Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19.

In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site-specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies.     

Identification of polypeptide components of the Epstein-Barr virus early antigen complex with monoclonal antibodies.

Three monoclonal antibodies were produced against the Epstein-Barr virus-induced early antigen complex. These antibodies were shown to be specific for the early antigen complex by the fact that they only reacted with cells supporting a permissive or abortive Epstein-Barr virus infection and their synthesis was not affected by inhibitors of viral DNA synthesis. One monoclonal antibody, designated R3, was directed against a diffuse component of the early antigen complex since it reacted by immunofluorescence with cells fixed in acetone or methanol. The other two monoclonal antibodies, designated K8 and K9, reacted with a methanol-sensitive restricted component of this complex. The appearance of the R3 antigen in P3HR-1 superinfected Raji cells occurred approximately 4 h earlier than the antigen detected by K8. By both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmunoelectrophoresis, it was determined that the R3 monoclonal antibody recognized two major polypeptides with molecular weights of approximately 50,000 to 52,000, whereas K8 and K9 precipitated a protein of approximately 85,000. The R3 monoclonal antibody also immunoprecipitated an in vitro primary translation product. It was, therefore, possible to map this product to the Epstein-Barr virus DNA BamH1 M fragment. These in vitro products were slightly smaller than the in vivo proteins, suggesting that these proteins probably undergo posttranslational modification during the virus replication cycle.     

Identification of a 37 kDa plant protein that interacts with the turnip mosaic potyvirus capsid protein using anti-idiotypic-antibodies

Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV). The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies. A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies. The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced. These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa. Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein. The 37 kDa protein was localized in chloroplasts and was detected in other plant species.     

Analysis of three late varicella-zoster virus proteins, a 125,000-molecular-weight protein and gp1 and gp3

Two monoclonal antibodies were prepared against varicella-zoster virus proteins. One of the monoclonal antibodies (10.2) reacted only with the nuclei of infected cells and immunoprecipitated one nonglycosylated late viral protein (125,000 molecular weight). The other monoclonal antibody (19.1) with neutralizing activity, reacted with membrane antigens of infected cells and with the varicella-zoster virus envelope and immunoprecipitated two late major viral glycoproteins (gp1 and gp3). Synthesis of the 125,000-molecular-weight protein, gp1, and gp3 began at 20 to 22 h postinfection, 2 h after the peak of viral DNA synthesis, and continued until 29 h postinfection, when the first progeny virus appeared in infected cells. Pulse-chase experiments showed that during pulse-labeling, only gp1 was detected, whereas during the chase period, gp1 as well as gp3 was detected in infected cells. Under nonreducing conditions, gp3 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 130,000-molecular-weight protein as compared with the 62,000-molecular-weight species obtained when gels were resolved under reducing conditions. This finding indicates that gp3 is a dimer that is disulfide linked.     

Synthesis and Assembly of Simian Virus 40 II. Synthesis of the Major Capsid Protein and Its Incorporation into Viral Particles

African green monkey kidney cells infected by simian virus 40 were analyzed for the presence of the major capsid protein (capsid protein I) by immunological and radiolabeling techniques. Antisera with different specificities were prepared by immunization with intact or denatured viral particles. Antisera prepared against intact virus reacted by complement fixation with viral particles and with an 8S subunit containing the capsid protein I. Antisera prepared against denatured viral particles reacted with unassembled capsid protein(s) as well as with viral particles. These antisera were used to detect 8S viral subunits or unassembled viral capsid protein in soluble extracts of infected cells after centrifugation at 100,000 x g to remove viral particles. The soluble antigen pool was found to be small during infection with wild-type virus or a temperature-sensitive mutant deficient in the synthesis of viral particles. Pulse-chase experiments, performed at a high multiplicity of infection, also indicated a small pool of nonparticle capsid protein I. Radioactive lysine was incorporated into capsid protein I of virus particles during a 2-hr pulse. A subsequent chase with excess unlabeled lysine resulted in only a slight increase in the radio-activity found in capsid protein I of viral particles. Furthermore, in the same experiments, capsid protein I was incorporated preferentially into empty shells during the pulse with a shift in radioactivity to intact virions during the chase period, indicating a possible precursor relationship between the two types of virus particles.     

