotic treatment required for incorporation.     

Inhibitory effects of anti-interleukin 2 receptor and anti-L3T4 antibodies on delayed type hypersensitivity: the role of complement and epitope

Although it is often assumed that anti-T cell antibodies mediate immunosuppression by targeting T cells for destruction, other activities should be considered. To dissect the mechanisms by which anti-L3T4 and anti-interleukin 2 receptor (IL 2R) monoclonal antibodies (Mab) mediate immunosuppression, the effects of anti-L3T4 and two complement-fixing anti-IL 2R Mab of the same isotype, but defining functionally distinct epitopes, were probed in a delayed type hypersensitive (DTH) model using BALB/c as well as two C5-deficient mouse strains. Low doses of anti-L3T4 and the M7/20 anti-IL 2R Mab, which competitively blocks IL 2 binding, inhibit DTH in BALB/c mice whereas an anti-receptor antibody which does not block the IL 2 binding site did not effectively abrogate DTH. Interestingly, anti-L3T4, but not M7/20 anti-IL 2 Mab treatment blocked DTH in the C5-deficient strains. On the other hand, M7/20 does not cause immunosuppression solely by blocking the IL 2R from occupancy by IL 2 because binding to T blasts by M7/20 is equivalent in BALB/c and C5-deficient strains. Consequently, immunosuppression mediated by anti-IL 2R Mab is dependent on both IL 2 receptor site blockade and C5. Clearly, anti-L3T4 and M7/20 have disparate requirements for C5 in mediating immunosuppression. There can be no doubt that factors other than the cellular targeting patterns influence the immunosuppressive activities of Mab. Ideally, anti-T cell Mab should fix complement and inhibit T cell function.     

Monoclonal antibodies specific for bovine pancreatic asparagine synthetase. Production and use in structural studies

Thirteen stable hybridoma cell lines producing monoclonal antibodies specific for asparagine synthetase were established and one monoclonal antibody was chosen to produce an immunoaffinity resin for the purification of asparagine synthetase. Bovine pancreatic asparagine synthetase was purified to a specific activity of 395 nmol of Asn produced/min/mg. Electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 54,000 polypeptide. Prior cross-linking with dimethyl suberimidate resulted in a band at Mr = 52,500 (monomer) and two additional bands at Mr = 97,000 and 98,000 (dimers), suggesting the possibility of a heterogeneous enzyme population with slight differences in subunit composition. The ratio of Gln-dependent and NH3-dependent asparagine synthetase activities was constant for immunoaffinity-purified enzyme, but the ratios of glutaminase activity to synthetase activities varied, suggesting separate aspartate and glutamine binding sites. The monoclonal antibodies were tested as inhibitors of the Gln-dependent and NH3-dependent asparagine synthetase activities as well as for inhibition of the glutaminase activity of the enzyme. Two antibodies inhibited Gln- and NH3-dependent synthesis of asparagine, but did not affect the glutaminase activity of immunoaffinity-purified asparagine synthetase. A third monoclonal antibody inhibited Gln-dependent synthesis of asparagine and glutaminase activity, but activated NH3-dependent asparagine synthetase activity. These data are discussed in terms of multiple substrate binding domains within the asparagine synthetase molecule.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

Monoclonal antibodies directed against plasmid-encoded released proteins of enteropathogenic Yersinia

Abstract Yersinia enterocolitica of serotypes O:3, O:8, O:9 and O:5,27 and Yersinia pseudotuberculosis of serotypes I and III release plasmid-encoded proteins into calcium-deficient medium. Mouse monoclonal antibodies were elicited against plasmid-encoded released proteins of Y. enterocolitica of serotype O:9. As shown by immunoblot analysis the monoclonal antibody Mab9–200 recognized the 46-kDa protein of Y. enterocolitica of serotypes O:3, O:9 and O:5,27, the 58-kDa protein of Y. enterocolitica of serotype O:8 and the 67-kDa protein of Y. pseudotuberculosis of serotypes I and III. Mab9–15 reacted with the 36-kDa protein of Y. enterocolitica of serotypes O:9, O:3 and O:8, and the 34-kd protein of Y. enterocolitica of serotype O:5,27 and Y. pseudotuberculosis of serotypes I and III. The 25-kDa proteins of Y. enterocolitica of serotypes O:3, O:9, O:8 and O:5,27, but not those of Y. pseudotuberculosis were recognized by the monoclonal antibody Mab-128. This species-specific recognition of epitopes could not be achieved by mouse polyclonal antibodies.     

