Monoclonal antibodies to monoamine oxidase B and another mitochondrial protein from human liver.

A monoclonal antibody has been generated to human liver monoamine oxidase (MAO) B by fusion of mouse myeloma cells with spleen cells from a mouse immunized with a mixture of semi-purified MAO A and MAO B. The antibody, 3F12/G10, an immunoglobulin G1, reacts with its antigen in cryostat sections of human liver, showing an intracellular particulate distribution as demonstrated by immunoperoxidase staining. The antibody indirectly precipitates [3H]pargyline-labelled human MAO B both from liver and platelet extracts but fails to precipitate MAO A from liver extracts. The antibody does not recognise rat liver MAO B, showing that the determinant is not universally expressed on MAO B. The antibody has no effect on the catalytic activity of MAO B. Other monoclonal antibodies were generated but they are directed to a protein with a subunit Mr of 54 000, a contaminant of the MAO preparation. One of these antibodies, A8/C2, an IgG2a, reacts with the same protein in both rat and human liver extracts.     

A Mouse Monoclonal Antibody against Dengue Virus Type 1 Mochizuki Strain Targeting Envelope Protein Domain II and Displaying Strongly Neutralizing but Not Enhancing Activity

Dengue fever and its more severe form, dengue hemorrhagic fever, are major global concerns. Infection-enhancing antibodies are major factors hypothetically contributing to increased disease severity. In this study, we generated 26 monoclonal antibodies (MAbs) against the dengue virus type 1 Mochizuki strain. We selected this strain because a relatively large number of unique and rare amino acids were found on its envelope protein. Although most MAbs showing neutralizing activities exhibited enhancing activities at subneutralizing doses, one MAb (D1-IV-7F4 [7F4]) displayed neutralizing activities without showing enhancing activities at lower concentrations. In contrast, another MAb (D1-V-3H12 [3H12]) exhibited only enhancing activities, which were suppressed by pretreatment of cells with anti-FcγRIIa. Although antibody engineering revealed that antibody subclass significantly affected 7F4 (IgG3) and 3H12 (IgG1) activities, neutralizing/enhancing activities were also dependent on the epitope targeted by the antibody. 7F4 recognized an epitope on the envelope protein containing E118 (domain II) and had a neutralizing activity 10- to 1,000-fold stronger than the neutralizing activity of previously reported human or humanized neutralizing MAbs targeting domain I and/or domain II. An epitope-blocking enzyme-linked immunosorbent assay (ELISA) indicated that a dengue virus-immune population possessed antibodies sharing an epitope with 7F4. Our results demonstrating induction of these antibody species (7F4 and 3H12) in Mochizuki-immunized mice may have implications for dengue vaccine strategies designed to minimize induction of enhancing antibodies in vaccinated humans.     

Characterization of a monoclonal antibody reacting with histone H3

A hybridoma cell line, 1GB3, has been obtained from a fusion between SP/O-Ag 14 myeloma cells and lymphocytes from BALB/c mice immunized with rat liver nuclear proteins. This hybridoma secreted a monoclonal antibody of the IgG2b class which reacted specifically with histone H3 in enzyme-linked immunosorbent assay (ELISA) as well as in immunoblotting and immunodot assays. Stringent test conditions were necessary to eliminate the presence of nonspecific or contaminating reactions with other histones than H3. The monoclonal antibody appears to recognize an epitope situated in the N-terminal residues 20-50 of histone H3; it recognizes this epitope in the octamer aggregate of core histones but not in the core particle.     

