Characterization of mouse-hamster-human cross-reacting antioocyte monoclonal antibodies produced by intrasplenic immunization of mice with 12 zona-free mouse oocytes

Several intrasplenic immunizations with batches of ～15 or ～30 zona-free, unfertilized mouse oocytes resulted in 200–300 hybrids, respectively, among which about 20 positive clones were selected from each fusion between splenic plasma cells and SP2/0 myeloma cells. When nonimmunized splenic plasma cells were used, only one antibody, showing weak immunoreaction, was obtained from ～370 hybrids collected from 2 fusions. From one immunization with a total of 12 zona-free, unfertilized mouse oocytes, 15 positive clones were selected for further study. Eleven of these 15 antibodies reacted with antigens only in unfertilized oocytes but not in fertilized, pronuclear stage oocytes. Three antibodies, which recognized antigens in paraffin-embedded oocyte sections, did not label growing ovarian oocytes, indicating that the antibodies were specific to ovulated, unfertilized oocytes. These antibodies did not detect any antigen epitopes in the panel of tissues examined. The molecular weight of one antigen, corresponding to a IgM antibody that is present both in ooplasma and zona pellucida, was ～116 kDa. Cross-reactivity to blots of unfertilized zona-free hamster oocytes was demonstrated by 6 antibodies and to unfertilized human oocytes by 7 antibodies. Three antibodies cross-reacted with both hamster and human oocytes. The study indicates that the intrasplenic immunization is an appropriate means of raising antibodies against unfertilized, zona-free mouse oocytes and that the method applied offers an easy way to select antibodies against human oocytes for functional studies. © 1994 Wiley-Liss, Inc.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Protective monoclonal antibody against Schistosoma mansoni: antigen isolation, characterization, and suitability for active immunization

Monoclonal antibodies that bind to the surface of developing schistosomula were generated from the spleens of chronically infected mice that were boosted with cercarial glycoproteins. The two most reactive monoclonal antibodies, denoted 152-66-9B and 152-66-1C, were used for identification of surface antigens. The antigen detected by these monoclonal antibodies persisted on the surface of the developing larva for 72 hr posttransformation. This monoclonal antibody effected complement-mediated killing of schistosomula in vitro as efficiently as infected mouse sera. It was also very efficient in inhibiting the infectivity of both cercariae and schistosomula. The antigen reactive with the 152-66-9B monoclonal antibody contains two major polypeptides (45 and 30 KD). These polypeptide chains might have originated from the same protein, because they have the same isoelectric point in two-dimensional gel electrophoresis. Moreover, the affinity-purified antigen migrated as only one protein band of approximately 200 KD in SDS-PAGE in nonreducing conditions. The 9B antigen was isolated, purified, and used for immunization, resulting in an antigen dose-dependent partial protection against S. mansoni infection.     

Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies

Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied. Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol. The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable. Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature. Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes.     

Built-in RNA-mediated chaperone (chaperna) for antigen folding tailored to immunized hosts

High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the “self” RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation “universal” influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.     

Murine red blood cells as efficient carriers of three bacterial antigens for the production of specific and neutralizing antibodies.

Three bacterial toxoids, CRM 197 (mutagenized diphtheria toxin), tetanus toxoid (formaldehyde-treated tetanus toxin), and PT-9K/129G (double mutant of pertussin toxin) were encapsulated within red blood cells (RBCs) of B6D2F1 and Balb/C mice according to a mild procedure based on hypotonic dialysis-isotonic resealing that yielded undamaged RBCs. The toxoid-loaded RBCs were injected intravenously in order to immunize animals and their effects were compared to those of identical amounts (30-95 micrograms per mouse subdivided into multiple injections) of the corresponding free toxoids injected intravenously in saline. Sera from treated mice were collected and tested for titers of specific antibodies against each of the three antigens and also for titers of neutralizing antibodies, i.e., affording protection from toxic effects induced by the corresponding native toxins. In all experiments, significant seroconversion was observed with both immunization systems. Titers of both specific and neutralizing antibodies against CRM 197 and tetanus toxoid were several-fold higher upon immunization with the RBC-encapsulated toxoids, than with the free toxoids. These differences were not due to qualitatively different recognition patterns of antigenic determinants by the two types of sera. Conversely, intravenous immunization with pertussis toxoid either as RBC-encapsulated or as free antigen elicited a comparably high production of specific and of neutralizing antibodies. These data demonstrate that properly engineered RBCs behave as natural carriers and possibly adjuvants for antigens of vaccinal interest.     

Generation of hybridomas producing human monoclonal antibodies against human cytomegalovirus

In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens.     

Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: effects on fertilization and early development

During development and maturation, mammalian oocytes are surrounded by the zona pellucida which in the mouse is comprised of three sulfated glycoproteins, ZP-1, ZP-2, and ZP-3. Previously, monoclonal antibodies to ZP-2 have been isolated. The isolation and characterization of monoclonal antibodies specific for ZP-3, the zona protein with sperm receptor activity are now reported. Following passive immunization, these monoclonal antibodies localize to the intraovarian zonae pellucidae and their presence precludes both in vivo and in vitro fertilization of subsequently ovulated eggs. Monoclonal antibodies specific for either ZP-2 or ZP-3 also completely block in vitro fertilization at relatively low concentration ranging from 0.4 to 75 micrograms/ml. The contraceptive effect requires the presence of the zona and appears to inhibit the penetration of the zona pellucida by sperm rather than by blocking the sperm binding site. Neither antibody interferes with in vitro development from the two-cell to the blastocyst stage or with subsequent hatching from the enveloping zona pellucida.     

Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells

Monoclonal antibodies against the highly conserved ubiquitous calcium- binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.     

A strategy for generating monoclonal antibodies against recombinant baculovirus-produced proteins: application to the Bcl-2 oncoprotein.

A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.     

Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens

Intranasal immunization with inactivated influenza virus vaccine can provide protective immunity, whereas many other antigens are less effective when used for mucosal immunization. To determine whether influenza virus could enhance immune responses to an antigen coadministered to a mucosal surface, we studied the intranasal immunization of mice with a mixture of simian-human immunodeficiency virus (SHIV) virus-like particles (VLPs) and inactivated influenza virus. Compared to mice immunized with SHIV VLPs alone, mice coimmunized with SHIV VLPs and inactivated influenza virus showed significant increases in serum immunoglobulin G (IgG) and mucosal IgA antibodies specific to the human immunodeficiency virus envelope protein, neutralizing activities, numbers of gamma interferon- and interleukin 4-secreting lymphocytes, and cytotoxic-T-lymphocyte activities. The levels of enhancement of immune response by coimmunization with inactivated influenza virus were equivalent to those induced by inclusion of immunostimulatory CpG oligodeoxynucleotides (CpG DNA). We also observed that SHIV VLPs bind to influenza virus virions, forming mixed aggregates. These results indicate that inactivated influenza virus can play a role as a mucosal adjuvant to coadministered antigens.     

Generation of a Human Anti-Tumor Necrosis Factor-α Monoclonal Antibody by in Vitro Immunization with a Multiple Antigen Peptide

We developed the in vitro immunization method to induce antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). However, when we used a peptide as sensitizing antigen, the antigen-specific immune response was found to be weak, and hence, we could not effectively obtain the antigen-specific antibody gene. In the present study, we attempted to improve the in vitro immunization method by augmenting the immune response to the peptide antigen. We used a multiple antigen peptide for sensitization. In vitro immunization of the multivalent antigen elicited a strong antigen-specific immune response in the PBMCs, and we succeeded in obtaining antigen-specific antibody genes by the phage-display method. Further, by combining the variable-region genes and constant-region genes of human IgG, we obtained four independent human monoclonal antibodies specific for tumor necrosis factor-α. This might be a good strategy for generating antigen-specific human monoclonal antibodies using a peptide antigen.     

Syngeneic monoclonal antimelanoma antibodies and their application for analysis of tumor antigens, gene cloning, and in vitro/in vivo diagnosis

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.     

Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

Development, Characterization, and Use of Monoclonal and Polyclonal Antibodies against the Myxosporean, Ceratomyxa shasta

Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.     

Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.     

Monoclonal antibodies to tissue-specific chromatin proteins

Antisera raised in mice to chromatins from different tissues of the chicken reacted preferentially with the chromatin type that was used for immunization. This tissue specificity was also evident in the spectrum of monoclonal antibodies generated when mice were immunized with erythrocyte chromatin. Three erythroid-specific antigens and one antigen that was present in a number of chicken tissues were characterized in further detail. These antigens, which comprised less than 0.1% of the erythrocyte chromatin proteins, were nuclear localized although three were also detected in the cytoplasm. Two of the erythroid-specific antigens existed as multiple polypeptides in isolated chromatin. The multiple chromatin forms of one antigen were derived from a precursor protein that was selectively cleaved within 1 min after erythrocyte lysis. Analysis of this antigen in extracts from erythrocytes and reticulocytes indicated that the cleavage of the precursor protein was developmentally regulated in vivo.     

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.     

Immunobiological characteristics of impurities in corpuscular influenza vaccines

Admixtures of free antigens have been shown to play the main role in the anaphylactogenic danger of vaccines. The immunogenic and anaphylactogenic action of such antigenic admixtures in corpuscular influenza vaccine can be observed after the immunization of animals in 2 or 3 injections. Host antigens incorporated into viral particles induce no anaphylactic reaction in guinea pigs after their immunization in 3 injections.     

IgG-mediated systemic anaphylaxis to protein antigen can be induced even under conditions of limited amounts of antibody and antigen

Systemic anaphylaxis is an acute, severe, and potentially fatal allergic reaction. Two classes of antibodies, IgE and IgG, contribute to the development of anaphylaxis in mice, through different mechanisms with distinct usage of effector cells and chemical mediators. Larger quantities of antibody and antigen are reportedly required to induce IgG-mediated anaphylaxis than IgE-mediated one, suggesting that the former may not happen as frequently as the latter in real life. To readdress this issue, we established in the present study a novel mouse model of passive IgG-mediated systemic anaphylaxis to a native protein antigen, ovalbumin (OVA), rather than artificially haptenated protein antigens used in previous studies. Passive sensitization of mice with a cocktail of but not individual IgG1 mAbs specific to distinct OVA epitopes elicited systemic anaphylaxis in response to OVA challenge. Importantly, much smaller doses of antibody and antigen than previously reported were sufficient for the induction of IgG-mediated systemic anaphylaxis. Moreover, a relatively small dose of antigen could induce severe anaphylaxis through both IgE- and IgG-mediated mechanisms when mice had been passively sensitized with antigen-specific IgE and IgG. These results strongly suggest that IgG-mediated systemic anaphylaxis is not rare among antibody-mediated systemic anaphylaxis, in contrast to previous thought, and significantly contributes to active systemic anaphylaxis in real life, at least in mice.