Specific immuno-detection of benzoate-para-hydroxylase with antibodies raised against synthetic peptides.

As a model system for the industrial use of fungal cells in the enzymatic conversion of chemicals, the parahydroxylation of benzoate was studied. To increase the amount of benzoate-para-hydroxylase (BPH, EC 1.14.13.12.) in the cell the gene coding for the enzyme (bphA) was cloned and expressed in Aspergillus niger. Detection of the enzymatic activity of the protein was not reproducible. It was decided to raise an antiserum for immuno-detection purposes. Sufficient benzoate-para-hydroxylase for immunization could not be obtained; therefore the synthetic-peptide strategy was used. We demonstrate that synthesis of antigenic determinants, can be useful in the production of highly specific reagents for the detection of proteins. The availability of monospecific polyclonal sera opens new possibilities in functional studies and purification of benzoate-para-hydroxylase.     

Characterization of cDNA clones for the human c-yes gene.

Three c-yes cDNA clones were obtained from poly(A)+ RNA of human embryo fibroblasts. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-yes mRNA, which could encode a polypeptide of 543 amino acids with a relative molecular weight (Mr) of 60,801. The predicted amino acid sequence of the protein has no apparent membrane-spanning region or suspected ligand binding domain and closely resembles pp60c-src. Comparison of the sequences of c-yes and v-yes revealed that the v-yes gene contains most of the c-yes coding sequence except the region encoding its extreme carboxyl terminus. The region missing from the v-yes protein is the part that is highly conserved in cellular gene products of the protein-tyrosine kinase family.     

Characterization of immune response to synthetic peptides derived from the hemagglutinin of measles virus

We describe here four synthetic peptides derived from the hemagglutinin of measles virus. The peptides were predicted by a computer program combining hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus hemagglutinin. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using immunostimulating complex as adjuvant. Anti-peptide antisera raised in rabbits against the peptide conjugates reacted well with the homologous peptides and with measles virus antigen as tested with plate ELISA. None of these sera had either neutralizing or hemagglutination inhibiting antibody or reacted with measles hemagglutinin protein in Western blot and reacted weakly in immunofluorescence. Human sera positive for measles virus antibody reacted with the synthesized peptides indicating that the selected locations function as partial antigenic sites.     

Antagonistic action between antibodies directed against 7-methylguanosine and polyamines on translation in vitro of RNA induced by measles virus

Antibodies directed against N7-methylguanosine (m7Guo) were prepared and added to a wheat germ cell-free protein-synthesizing system programmed with RNA extracted from monkey cells persistently infected with measles virus. A dose-dependent inhibition of [35S]methionine incorporation was observed when RNA was preincubated with anti-m7Guo immunoglobulins. Antibodies preincubated with m7Guo did not show any inhibiting activity. The inhibitory effect of antibodies was abolished when RNA was preincubated with immunoglobulins in the presence of spermine and spermidine. When polyamines were added to the assay programmed with the IgG-RNA complex, no inhibition was observed.     

Mapping of linear B cell epitopes on open reading frames 2- and 3-encoded proteins of hepatitis E virus using synthetic peptides

Abstract Hepatitis E virus (HEV) is the causative agent of non-A, non-B hepatitis which is transmitted by the fecal-oral route and occurs principally in the form of large epidemics and outbreaks in developing countries. Two overlapping synthetic peptides corresponding to overlapping DNA sequences of the ORF 3 of HEV genome were found to be immunoreactive with sera from patients involved in two epidemics of enterically transmitted non-A, non-B hepatitis. The results suggested the existence of two distinct epitopes. The four synthetic peptides representing these two epitopes from Burma and Mexico strains of hepatitis E virus, were used to investigate anti-HEV reactivities. HEV antibodies were detected in 84–88% of HEV-infected individuals according to the peptide used. The results suggest that a peptide-based ELISA can provide an accurate tool for the diagnosis of acute hepatitis type E.     

Antibodies directed against synthetic peptides distinguish between the catalytic subunits of protein phosphatases 1 and 2A.

