Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.     

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.     

Immunoprotective human monoclonal antibodies against five major serotypes of Pseudomonas aeruginosa

Human monoclonal antibodies (Mabs) against the O antigens of Pseudomonas aeruginosa lipopolysaccharides (LPS) were produced by cell fusion between human tonsillar lymphocytes and P3-X63-Ag8-U1 (P3U1) mouse myeloma cells. To obtain human Mabs efficiently, 6 d culture supernatants of pokeweed-mitogen-stimulated lymphocytes (21 cultures from peripheral blood and 76 from tonsils) were assayed by ELISA. Five tonsillar lymphocytes which produced IgG antibody specific for P. aeruginosa LPS were preselected for fusion. The human Mabs, named P1-1 (IgG2, kappa), P5-1 (IgG2, lambda), P7-1 (IgG2, lambda), P8-1 (IgG2, lambda) and P10-1 (IgG2, kappa), bound with high specificity to Homma standard serotype strains A, E, B, G and I, respectively, and recognized O antigens. Each Mab showed opsonophagocytic killing activity of the corresponding serotype strain. Four of the Mabs caused agglutination at a very low concentration; a rather higher concentration of P7-1 was required for this effect. Although all the Mabs conferred type-specific protection against peritoneal infection, the strongly agglutinating Mabs provided better protection than the moderately agglutinating P7-1. The protective activity of P8-1 was estimated in compromised mice. A low dose (PD50 0.5-0.6 microgram per mouse) of P8-1 prevented subcutaneous infection in burned mice and peritoneal infection in leucopenic mice. All the hybridomas described here could be cultured in serum-free medium, and they have continued to secrete human Mabs for more than 14 months at rates of 10-20 micrograms per 10(6) cells in 24 h. These results suggested that these five human Mabs specific for O antigens might be useful in the prophylaxis and treatment of P. aeruginosa infections.     

Neutralizing monoclonal antibodies to Theiler''s murine encephalomyelitis viruses.

Theiler's murine encephalomyelitis viruses (TMEV) are serologically related picornaviruses which cause both enteric and neurological disease in mice. The biological activities of TMEV vary between the two different TMEV subgroups (TO and GDVII) and with different passage histories of the same TMEV strain (e.g., mouse brain-passed versus tissue culture-passed DA strain of the TO subgroup). We raised neutralizing monoclonal antibodies (mAbs) against tissue culture-passed DA and GDVII strains of TMEV. We produced two mAbs against the DA strain which neutralized all members of the TO subgroup, but not the GDVII subgroup strains (GDVII and FA); these two DA mAbs reacted similarly with both mouse brain-passed DA and tissue culture-passed DA. Of six neutralizing GDVII mAbs, four reacted only to GDVII and FA, whereas two neutralized TO strains as well. These mAbs demonstrate the presence of TMEV group-specific as well as subgroup-specific neutralization and substantiate the division of TMEV into two distinct subgroups. On Western immunoblots one of the two DA mAbs reacted against isolated DA VP1, two GDVII mAbs (which were TMEV group specific) reacted against isolated GDVII VP1 and DA VP1, and the other DA mAb and four other GDVII mAbs required an intact virion conformation for reactivity. An analysis of the epitopes recognized by these mAbs may elucidate sites important in TMEV biological activities.     

Cell-protective monoclonal antibodies to bovine enterovirus-3 and partial or no activity against other serotypes.

Preparation of monoclonal antibodies to bovine virus diarrhea virus (BVDV) yielded some hybridoma cells that secreted monoclonal antibodies against the Madin-Darby bovine kidney cells. The anti-cellular monoclonal antibodies reacted with other bovine cells (bovine turbinate and testicle) but not with cell lines derived from other animal species. Subclones derived from one hybridoma partially blocked the infectivity of BVDV, possibly through the binding of the monoclonal antibodies with an epitope close to the receptor site of BVDV and not by way of steric hindrance. Unexpectedly, these same subclones completely blocked the infectivity of bovine enterovirus-3 (BEV-3) strain 240A and partially blocked the infectivity of BEV-2 and BEV-3 (ATCC strain) but not that of other serotypes. Other subclones derived from two other hybridomas, although cell membrane specific, did not have a protective activity against BEV or BVDV.     