A monoclonal antibody which inhibits epidermal growth factor binding has opposite effects on the biological action of epidermal growth factor in different cells

An epidermal growth factor (EGF) receptor-interactive monoclonal antibody (151-IgG) that inhibits EGF binding to PC12 rat pheochromocytoma cells and to various other cell types has been produced. The hybridoma clone was obtained by fusing Sp2/O-Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside-solubilized protein from isolated PC12 cell plasma membranes. The antibody is an IgG which binds to protein A. 151-IgG did not bind EGF. At 0.5 degrees C 151-IgG was directly competitive for EGF binding to PC12 cells. It also inhibited EGF binding to bovine corneal endothelial cells, rabbit corneal fibroblasts, human foreskin fibroblasts, and normal rat kidney cells, and it slightly enchanced EGF binding to SW 3T3 cells. PC12 cells have the same number of binding sites for 151-IgG as for EGF (approximately 27,000 sites/cell). 151-IgG inhibited the photoactivatable cross-linking of EGF to a protein of Mr 170,000 in PC12 cells. 151-IgG inhibited the EGF-stimulated incorporation of [3H]thymidine into quiescent bovine corneal endothelial cells, rabbit corneal endothelial cells, epithelial normal rat kidney cells, and SW 3T3 cells while it enhanced the EGF-stimulated [3H]thymidine incorporation into quiescent human foreskin fibroblasts. 151-IgG by itself possessed intrinsic EGF-like activity for human fibroblasts but not for the other cells tested. This suggests that there is a difference in EGF receptors and/or processing in these normal cell types.     

Identification of circulating antigens, including an immunoglobulin binding protein, from Toxoplasma gondii tissue cyst and tachyzoites in murine toxoplasmosis

Here we describe the identification of Toxoplasma gondii circulating antigens in sera of BALB/c mice experimentally infected with either the virulent RH strain, or the cystogenic WTD1 strain or with an isolate from a human patient. The circulating antigens were identified by immunoblot in tachyzoite (RH strain) and in tissue cyst (ME-49 strain) crude antigens, using antibodies produced by immunisation of BALB/c mice with homologous sera from infected animals. The most relevant tachyzoite antigen identified are in the following four clusters of 109-94, 67-57, 35-31 and 28-21 kDa. Tissue cyst-specific circulating antigens, like the 18 kDa one, were detected in sera from mice infected with the cystogenic strains. These immune sera, after depletion of tachyzoite specific antibodies, recognised three tissue cysts antigens with Mr of 120, 79 and 48 kDa, and a cluster of antigens in the range of 68-53 kDa. We produced monoclonal antibodies by fusion of myeloma cells with lymphocytes from the mouse immunised with circulating antigens from the RH strain. One of the clones (3A11/H12) obtained, secretes IgG(1) and recognises a peptide epitope from a tachyzoite 67 kDa protein. This parasite protein also binds irrelevant mouse IgG(1) as well as immunoglobulins from other species. The reactivity with non-specific antibodies was inhibited by preincubation with 2% normal mouse and goat serum, while the reaction with the monoclonal antibody 3A11/H12 was not. Furthermore, a biotinylated F(ab')(2) of an irrelevant mouse IgG(1) did not show any reactivity while the F(ab')(2) of the monoclonal antibody 3A11/H12 reacts specifically with the 67 kDa antigen suggesting that this circulating antigen is a putative Fc binding protein.     

A murine–human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro

Vascular endothelial growth factor receptor 2 (VEGFR2) has been reported to play an important role in angiogenesis and tumorigenesis. A murine anti-VEGFR2 mAb (A8H1) has been established in a previous study. To reduce the incompatibility of the murine mAb for human use, the chimeric anti-VEGFR2-IgG was developed by genetic recombination of the variable regions of the A8H1 antibody and the constant regions of human IgG, and was expressed in Sp2/0 cells transfected with the two recombinant vectors containing the heavy chain and the light chain regions. After screening, clone 2F12 was selected and was found to stably secrete the murine–human chimeric anti-VEGFR2-IgG (coded 2F12). This chimeric IgG maintained the specificity and the affinity of the parental murine antibody against VEGFR2, and effectively identified VEGFR2 expressed on the surface of HUVECs and BEL-7402 cells. Furthermore, the 2F12 antibody demonstrated inhibition of angiogenesis in vitro, such as proliferation, migration, invasion and tube formation of HUVECs. This murine–human chimeric IgG may be considered for further development as an anti-angiogenesis and anti-tumor agent.     

An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection.