Two antipeptide antibodies (designated type 1 antibody and type 2A antibody) were raised against synthetic peptides, Cys-Thr-Pro-Pro-Arg-Asn-Ser-Ala-Lys-Ala-Lys-Lys and Cys-Val-Thr-Arg-Arg-Thr-Pro-Asp-Try-Phe-Leu, corresponding to the carboxyl termini of the catalytic subunits of protein phosphatase 1 and phosphatase 2A (Cys was added for specific coupling to carrier protein). These antipeptide antibodies were highly specific and were useful in discriminating between protein phosphatase 1 and phosphatase 2A in crude extracts or purified preparations. Type 2A antibody reacted with both native and denatured protein phosphatase 2A whereas under the same condition type 1 antibody reacted only with denatured protein phosphatase 1.     

Analysis of antibody response to synthetic peptides derived from the fusion protein of measles virus

A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.     

Formation of an infectious virus-antibody complex with Rous sarcoma virus and antibodies directed against the major virus glycoprotein.

Preparations of Rous sarcoma virus (RSV) can form an infectious viral-antibody complex with antibodies raised against the major glycoprotein, gp85, isolated from avian myeloblastosis virus and Prague-RSV subgroup C. Binding of anti-gp85 antibodies to RSV can be demonstrated by the inhibition of focus-forming activity after addition of goat anti-rabbit immunoglobulin and by a shift in density of virions treated with anti-gp85 serum. Group- rather than subgroup- specific regions of viral gp85 appear to be the site of binding for infectious complex.     

Antibodies against synthetic peptides reveal that the unidentified reading frame A6L, overlapping the ATPase 6 gene, is expressed in human mitochondria

Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to detect human mitochondrial gene products. In particular, antibodies directed against either the NH2-terminal decapeptide or the COOH-terminal undecapeptide of cytochrome c oxidase subunit II (COII) were both very effective in immunoprecipitating the previously identified COII polypeptide from an SDS lysate of mitochondria from HeLa cells. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the unidentified reading frame A6L, which overlaps the ATPase 6 gene, immunoprecipitated specifically a component (#25) of the HeLa cell mitochondrial translation products; antibodies directed against the NH2-terminal octapeptide also precipitated protein 25, although less efficiently. The size of protein 25, as estimated from its electrophoretic mobility, is compatible with its being the unidentified reading frame A6L product. Furthermore, a fingerprinting analysis of this protein after trypsin digestion has given results consistent with this identification.     

C-terminal-specific monoclonal antibodies against the human red cell glucose transporter. Epitope localization with synthetic peptides

Mice were immunized with human red cell glucose transporter for production of monoclonal antibodies. Four peptides were synthesized that correspond to relatively hydrophilic segments of the human HepG2 glucose transporter (Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W. J., Lienhard, G.E., and Lodish, H.F. (1985) Science 229, 941-945), including a C-terminal segment. After identification of hybridomas that were positive for the red cell glucose transporter, enzyme-linked immunosorbent assays were done with the synthetic peptides in solution to detect peptide-binding monoclonals. The very hydrophilic C-terminal peptide 478-492 (P2), but no other peptide, gave strong and selective inhibition of antibody binding to the glucose transporter. Two C-terminal-specific monoclonal antibodies were selected. The binding of these two antibodies to immobilized inside-out vesicles of human red cell membranes could be inhibited with the peptide P2. The antibodies did not react with right-side-out vesicles. The binding of these C-terminal-specific antibodies to the glucose transporter, to immobilized vesicles, and to the peptide P2 was enhanced by the presence of the peptide 218-232 (P1), although the peptide P1 alone showed no reaction with these antibodies. This suggests that the C terminus and the segment 218-232 of the red cell glucose transporter are exposed at the cytoplasmic face of the membrane and interact in the transporter. The C-terminal-specific monoclonal antibodies reacted strongly in Western blotting with the human red cell glucose transporter.     

Immunoprecipitation of some forms of simian virus 40 large-T antigen by antibodies to synthetic peptides.

