Generation of monoclonal antibodies against human prion proteins in PrP0/0 mice.

BACKGROUND: Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available. MATERIALS AND METHODS: We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared. RESULTS: Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. CONCLUSIONS: These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.     

Generation of antibodies against membrane proteins

The monoclonal antibody has become an important therapeutic in the treatment of both hematological malignancies and solid tumors. The recent success of antibody-drug conjugates (ADCs) has broadened the extent of the potential target molecules in cancer immunotherapy. As a result, even molecules of low abundance have become targets for cytotoxic reagents.     

Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen.

Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells. The proteins were different from each other, PAb419 reacting with a 35,000-molecular-weight protein, PAb427 reacting with a 75,000-molecular-weight phosphoprotein, PAb405 reacting with a 150,000-molecular-weight phosphoprotein, and PAb204 reacting with a 68,000-molecular-weight protein. It is suggested that although some of these cross-reactions may be fortuitous, they may, as an alternative, reflect similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

Generation of monoclonal antibodies directed against organ-specific endothelial cell surface determinants.

Organ-specific determinants expressed on the luminal surface of vascular endothelia are often unstable when cells are removed from their normal tissue environment and grown in culture. Unspecific endothelial cells of large vessel origin [e.g., bovine aorta (BAEC)] can be modulated to express and preserve such determinants when they are grown on the extracellular matrix of the desired organ. Lung matrix-modulated BAEC were used here to generate MAb against lung-specific vascular endothelia. Immunization was accomplished with outside-out membrane vesicles obtained by incubating BAEC monolayers grown on lung matrix with a low-strength paraformaldehyde solution. In four of the six fusions performed, this active immunization was preceded by passive immunization with mouse antiserum directed against membrane vesicles from BAEC grown on plastic. Among the growing hybrids, 7.6% secreted MAb that bound efficiently to both BAEC grown on lung-derived matrix and BAEC grown on plastic, while 3.5% (50) secreted MAb that bound primarily to BAEC grown on lung matrix. The fusion data show that only a passive/active immunization protocol yielded MAb directed against lung-specific endothelia. For example, MAb 6D3 and 5F5 selectively recognized endothelia from small- and medium-sized venules of bovine lungs, but failed to react with endothelial cells in other organs and tissues.     

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.     

Three monoclonal antibodies against measles virus F protein cross-react with cellular stress proteins.

In a group of 11 monoclonal antibodies specifically reacting with the measles virus fusion protein, three antibodies also immunoprecipitated other proteins, in particular a 79,000-molecular-weight protein from virus-infected cells. The cross-reacting 79,000-molecular-weight protein was shown to be a virus-induced host stress protein. This protein could be induced by (i) different paramyxoviruses, (ii) heat shock of uninfected HeLa cells, and (iii) 2-deoxyglucose, tunicamycin, or L-canavanine treatment of different mammalian cell lines. Immunofluorescence of stressed HeLa cells localized the cross-reacting host protein(s) mainly in the cytoplasm. The significance of these results in relation to autoimmunity is discussed.     

抗鸡白细胞介素4单克隆抗体的制备及鉴定

为了制备鸡白细胞介素4(chIL-4)单克隆抗体,将成熟的chIL-4基因亚克隆至原核表达载体pET-28a和pGEX-6P-1上,然后在大肠杆菌中分别诱导重组蛋白His-chIL-4和GST-chIL-4的表达,并纯化。将纯化后的His-chIL-4作为免疫原免疫BALB/c小鼠,经4次免疫后,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合。将纯化后的GST-chIL-4作为筛选抗原,利用间接ELISA筛选阳性克隆。阳性细胞株经3次亚克隆后,获得3株稳定分泌抗chIL-4蛋白的杂交瘤细胞株,分别命名为1G11-3B、2E5-3D和1G11-5H。经ELISA检测,3株单克隆抗体的亚型均为IgG1,亲和力解离常数(Kd)分别为1.79×10~(–9)、1.61×10~(–9)和2.36×10~(–9)。经Western blotting及间接免疫荧光试验鉴定,3株单克隆抗体均能特异性识别原核和真核表达的chIL-4蛋白。Western blotting试验证明1G11-3B、2E5-3D和1G11-5H识别的抗原表位区域分别为chIL-4蛋白N端的第1–40、80–112和40–80位氨基酸。该单克隆抗体的制备为chIL-4的检测和生物学功能研究奠定了基础。     

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.     