A human mAb (HmAb) termed F105 was obtained by fusion of antibody-producing EBV-transformed cells with the HMMA2.11TG/O cell line. F105 is an IgG1 kappa antibody that binds to the surfaces of cells infected with all HIV-1 strains tested: MN, RF, IIIB, and SF2, but not uninfected cells. The HmAb immunoprecipitates GP120 from all four strains. F105 does not react with denatured GP120 on Western blots, but does react with viral lysates and purified GP120 dotted onto nitrocellulose filter paper under nondenaturing conditions. rGP120 from SF2 and soluble rCD4 inhibit antibody binding to infected cells in a dose-dependent manner. F105 inhibits the binding of free, infectious virions to uninfected HT-H9 cells with 50% of maximal (100%) inhibition at approximately 1 microgram/ml. F105 inhibits infection of HT-H9 cells by 100 tissue culture infective dose 50% units of MN and IIIB strains with 50% inhibition at concentrations of HmAb readily achievable in man. It appears that the F105 HmAb reacts with a conformationally defined epitope on HIV-1/GP120 that is exposed on the free virion and is important for binding to the cell surface by the virion. The epitope, which is immunogenic in humans, appears to be within, or topographically near, the CD4-binding site. F105 and the F105 epitope are potentially useful in therapy and in the design of peptide or anti-Id based vaccines; monitoring of the expression of the Id may prove useful in evaluating immune responses in infected individuals or vaccinated volunteers.     

Fliv-1: a surface membrane differentiation antigen of fetal rat hepatocytes

A monoclonal antibody of IgG1 subclass was raised against 15 day fetal hepatocytes. The antibody appears to recognize a surface membrane-associated fetal hepatocyte antigen. Preliminary experiments suggest that the antibody recognizes a protein epitope. The tissue distribution of the antigen, designated Fliv-1, was studied by immunoperoxidase staining on whole body sections from fetal rats of different gestational ages. In 15 day fetal rats the antigen was detected on Rathke's pouch, brain infundibulum, tongue, liver, pancreatic acini, adrenal gland, vertebral myotome, and some developing muscle tissue. All normal adult tissues that were examined, two rat hepatoma cell lines, and hepatomas induced in rats by a single injection of diethylnitrosamine, were negative for the Fliv-1 antigen. On the basis of its tissue distribution, it seems unlikely that Fliv-1 is a previously described fetal hepatocyte marker.     

Drosophila CTLA-2-like protein (D/CTLA-2) inhibits cysteine proteinase 1 (CP1), a cathepsin L-like enzyme

In this study, we present a propeptide-like cysteine proteinase inhibitor, Drosophila CTLA-2-like protein (D/CTLA-2), a CG10460 (crammer) gene product, with an amino acid sequence significantly similar to the proregion of Drosophila cysteine proteinase 1 (CP1). Recombinant D/CTLA-2, expressed in E. coli, strongly inhibited Bombyx cysteine proteinase (BCP) with a Ki value of 4.7 nM. It also inhibited cathepsins L and H with Ki values of 3.9 (human liver) and 0.43 (rabbit liver) nM, and 7.8 nM (human liver), respectively. Recombinant D/CTLA-2 exhibited low but significant inhibitory activities to cathepsin B with Ki values of 15 nM (human liver) and 110 nM (rat liver), but hardly inhibited papain. We attempted to purify cysteine proteinases inhibited by D/CTLA-2 from total bodies of adult Drosophila. Recombinant D/CTLA-2 significantly inhibited CP1 with a Ki value of 12 nM, indicating that CP1, a cognate enzyme of D/CTLA-2, is a target enzyme of the inhibitor in Drosophila cells. These results indicate that D/CTLA-2 is a selective inhibitor of cathepsin L-like cysteine proteinases similar to other propeptide-like cysteine proteinase inhibitors such as Bombyx cysteine proteinase inhibitors (BCPI) and cytotoxic T-lymphocyte antigen-2 (CTLA-2). D/CTLA-2 was expressed over the whole life cycle of Drosophila. Strong expression was observed in the garland cells and prothoracic gland in the late stages of embryonic development. These results suggest that D/CTLA-2, implicated in intra- and extra-cellular digestive processes, functions in these tissues by suppressing uncontrolled enzymatic activities of CP1.     