Antibodies were raised against six synthetic peptides corresponding to overlapping amino acid sequences (106 through 145) from a putative DNA binding domain in simian virus 40 (SV40) large-T antigens. All six antipeptide sera immunoprecipitated large-T from crude extracts of SV40-transformed cells, but the efficiency varied widely; in general, antibodies to the longer peptides produced the strongest anti-large-T activity. Antisera were purified by immunoaffinity chromatography on immobilized peptide. The purified antisera recognized only some forms of large-T; full-sized large-T from transformed cells, super-T from SV3T3 C120 cells, and 70,000-dalton T-antigen from Taq-BamHI cells were immunoprecipitated, whereas large-T from productively infected cells reacted irreproducibly, and the full-sized protein, synthesized in vitro or eluted from sodium dodecyl sulfate-containing gels, and the 33,000- and 22,000-dalton truncated large-Ts from Swiss SV3T3 and MES2006 cells, respectively, were not immunoprecipitated. This pattern of reactivity was explained when extracts were fractionated by sucrose density centrifugation, and it was found that only rapidly sedimenting forms of large-T were immunoprecipitated by the antipeptide sera; that is, large-T complexed with nonviral T antigen was detected, whereas lighter forms were not detected. Cascade immunoprecipitations did not support the view that this result was caused by the low affinity of the peptide antisera for large-T, and Western blotting experiments confirmed that the peptide antisera react directly with immobilized, monomeric large-T but not with nonviral T antigen. Immunoprecipitation assays to detect large-T:nonviral T antigen complexes bound specifically to fragments of SV40 DNA showed that under conditions of apparent antibody excess, DNA still bound to the complex.     

Biological activities of rabbit antibodies against synthetic human thyrotropin receptor peptides representing thyrotropin binding regions.

Recently, we have shown that the thyrotropin (TSH) binding regions of human thyrotropin receptor (TSHR) reside in two areas within residues 12-44 and 308-344. Serial antisera were raised against four overlapping synthetic peptides representing these two regions of TSHR (peptides 12-30, 24-44, 308-328, and 324-344) and were investigated for their ability to stimulate or block the cultured porcine thyroid cells. In addition, serum concentrations of triiodothyronine (T3) and thyroxine (T4) in serial sera obtained from each rabbit were examined. It was shown that residues of 12-30 and 324-344 of TSHR, respectively, are the site (at least a part of the site) where stimulating (TSAb) and blocking type (TSBAb) immunoglobulins are directed.     

Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.     

Use of synthetic peptides for the detection and quantification of autoantibodies

Summary and conclusions The rapid progress made over the last 10 years in the identification of individual autoantigens and in the localization of the epitopes involved, has resulted in a parallel reduction in the complexity of the antigen required for the detection of autoantibodies. The ability to use synthetic peptides as antigens is a remarkable culmination of this process considering that many antigenic particles contain multiple proteins (eg. Sm consist of 8 or more individual proteins).Despite the fact that patients with SLE have a polyclonal hypergammaglobulinemia, excellent correlations between ELISAs utilizing the P2 or SmB/B synthetic peptides, ELISAs utilizing r proteins and immunoblotting were obtained [28, 38, 50]. However, false positive/non-specific binding to a P2-BSA-glutaraldehyde conjugate has been observed with serum from old MRL/lpr mice (unpublished observations). In addition, some of the results obtained in human autoimmune diseases suggest that non-specific binding may be problematic in some instances. It is difficult, at present, to know whether the higher frequencies of detection of autoantibodies to certain synthetic peptide antigens reflect increased sensitivity or decreased specificity.Synthetic peptide antigens have beeen used to detect autoantibodies in both organ specific and multisystem autoimmune diseases. In only a small number of cases have these reagents been rigorously tested for sensitivity and specificity. Despite this, synthetic peptides have been shown to be valuable for detection and quantification of autoantibodies in certain clinical situations. Undoubtedly, further progress in epitope mapping of autoantigens coupled with technological advances in protein synthesis and improved prediction of protein structure will lead to a large number of synthetic peptide antigens for research and clinical applications. It is unlikely that short synthetic peptides will substitute for native proteins in all instances since some autoantibodies show a striking preference for conformational epitopes.Abbreviations r recombinant - SLE systemic lupus erythematosus     

Monoclonal antibodies directed against a synthetic peptide corresponding to the alpha-bungarotoxin binding region of the acetylcholine receptor

Murine monoclonal antibodies have been produced against a 32 amino acid synthetic peptide corresponding to residues 173-204 on the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica. All of the monoclonal antibodies were of the IgM subtype and most cross-reacted with the purified native receptor. None of the antibodies were effective in blocking alpha-bungarotoxin binding to the receptor nor, conversely, did alpha-bungarotoxin interfere with antibody binding. However, two monoclonal antibodies, previously shown to bind near the ligand binding site on the native receptor, did compete partially (50%) with the binding of one of the IgM monoclonal antibodies.     