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.     

Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance.

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.     

Neutralizing monoclonal antibodies that distinguish three antigenic sites on human cytomegalovirus glycoprotein H have conformationally distinct binding sites.

Seven neutralizing murine monoclonal antibodies specific for the glycoprotein H of human cytomegalovirus were produced and used to construct a topological map of two nonoverlapping antigenic sites that are bridged by a third antigenic site. Neutralization assays with 15 laboratory or clinical human cytomegalovirus strains indicated that the monoclonal antibodies recognize three antigenically variable and three conserved epitopes within the three antigenic sites. The variable-domain genes encoding monoclonal antibodies representing each of the three antigenic sites were cloned and sequenced, and molecular models of their binding sites were generated. Conformational differences in the antibody-binding sites suggested a structural basis for experimentally observed differences in gH epitope recognition.     

Production and characterization of monoclonal antibodies against particulate guanylate cyclase in porcine kidney

Three monoclonal antibodies (Ig G1 type) to particulate guanylate cyclase from porcine kidney cortex have been produced by fusing spleen cells from immunized BALB/c mouse with P3X63 myeloma cells. The antibodies were detected by their ability to bind immobilized antigen and by immunoprecipitation of enzyme activity. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid. The antibodies recognized a 180,000 dalton protein in Lubrol-PX extract of porcine kidney cortex membrane, and when immobilized on Sepharose 4B, they co-precipitated both [125I]human atrial natriuretic peptide (ANP)-receptor complex and guanylate cyclase activity. The antibodies caused a greater increase in generation of cGMP than that of ANP.     

Neutralizing single-chain antibodies against the tick-borne encephalitis virus

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the scl3D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified scl3D6 was (3.0 ± 0.2) × 10⁷ M^?1 for the equilibrium state and (2.8 ± 0.3) × 10⁷ M^?1 in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC₅₀ value for scl3D6 was 16.7 μg/ml.     

Isolation of human monoclonal antibodies that neutralize human rotavirus

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones-1-2H, 2-3E, and 2-11G-were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 micro g/ml.     

Monoclonal anti-idiotype induces antibodies against bovine Q17 rotavirus.

This study describes, for the first time, the production and use of an "internal-image" anti-idiotypic monoclonal antibody (MAb) to elicit a rotavirus-specific antibody response. An immunoglobulin G2a MAb, designated RQ31 (MAb1), specific for the outer capsid protein VP4 of bovine Q17 rotavirus and capable of neutralizing viral infection in vitro was used to generate an anti-idiotypic MAb (MAb2). This MAb2, designated RQA2, was selected by enzyme-linked immunosorbent assay (ELISA) using F(ab')2 fragments of RQ31. RQA2 (MAb2) inhibited the binding of RQ31 (MAb1) to the virus but had no effect on the binding of other rotavirus-specific MAbs. The MAb2 also inhibited virus neutralization mediated by MAb1 in a dose-dependent fashion. Naive guinea pigs immunized with the MAb2 produced anti-anti-idiotypic antibodies (Ab3) that reacted with bovine Q17 rotavirus in an ELISA and neutralized rotavirus infection in vitro. The Ab3 response was characterized as MAb1-like because the Ab3 recognizes only the Q17 and neonatal calf diarrhea virus rotavirus strains in ELISA, as did RQ31 (MAb1). The Ab3 response also possessed two other characteristics of RQ31: the abilities to bind the 1.36 (double-capsid) but not the 1.38 (single-capsid) purified rotavirus fraction in ELISA and to immunoprecipitate the VP4 rotavirus protein.     