A monoclonal antibody to glucosylceramide inhibits the growth of Fonsecaea pedrosoi and enhances the antifungal action of mouse macrophages

Fungal glucosylceramides (GlcCer) are conserved lipid components in a large variety of pathogenic and non-pathogenic fungal species, but their biological functions are still unclear. Recent studies demonstrate that GlcCer are immunologically active components inducing the production of antifungal antibodies. In this work, a major GlcCer was purified and characterized from mycelial forms of Fonsecaea pedrosoi, the most frequent causative agent of chromoblastomycosis. As determined by fast atom bombardment mass spectrometry (FAB-MS) analysis, the purified molecule was identified as the conserved structure N-2'-hydroxyhexadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. A monoclonal antibody (Mab) against this structure was used in indirect immunofluorescence with the different morphological stages of F. pedrosoi. Only the surface of young dividing cells was recognized by the antibody. Treatment of F. pedrosoi conidia with the Mab against GlcCer resulted in a clear reduction in fungal growth. In addition, the Mab also enhanced phagocytosis and killing of F. pedrosoi by murine cells. These results suggest that, in F. pedrosoi, GlcCer seem to be cell wall components targeted by antifungal antibodies that directly inhibit fungal development and also enhance macrophage function, supporting the use of monoclonal antibodies to GlcCer as potential tools in antifungal immunotherapy.     

Topographical separation of the catalytic sites of asparagine synthetase explored with monoclonal antibodies

Monoclonal antibodies which inhibited the enzymatic activity of bovine pancreatic asparagine synthetase were mapped to two topographically separate regions of the enzyme surface using competitive binding assays. Three antibodies which all inhibited glutamine- and NH3-dependent synthesis of asparagine bound to a common antigenic region. A fourth monoclonal antibody which interfered with glutamine binding or cleavage but not with NH3-dependent synthesis of asparagine was mapped to a separate region of the enzyme surface. These findings suggest a topographical separation between the aspartyl-AMP and glutamine-binding sites of bovine pancreatic asparagine synthetase. Three noninhibitory antibodies exhibited conformation-dependent binding and were mapped to a third region of the enzyme. Binding assays were used to demonstrate extensive cross-reaction of these antibodies with asparagine synthetases isolated from bovine liver and sheep pancreas. Substantial cross-reactions were also seen with the enzyme isolated from rat liver or pancreas, a human tumor cell line, and a mouse tumor cell line. Of the four antibodies that inhibited glutamine- and NH3-dependent synthesis of asparagine from ruminant species, only one bound to and inhibited the enzyme from rat liver or mouse cells, which suggests significant structural differences between the ruminant and rodent enzymes.     

Neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates.

Cells producing neutralizing monoclonal antibodies to a serotype 3 human neonatal rotavirus strain RV-3 were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. As ascites fluid, three rotavirus-neutralizing monoclonal antibodies were characterized by hemagglutination inhibition and reacted with 17 cultivable mammalian rotaviruses representing five virus serotypes, by fluorescent focus neutralization and enzyme immunoassay. Two antibodies, Mab RV-3:1 and Mab RV-3:2, reacted with the seven serotype 3 rotaviruses only. Mab RV-3:1 was shown to bind to the outer capsid glycoprotein gp34 of rotavirus when variants of SA 11 rotavirus were used, and it therefore appears to react with the major neutralization epitope of serotype 3 rotaviruses. The antibody Mab RV-3:3 was specific for an epitope of RV-3 rotavirus not present on any other rotavirus of any serotype tested, including another neonatal isolate of identical RNA electropherotype isolated from the same ward of the same hospital as RV-3 3 months earlier. These two viruses were also distinguishable by fluorescent focus neutralization, using antiserum to RV-3 virus. Western blot analysis showed binding of Mab RV-3:3 to the trypsin cleavage product of the outer capsid protein p86 of RV-3. This suggests that antigenic drift may have occurred among neonatal rotaviruses in Melbourne. These monoclonal antibodies will be useful in serotyping assays of rotaviruses directly in stool samples, and in further analysis of antigenic variation within the serotype.     