Passive immunization against murine malaria with an IgG3 monoclonal antibody

Spleen cells of BALB/c mice that were immune to the 17X strain of P. yoelii were fused with P3X63Ag8 myeloma cells. Two hundred fifty-three of 1053 hybrid cells produced antibodies reactive with disrupted 17X parasites in a solid phase radioimmunoassay. One of these antibodies, McAb 302, reacted with the merozoites of the 17X (nonlethal) and 17XL (lethal) variants of P. yoelii. Of greater significance, McAb 302 passively protected mice against challenge infection with the lethal variant. Mice treated with this antibody before infection developed low-grade parasitemia (less than 0.3%) of short duration when challenged with P. yoelii 17XL . In contrast, control mice that had been untreated or injected with ascites fluid lacking McAb 302 uniformly died with fulminating malaria upon challenge with the same parasite. In other experiments, McAb 302 was shown capable of controlling blood parasite levels when administered to mice with patent P. yoelii 17XL infections. Although all control mice died, mice protected with a single dose of McAb 302 ultimately cleared their infections. Regardless of how passive immunization was performed, mice given McAb 302 were resistant to subsequent challenge with P. yoelii 17XL , indicating they had developed significant immunity during their initial controlled infections. McAb 302 also showed pronounced passive protective activity against the nonlethal 17X strain of P. yoelii, which is a parasite of reticulocytes. The protection afforded by McAb 302 was specific, because mice passively immunized with this antibody died when challenged with the unrelated P. vinckei. McAb 302 was shown to possess the IgG3 isotype and precipitated a 230-kd protein plus several smaller polypeptides from metabolically labeled parasite antigen preparation derived from both variants of P. yoelii. It did not react with similar preparations of other murine plasmodial species.     

Generation of recombinant antibodies against orchardgrass Acidic nsLTP-like proteins

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non-embryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naive phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.     

Establishment of bovine prion peptide-based monoclonal antibodies for identifying bovine prion

To obtain high titer monoclonal antibodies (McAbs) which can react with mammalian prion protein (PrP), Balb/C mice were immunized with bovine (Bo) PrP peptide (BoPrP 209—228 aa) coupled to keyhole limpet hemocyanin (KLH). The hybridoma cell lines secreting monoclonal antibodies against the pep-tide were established by cell fusion and cloning. The obtained McAbs were applied to detect recombi-nant human, bovine and hamster PrP, cellular prion protein (PrP^c) in normal bovine brain and patho-genic scrapie prion protein (PrP^Sc) accumulated in the medulla oblongata of bovine spongiform en-cephalopathy(BSE)specimen with Western blot and immunohistochemical detection, respectively. The current procedure might offer a simple, feasible method to raise high titer antibodies for studying bio-logical features of PrP in mammals, as well as detection of transmissible spongiform encephalopathy (TSE) and diagnosis of BSE, in particular.     

Generation and characterization of chicken monoclonal antibodies against human LOX-1

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor for oxidized LDL (oxLDL), and plays a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. Monoclonal antibodies (mAbs) specific for human LOX-1 (hLOX-1) were generated by a phage display technique using chickens immunized with recombinant hLOX-1 (rhLOX-1). A total of 53 independent scFv clones reactive for rhLOX-1 were obtained. Of the 53 clones, 49 recognized the C-type lectin-like domain (CTL domain), which contributes to the binding of oxLDL. Of these, 45 clones inhibited oxLDL-binding with LOX-1. Furthermore, some of these clones cross-reacted with rabbit, pig and/or mouse LOX-1. For possible application as therapeutic agents in the future, two cross-reactive mAbs were re-constructed as chicken-human chimeric antibodies. The chimeric antibodies showed similar characteristics compared to the original antibodies, and inhibited oxLDL binding to LOX-1 expressed on CHO cells. The results obtained in this study indicate that anti-LOX-1 mAbs might be useful tools for functional analyses and development of therapeutic agents for cardiovascular indications such as atherosclerosis.     