Characterization of murine monoclonal antibodies directed against the core proteins of human immunodeficiency virus types 1 and 2.

Monoclonal antibodies (MAbs) raised against the core proteins of human immunodeficiency virus type 1 (HIV-1; laboratory strain HTLV-IIIB) and HIV-2 (strain ROD) were investigated in a variety of tests, e.g., enzyme-linked immunosorbent assay (ELISA), immunostaining of Western immunoblots, immunofluorescence, immunoprecipitation, and alkaline phosphatase anti-alkaline phosphatase assay. The MAbs were grouped according to their cross-reactions. Seven HIV-1-specific MAbs reacted exclusively with HIV-1, and five showed cross-reactivity with HIV-2 and simian immunodeficiency virus of macaques in ELISA. Four of the 15 MAbs against HIV-2 reacted only with the HIV-2 protein p26. Six showed cross-reactivity with HIV-1, and five showed a broad reaction with all three viruses. Overlapping 30-amino-acid-long peptides derived from the p24 protein sequence of HIV-1 were used in an epitope-mapping system. Three different immunogenic regions (A, B, and C) could be defined. Specific regions where anti-HIV-1 and -HIV-2 MAbs cross-reacted were mapped with shorter oligopeptides.     

Use of synthetic peptides to map the antigenic determinants of glycoprotein D of herpes simplex virus.

The predictive algorithm Surfaceplot (J.M.R. Parker, D. Guo, and R.S. Hodges, Biochemistry 25:5425-5432, 1986) was used to examine glycoprotein D of herpes simplex virus type 1 (HSV-1) for amino acid residues with a high probability of being exposed on the molecular surface. Based on these data, 11 different peptides corresponding to 10-residue segments in the primary sequence of glycoprotein D and one 20-residue segment were synthesized, conjugated to carrier proteins, and used to generate specific antisera in rabbits. Two synthetic peptides predicted not to be on the surface of glycoprotein D were included as negative controls. The polyclonal antisera against individual synthetic peptide conjugates were in turn evaluated for their ability to recognize both isolated glycoprotein D and intact HSV-1 virions in an enzyme-linked immunosorbent assay. Based on Surfaceplot predictions, eight linear antigenic sites on glycoprotein D were thereby defined from the 12 antipeptide antisera prepared. Four of these sites contained epitopes to which complement-independent neutralizing antibodies could be generated. The latter sites corresponded to sequences 12 to 21, 267 to 276, 288 to 297, and 314 to 323 of the mature protein. An additional peptide sequence, 2 to 21, was found to generate antisera which had potent virus-neutralizing capacity in the presence of complement. Identification of a neutralizing epitope in the sequence 314 to 323 makes it likely that the membrane-spanning region of glycoprotein D is within the subsequent sequence, 323 to 339. Antipeptide antisera prepared in this study from 12 synthetic peptides contained 13 surface sites predicted by Surfaceplot, of which 7 were not predicted by the parameters of Hopp and Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981). Of these seven sites not predicted by the Hopp and Woods plot, all generated antipeptide antibodies that bound to HSV-1 virions and three of these seven sites generated neutralizing antibodies. In total, 8 of 12 synthetic peptides containing surface regions produced antipeptide antibodies that bound to HSV-1 virions and 5 of these generated neutralizing antibodies. These results suggest the advantages of Surfaceplot in mapping antigenic determinants in proteins.     

VIDA: a virus database system for the organization of animal virus genome open reading frames

VIDA is a new virus database that organizes open reading frames (ORFs) from partial and complete genomic sequences from animal viruses. Currently VIDA includes all sequences from GenBank for Herpesviridae, Coronaviridae and Arteriviridae. The ORFs are organized into homologous protein families, which are identified on the basis of sequence similarity relationships. Conserved sequence regions of potential functional importance are identified and can be retrieved as sequence alignments. We use a controlled taxonomical and functional classification for all the proteins and protein families in the database. When available, protein structures that are related to the families have also been included. The database is available for online search and sequence information retrieval at http://www.biochem.ucl.ac.uk/bsm/virus_database/ VIDA.html.