Production of monoclonal antibody to bovine leukemia virus glycoprotein gp51 by in vitro immunization

The monoclonal antibody against glycoprotein gp51 of bovine leukemia virus (BLV) envelope antigen was produced by in vitro immunization. This monoclonal antibody reacted with viral antigen was observed at the 69 kilodalton (kDa) glycoprotein. However, this monoclonal antibody was not involved in neutralizing. It was shown that in comparison with in vivo immunization, in vitro immunization has some advantages, namely a short immunization period and a small antigen quantity.     

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

Background

Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein.

Results

In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody.

Conclusion

The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.     

Characterization of a monoclonal antibody against active pectate lyase from Erwinia carotovora

A monoclonal antibody (2E2) produced against pectate lyase from Erwinia carotovora ssp. carotovora reacted with a 41- and a 44-kilodaltion protein on Western blots of concentrated Erwinia culture supernatants resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. It was unequivocally shown that monoclonal 2E2 reacted with an active form of pectate lyase by affinity purifying the antigen with the monoclonal. The affinity-purified antigen was enzymatically active and moved as a single protein band in a nonequilibrium isoelectric focusing gel. Monoclonal 2E2 reacted with the pectate lyases of a diverse range of E. carotovora ssp. carotovora, ssp. atroseptica, and ssp. betavasculorum strains, as well as with one of three strains of E. chrysanthemi. The electrophoretic mobility of the major protein (44 kilodaltons) that reacted with 2E2 was identical within a subspecies but differed among subspecies.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies

A systematic approach for the determination of epitope specificities of monoclonal antibodies to a complex antigen system is described. After initial screening to identify antigen-binding monoclonal antibodies, one or more of the clones are isolated by limiting dilution cloning, grown in ascites, and the resulting antibodies secreted into the ascitic fluid are affinity purified on Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice were immunized with either purified carcinoembryonic antigen (CEA) or the CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned, resulting in 33 different clones. The antibodies produced by the different cloned hybrids and the remaining uncloned hybrids recognized a total of five different epitopes on CEA. All of the epitopes reside on the protein moiety of the molecule as determined by antibody binding to deglycosylated CEA. The monoclonal antibodies with five different epitope specificities were reacted with tissue sections of normal and cancerous tissues and with peripheral blood smears. Each of the five monoclonal antibodies reacted with tissue sections from colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted with normal liver tissue. Granulocytes in peripheral blood smears bound three antibodies strongly and one antibody weakly, and one antibody was not bound. One monoclonal antibody that reacted with normal liver tissue was not bound by granulocytes. The ability of these five monoclonal antibodies to differentially detect three different CEA-related antigens in normal and malignant tissues may have clinical utility.     

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.     

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon

A monoclonal antibody specific to yellow head virus (YHV) was produced from a mouse immunized with gill extracts prepared from laboratory-reared Penaeus monodon dually infected with YHV and white spot syndrome virus (WSSV). One clone designated V3-2B specifically bound to native and SDS-treated viral specific antigens. Immunocytochemical studies of infected gills revealed viral specific immunoreactivities in the cytoplasm of gill tissue and in haemocytes. No antibody binding was observed in gills from non-infected shrimp. In addition, immunocytochemical examination of tissues from shrimp experimentally infected with YHV gave a positive reaction, while tissues from uninfected control shrimp or shrimp experimentally infected with WSSV did not. Western blot analysis indicated that the antibody reacted with a protein of approximately 135 kD that was present only in shrimp infected with YHV. In dot-blot indirect immunoperoxidase assays, the antibody was able to detect viral associated antigen in diluted haemolymph up to 1:50 dilution and in an ammonium sulfate precipitate of haemolymph up to 1:1000 dilution. The results suggested that this antibody might be useful for development of effective diagnostic techniques for both heavy and mild YHV infections in shrimp.     

Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with ionic character, and van der Waals contacts with antibody residues from the six hypervariable loops as well as several framework amino acids. The conformation adopted by the peptide is closely related to the corresponding region of the viral protein VP2 on the surface of human rhinovirus 1A whose three-dimensional structure is known. Implications for the cross-reactivity between peptides and the viral capsid are discussed. The peptide-antibody interactions, together with the analysis of mutant viruses that escape neutralization by 8F5 suggest two different mechanisms for viral escape. The comparison between the complexed and uncomplexed antibody structures shows important conformational rearrangements, especially in the hypervariable loops of the heavy chain. Thus, it constitutes a clear example of the 'induced fit' molecular recognition mechanism.     