Generation of neutralizing monoclonal antibodies against Enterovirus 71 using synthetic peptides

Enterovirus 71 (EV71) has led to recent outbreaks of hand, foot and mouth disease (HFMD) in China, resulting in high mortality. In this study, several monoclonal antibodies were generated by immunizing mice with two synthetic peptides, SP55 and SP70, containing amino acids 163-177 and 208-222 of VP1. The specificities of the anti-EV71 peptide monoclonal antibodies were confirmed by Western blot analysis and immunocytochemistry against EV71 virus. Most importantly, we have identified a monoclonal antibody, clone 22A12, which shows strong neutralizing activity against EV71 in an in vitro neutralization assay. Because there is no vaccine available and treatment is very limited, mouse anti-EV71 monoclonal antibody, clone 22A12, could be a promising candidate to be humanized and used for treatment of EV71 infection.     

Mapping nucleolar proteins with monoclonal antibodies

Using monoclonal antibodies as probes, we have characterized three antigens with respect to localization in the nucleolus, molecular weight and solubility. Two proteins, of 110,000 and 94,000 apparent molecular weight, were found associated with the ribonucleoprotein fibers. A third protein, with a molecular weight of 40,000, was accumulated at the nucleolar periphery, was present in the nucleoplasm, and may be involved in pre-ribosome maturation and transport.     

Generation and characterization of chicken monoclonal antibodies against human LOX-1

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor for oxidized LDL (oxLDL), and plays a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. Monoclonal antibodies (mAbs) specific for human LOX-1 (hLOX-1) were generated by a phage display technique using chickens immunized with recombinant hLOX-1 (rhLOX-1). A total of 53 independent scFv clones reactive for rhLOX-1 were obtained. Of the 53 clones, 49 recognized the C-type lectin-like domain (CTL domain), which contributes to the binding of oxLDL. Of these, 45 clones inhibited oxLDL-binding with LOX-1. Furthermore, some of these clones cross-reacted with rabbit, pig and/or mouse LOX-1. For possible application as therapeutic agents in the future, two cross-reactive mAbs were re-constructed as chicken-human chimeric antibodies. The chimeric antibodies showed similar characteristics compared to the original antibodies, and inhibited oxLDL binding to LOX-1 expressed on CHO cells. The results obtained in this study indicate that anti-LOX-1 mAbs might be useful tools for functional analyses and development of therapeutic agents for cardiovascular indications such as atherosclerosis.Key words: LOX-1, oxLDL, chicken monoclonal antibody, chimeric antibody, neutralizing antibody     

Protection against Streptococcus equi infection by monoclonal antibodies against an M-like protein.

We have developed an in vivo passive transfer assay using mice to identify monoclonal antibodies (mAbs) which offer protection against Streptococcus equi infection. The assay was developed using serum antibodies collected from horses convalescing from strangles. In this study, we show that a preparation of M-like protein, acid-extracted from S. equi, affords 80% protection to mice immunized with it. A number of mouse mAbs directed against a preparation of M-like protein were then assessed for their ability to passively protect mice against challenge with a lethal dose of the bacteria. Two mAbs, 1D10 and 2A6, were shown to be highly protective. It was also demonstrated, by means of a competitive enzyme immunoassay, that these mAbs recognized different epitopes in the preparation. Examination of a dose-response curve for mAbs 1D10 and 2A6 revealed that optimal levels of protection were achieved using 1 mg of either 1D10 or 2A6, or 0.5 mg 1D10 and 0.5 mg 2A6 given together. Immunological reactivity of these mAbs with a preparation of M-like protein showed that the antigens they recognized were comparable in size to some of the antigens recognized by convalescent horse serum antibodies. The role of immunoglobulin isotype in conferring protection is discussed.     

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.