Properties of three monoclonal antibodies that recognize an 80-kDa phytohemagglutinin-binding glycoprotein from porcine lymphocytes

Monoclonal antibodies (mAbs) directed against porcine splenocyte phytohemagglutinin receptor glycoproteins were produced in BALB/c mice. Three antibody-producing, stable hybridomas were cloned and expanded in the peritoneal cavity of BALB/c mice. The mAbs (A7, B1, and H3) were purified and belong to the IgG2 subclass of immunoglobulins (kappa light chain). Each 125I-labeled mAb bound to purified porcine splenocytes with an (apparent) affinity KA congruent to 10(8) M-1 (Scatchard analysis). The number of (apparent) binding sites was 5 x 10(4) sites/cell in the case of B1 and H3, and approximately 15 x 10(4) sites/cell for A7. Immunoprecipitation experiments showed that the three mAbs recognized a single antigenic protein of Mr 80 kilodaltons (gp80). In addition, each mAb recognized a different epitope of gp80, as observed by Western blot analyses. Assessment of the relative ability of anti-gp80 mAbs to stimulate porcine splenocytes as determined by [3H]thymidine incorporation showed weak (A7 and B1) or no (H3) mitogenic activity. Cross-linked anti-gp80 mAbs were not mitogenic, except in the case of B1. In contrast, each anti-gp80 mAb (cross-linked or untreated) showed synergistic mitogenic properties when used in combination with a suboptimal concentration of phytohemagglutinin. The mechanism involved in this synergistic effect is discussed.     

幽门螺杆菌尿素酶单克隆抗体的研制及鉴定

应用杂交瘤技术 ,建立稳定分泌抗幽门螺杆菌尿素酶单克隆抗体 (HPU McAb)的细胞系。用纯化幽门螺杆菌尿素酶 (HPU)抗原加福氏佐剂免疫BALB/c小鼠 ,按常规方法进行细胞融合 ,以纯化HPU抗原包被 ,间接ELISA方法筛选 ,并经多次有限稀释法克隆。获得 6株抗HPU的杂交瘤 ,腹水效价达 1∶6 .4× 10 4～ 1∶2 .5 6× 10 5,特异性专一 ,并对其进行体内外连续传代 3个月 ,分泌抗体能力稳定。IgG亚类分型主要为IgG1型。该细胞系能稳定分泌幽门螺杆菌尿素酶单抗。     

Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity.     

Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.     

Neutralizing antibodies against endogenous interferon in myasthenia gravis patients

Anti-cytokine antibodies (Abs) play an important role in the regulation of the immune response, both under normal conditions and in several autoimmune and neoplastic disorders. In the present study, we have investigated the occurrence and the clinical significance of natural neutralizing Abs (NAbs) against interferons (IFNs) alpha, beta, and gamma, as detected by bioassay, in 52 patients with myasthenia gravis (MG), and 43 sex- and age-matched healthy individuals. Patients showing titres > or = 1.3 Log t(1/10), confirmed in 2 consecutive samples collected two months apart, were considered positive. NAbs against any of the IFNs were not detected in healthy subjects. Of the 52 MG patients, 11 (21.1%) had NAbs against IFNalpha and three (5.8%) had NAbs against IFNbeta. None of these patients was found to be positive for NAbs against IFNgamma. Of the patients positive for NAbs against IFNalpha, eight (15.4%) had NAbs at titres > or =2 Log t(1/10). A positive association was observed between high titres of NAbs and the presence of thymoma. These data suggest the presence of a generalized activation of the humoral response in MG.     

Production of monoclonal antibodies against avidin

Monoclonal antibodies of the IgG1 subclass were generated against chicken avidin. These antibodies were shown to be as sensitive as polyclonal antiserum in detecting avidin by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) methods. Furthermore, the monoclonal antibodies were considerably more specific. Our results with a monoclonal anti-avidin RIA support previous findings that in inflammatory conditions avidin is synthesized also in other organs than the oviduct, although in the liver a major part of the activity detected by polyclonal anti-avidin RIA or biotin-bentonite assay was not due to avidin.