A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses

Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains.     

Immunolocalization of detergent-susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody

The A-type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA-2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA-2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T(1/2) whole cell lysates and nuclear lysates. The epitope for LA-2F9 was narrowed down to amino acid residues 268-278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell-cycle-dependent, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) phase of the cell-cycle. The intensity of staining increased 3.5-fold within 6 h of refeeding, when the maximum number of cells were labeled with LA-2F9. We also checked whether the LA-2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC-35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA-2F9 has a novel and specific reactivity towards detergent-susceptible lower order lamin structures that are likely to be assembly intermediates.     

Appearance of neoantigen in mouse IgG1 upon reduction of interchain disulfide bridges: assessment of local and general conformational rearrangements using monoclonal antibody and small-angle X-ray scattering

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)2 fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between CH and CL domains. The second produced monAb F6C2 reacts with CH1-CL region of reduced mouse IgG2. Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.     

Characterization of a conserved apoptotic marker expressed in Caenorhabditis elegans phagocytic cells

We have identified and characterized a monoclonal antibody, F2-P3E3, that recognizes a Caenorhabditis elegans apoptotic epitope expressed within phagocytic cells, which is conserved in four other nematode species. In C. elegans, F2-P3E3 staining requires both programmed cell death and phagocytosis. We show that the F2-P3E3 epitope is expressed within embryonic intestinal cells, which act as phagocytes but do not undergo programmed cell death. F2-P3E3 staining is present within LMP-1::GFP labeled organelles in the intestinal primordium and is coincident with persistent DNA that has been phagocytosed in nuc-1(-) embryos, suggesting that it labels phagosomes. While apoptotic events are typically isolated in C. elegans, F2-P3E3 staining is commonly found within adjacent cells. This observation suggests that F2-P3E3 might recognize an epitope expressed in multiple cells in response to signals from a single corpse. F2-P3E3 represents a new tool for studying cell death in C. elegans.     

Subcellular Distribution of Distinct Nucleolin Subfractions Recognized by Two Monoclonal Antibodies

Monoclonal antibodies binding to different domains of nucleolin have been used to localize nucleolin in tissue culture cells ofXenopus laevis.The monoclonal antibody b6-6E7 binds to an epitope in the N-terminal domain, which contains arrays of phosphorylation consensus sites. This monoclonal antibody binds to nucleolin of oocytes and of eggs with high affinity. In contrast, the monoclonal antibody Nu-1H6 binds poorly to the modified forms of nucleolin arising during meiosis and mitosis. In interphase cells, monoclonal antibody b6-6E7 preferentially stains the periphery of the nucleoli, where most of the rRNA accumulates. Staining by monoclonal antibody Nu-1H6 complements this pattern by staining mainly the center of the nucleoli. The epitope of monoclonal antibody Nu-1H6 is within the central domain of nucleolin, which contains the first two RNA binding domains. RNase treatment of cells results in loss of nucleolin from nucleoli. In mitotic cells, both monoclonal antibodies decorate the surface of condensing chromosomes in prophase. The periphery of the condensed chromosomes in metaphase and anaphase is preferentially stained by monoclonal antibody b6-6E7.     

Crystal structure of the complex between the F(ab)' fragment of the cross-neutralizing anti-HIV-1 antibody 2F5 and the F(ab) fragment of its anti-idiotypic antibody 3H6