Bovine pancreatic asparagine synthetase explored with substrate analogs and specific monoclonal antibodies.

Several substrate analogs were tested for their ability to inhibit bovine pancreatic asparagine synthetase. Of the substrate analogs tested both 6-diazo-5-oxo-L-norleucine (DON) and 5-chloro-4-oxo-L-norvaline (CONV) were shown to inhibit the enzyme strongly. DON inhibited the glutaminase and glutamine-dependent asparagine synthetase activities and CONV inhibited the ammonia-dependent activity as well. Both of these inhibitors appeared to be relatively tight binding since desalting failed to remove the inhibition. The inactivation of bovine pancreatic asparagine synthetase by DON is accompanied by a shift from a 47,000 molecular weight monomer to a 96,000 molecular weight dimer as observed by HPLC gel filtration chromatography. This DON-induced shift is prevented by the presence of the substrate glutamine. A monoclonal antibody known to inhibit specifically the ammonia-dependent and glutamine-dependent asparagine synthetase activities but not glutaminase (monoclonal antibody 2B4) binds to both the monomer and the dimer forms of untreated enzyme, as well as to the dimer form of the DON-inactivated enzyme. On the other hand, a monoclonal antibody known to inhibit specifically the glutaminase and glutamine-dependent activities and not the ammonia-dependent asparagine synthetase (monoclonal antibody 5A6) binds to both forms of untreated enzyme but cannot bind to the DON-inactivated enzyme. These data are used to describe the relation of regions of the active site of asparagine synthetase in relation to antibody binding sites.     

The topology of the glutamine and ATP binding sites of human asparagine synthetase.

Human asparagine synthetase was examined using a combination of chemical modifiers and specific monoclonal antibodies. The studies were designed to determine the topological relation between the nucleotide binding site and the glutamine binding site of the human asparagine synthetase. The purified recombinant enzyme was chemically modified at the glutamine binding site by 6-diazo-5-oxo-L-norleucine (DON), and at the ATP binding site by 8-azidoadenosine 5'-triphosphate (8-N3ATP). The effects of chemical modification with DON included a loss of glutamine-dependent reactions, but no effect on ATP binding as measured during ammonia-dependent asparagine synthesis. Similarly, modification with 8-N3ATP resulted in a loss of ammonia-dependent asparagine synthesis, but no effect on the glutaminase activity. A series of monoclonal antibodies was also examined in relation to their epitopes and the sites modified by the two covalent chemical modifiers. It was found that several antibodies were prevented from binding by specific chemical modification, and that the antibodies could be classified into groups correlating to their relative binding domains. These results are discussed in terms of relative positions of the glutamine and ATP binding sites on asparagine synthetase.     

Overproduction, preparation of monoclonal antibodies and purification of E. coli asparagine synthetase A.

In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E. coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E. coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E. coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E. coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.     

Characterization of murine TWEAK and its receptor (Fn14) by monoclonal antibodies

In the present study, we characterized murine TWEAK and its receptor (Fn14) by generating cDNA transfectants and specific monoclonal antibodies (mAbs). Recombinant murine TWEAK bound to murine Fn14-transfected L5178Y (mFn14/L5178Y) cells and induced cell death. Some anti-human Fn14 mAbs we previously generated strongly cross-reacted with murine Fn14 and induced cell death in mFn14/L5178Y cells. Murine TWEAK-transfected L5178Y cells expressed murine TWEAK on cell surface and secreted functional TWEAK, which were detected by a newly generated anti-murine TWEAK mAb (MTW-1). Although thioglycolate-elicited murine peritoneal macrophages did not express a detectable level of TWEAK on their surface, they secreted functional TWEAK that was cytotoxic against mFn14/L5178Y cells and neutralized by MTW-1. The anti-murine TWEAK and Fn14 mAbs will be useful for further investigating the physiological and pathological functions of TWEAK and Fn14.     

Localization of atrial natriuretic peptide in pig granulosa cells isolated from ovarian follicles of various size

The aim of the current study was to present the spatial distribution of atrial natriuretic peptide (ANP) in short-term cultures of pig granulosa cells obtained from small, medium, and large ovarian follicles. The specific immunoreactivity was detected by three monoclonal antibodies recognizing different epitopes of the ANP molecule (Mab 6C3, Mab 6F11, Mab5D3). The specific ANP immunoreactivity detected by Mab 6C3 and Mab 6F11 showed dense staining of cytoplasm and was similar in granulosa cells from small and medium follicles. The strongest ANP immunostaining was observed in GC obtained from large follicles. The ANP immunostaining detected by Mab 5D3 had granular appearance moderately expressed in the submembrane region of granulosa cells of all types of follicles. Since ANP and ANP receptors are present in reproductive organs, the three anti-ANP antibodies may be an useful tool in further studies concerning the role of ANP in granulosa cell differentiation and function.     

Cell cycle analysis using the monoclonal antibody Ki-67

We determined by flow cytometry the proportion of cells in cycle with the monoclonal antibody Ki-67 and also in S-phase after incorporation of bromodeoxyuridine (BrdUrd) with monoclonal antibody to BrdUrd. The monoclonal antibody Ki-67 was useful to detect a nuclear antigen present only in proliferating cells but not expressed in resting (Go) cells. Cell preparation to measure BrdUrd amount incorporated into cellular DNA was difficult but this anti-BrdUrd antibody was useful for measuring the rate of DNA synthesis and for the analysis of precious cell kinetics. These antibodies may provide useful information of cell kinetics.     

Immunoreactivity of the monoclonal antibody F89/160.1.5 for the human prion protein

Monoclonal antibodies (Mab) to the prion protein (PrP) have been critical to the neuropathological characterisation of PrP-related diseases in human and animals. Although PrP is highly evolutionary conserved, there is some sequence divergence among species. We have analysed the F89/160.1.5 Mab raised against the bovine prion protein for immunoreactivity with the human prion protein. The antibody recognised the IHFG epitope of the prion protein. An analysis of the Swiss Prot database confirmed conservation of the epitope in humans. Further immunohistochemical (IHC) analysis showed a highly sensitive (final concentration 55 ng/ml) and specific antibody for prion detection in humans. The observed immunoreactivity of the prion protein did not differ from that observed after staining with the well-known 3F4 (Senetek) monoclonal antibody.     

Anti-interleukin 2 receptor antibody suppresses delayed-type hypersensitivity to foreign and syngeneic antigens

Delayed-type hypersensitivity (DTH) requires stimulation of antigen-specific helper T cells (Th). Because de novo expression of the interleukin 2 receptor (IL 2R) is a necessary step in T cell activation, we tested the capacity of anti-mouse IL 2R monoclonal antibody (Mab) and anti-Th Mab (anti-L3T4) to block DTH. We examined the effect of these Mab on two distinct DTH systems, i.e., to foreign hapten (trinitrobenzenesulfonic acid) and to this hapten present on syngeneic blasts. Both anti-IL 2R and anti-L3T4 Mab suppress DTH. Therapy is as effective treating with one injection just before challenge with the hapten as giving six daily injections. These data indicate that DTH is dependent on a discrete subset of activated IL 2R-positive T cells, because anti-IL 2R therapy, which targets few cells, is as effective as anti-L3T4 Mab treatment, which targets the entire Th subset.     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H^* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10⁶ cells ml^–1 producing 76.6 mg l^–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C cholesterol - CD cyclodextrin - dhfr dihydrofolate reductase - F68 Pluronic F68 - GS glutamine synthetase - MSX methionine sulphoximine - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - ADCC Antibody-dependant cellular cytotoxicity