Studies of mammalian ornithine decarboxylase using a monoclonal antibody.

A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.     

Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins.

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gt11-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-beta-galactosidase. Serum of a rabbit immunized with BFRF3-beta galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-beta-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens.     

Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus.

A series of monoclonal antibodies was isolated which reacted with one of two major surface proteins of rhesus rotavirus. Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin. These monoclonal antibodies exhibited hemagglutination inhibition activity and neutralized rhesus rotavirus to moderate or high titer. Three monoclonal antibodies immunoprecipitated the 38-kilodalton outer capsid glycoprotein, the eighth or ninth gene product. These three monoclonal antibodies neutralized rhesus rotavirus to high titer and also inhibited viral hemagglutination.     

Identification and characterization of a filament-associated protein encoded by Amsacta moorei entomopoxvirus.

A novel protein which is expressed at high levels in insect cells infected with Amsacta moorei entomopoxvirus was identified by our laboratory. This viral gene product migrates as a 25/27-kDa doublet when subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. It is expressed at late times of infection and is present in infected cells but is absent in purified extracellular virions and occlusion bodies. The gene encoding this polypeptide was mapped on the viral genome, and cDNA clones were generated and sequenced. The predicted protein was shown to be phosphorylated and contained an unusual 10-unit proline-glutamic acid repeat element. A polyclonal antiserum was produced against a recombinant form of the protein expressed in Escherichia coli, and a monoclonal antibody which reacted with the proline-glutamic acid motif was also identified. Immunofluorescence and immunoelectron microscopy techniques revealed that this protein is associated with large cytoplasmic fibrils which accumulate in the cytoplasm between 96 and 120 h postinfection. We subsequently called this viral polypeptide filament-associated late protein of entomopoxvirus. The fibrils containing this polypeptide are closely associated with occlusion bodies and may play a role in their morphogenesis and maturation.     

猪圆环病毒2型(PCV2)核衣壳蛋白免疫原性的鉴定和应用及其多抗在PCV2感染诊断中的应用

猪圆环病毒2型ORF2编码与病毒毒力相关的结构蛋白--核衣壳蛋白(Cap),该蛋白可以用于PCV2感染的血清学调查,但不同区域的PCV2分离株的ORF2特别是其抗原表位序列存在一定的突变.本研究将PCV2浙江分离株ORF2的主要抗原表位以及PCV1 ORF2进行了原核表达,将分别纯化的融合蛋白Cap2s和Cap1s免疫SPF兔后制备多抗,并进一步分析了纯化蛋白的免疫原性和多抗的特性.Western blot结果表明无论Cap2s和Cap1s均能与两个多抗发生交叉反应,而PCV2或PCV1阳性猪血清只能分别特异性地识别Cap2s和Cap1s.IFA结果则证明两个多抗对于天然Cap蛋白无交叉反应性.利用Cap2s作为包被抗原对13个猪场的259份血清样品的PCV2抗体进行ELISA检测,平均阳性率为80.69%(209/259),而各猪场的阳性率差异较大(48.28%～100%).以上结果表明Cap2s可作为一个型特异性抗原用于浙江省本地猪场猪群血清中PCV2抗体的监控,而其多抗也可用于免疫组化对PCV2感染进行有效诊断.     

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.     

Purification of cross-reacting protein antigen shared by Yersinia enterocolitica and other gram-negative bacteria with monoclonal antibody

A monoclonal antibody against the Yersinia enterocolitica 60-kilodalton (kDa) antigen, designated cross-reacting protein antigen (CRPA), was obtained by cell fusion. The CRPA common to gram-negative bacteria was purified from Y. enterocolitica by the affinity chromatography with the monoclonal antibody (IgG1) thus obtained. The purified CRPA showed a single band of 60 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reacted with rabbit antisera against Y. enterocolitica, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei in Western blot analysis. The monoclonal antibody, however, reacted with a 60 kDa peptide from Y. enterocolitica, but not with the antigens from other gram-negative bacteria such as V. cholerae, E. coli, S. sonnei, Salmonella enteritidis, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa. The results suggested that both species-specific and cross-reactive epitopes were present on a CRPA molecule.     

Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis

Summary For analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 10³ and 30 × 10³ Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level. Offprint requests to: A.S. Suzuki