The monoclonal antibody 2F5 neutralizes a broad range of human immunodeficiency virus-1 isolates via a conserved epitope on the viral glycoprotein gp41. The conformation of the principal epitope is a type I β-turn centered on gp41 residues ⁶⁶⁴DKW⁶⁶⁶; in addition, binding studies indicate that residues N- and C-terminal to this core as well as structurally more distant parts of gp41 also contribute to the interaction. Ab2/3H6 is an anti-idiotypic antibody that inhibits the interaction between 2F5 and gp41 and as such, Ab2/3H6 may, in principle, possess a paratope that mimics the gp41 epitope. To establish the potential of Ab2/3H6 to serve as a guide for the design of vaccine components against human immunodeficiency virus, we investigated the crystal structure of the heterodimeric complex of Ab2/3H6 F_ab and 2F5 F_ab′. Ab2/3H6 F_ab binds to 2F5 F_ab′ via a helix-like protrusion formed by residues ^58(H)RYSPSLNTRL^67(H) of the 2F5 F_ab′ variable domain and proximal to but not overlapping with the gp41 ⁶⁶⁴DKW⁶⁶⁶ epitope-binding pocket. This defines Ab2/3H6 as an anti-idiotypic antibody of the Ab2γ class, i.e., an antigen-inhibitable idiotype that does not carry the internal image of the linear primary gp41 ⁶⁶²ELDKWAS⁶⁶⁸ epitope.     

Expression, Purification, and In Vivo Administration of a Promising Anti-Idiotypic HIV-1 Vaccine

To date only a few neutralizing antibodies against HIV-1 exist. Since these neutralizing antibodies are only rarely found in sera of HIV-1 infected individuals an active vaccine is required. We recently developed murine anti-idiotypic antibody Ab2/3H6 against monoclonal antibody (mAb) 2F5, which is one of the most prominent neutralizing antibodies. Anti-idiotypic antibody Ab2/3H6 has been partially humanized and expressed as whole immunoglobulin G in Chinese hamster ovary cells in order to minimize the human anti-mouse antibody response. Here we describe the expression, purification, and immunohistochemical characterization of the chimeric Ab2/3H6 Fab fragment, which was finally used beside the whole IgG1 as an antigen for immunization of guinea pigs. The crude sera were screened for specific antibodies against the epitope of mAb 2F5 ELDKWA as well as for reactivity against HIV-1 gp41.     

Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E

Summary A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 µg. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1—Tc1. The cytotoxic cell population was more than 90% CD4⁺. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.     

Role of the transferrin receptor in lymphocyte growth: a rat IgG monoclonal antibody against the murine transferrin receptor produces highly selective inhibition of T and B cell activation protocols

We have produced a new rat IgG monoclonal antibody against the murine transferrin receptor (TR). This antibody (C2F2) exhibits a surprisingly selective pattern of inhibition of murine lymphocyte activation protocols. C2F2 inhibits the mixed lymphocyte reaction (MLR) and the generation of cytotoxic T cells. Interestingly, although interleukin 1 (IL 1)-dependent thymocyte co-stimulatory activity is strongly inhibited by C2F2, interleukin 2 (IL 2)-dependent thymocyte co-stimulation is only marginally reduced. IL 2-dependent growth of CTLL cells is also not inhibited by C2F2. These data suggest that IL 1-dependent helper T cell activation is very sensitive to C2F2-mediated inhibition. Studies with phytohemagglutinin, Concanavalin A, and lipopolysaccharide induced activation also indicate that the inhibitory effects of C2F2 are selective, and T cell activation may be more sensitive to inhibition than B cell activation. Although there is little published information about the functional effects of other rat anti-mouse TR antibodies, the available data suggest that the patterns of inhibition produced by anti-TR antibodies may be individually distinct. Anti-TR antibodies may constitute a new set of highly selective probes for the study of lymphocyte activation.     

Appearance of Neoantigen in Mouse IgG1 Upon Reduction of Interchain Disulfide Bridges: Assessment of Local and General Conformational Rearrangements Using Monoclonal Antibody and Small-angle X-ray Scattering

Abstract

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)₂ fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between C_H and C_L domains. The second produced monAb F6C2 reacts with C_H1-C_L region of reduced mouse IgG2.

Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.     

A Cytotoxic Antibody Recognizing Lacto-N-fucopentaose I (LNFP I) on Human Induced Pluripotent Stem (hiPS) Cells

We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MSⁿ analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc). A critical role of the terminal Fucα1–2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1–2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis.