RESUMO
Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs) were evaluated in a competitive ELISA. The binding of one (F3) to purified BTV was only inhibited by antibodies against the homologous serotype. The binding of the other (F10) was blocked by antisera to each of the 24 BTV serotypes. F10 recognised VP7, a major structural protein of the BTV core, but not if the protein was directly adsorbed to a plastic surface. It did, however, bind to recombinant VP7 that had been captured from suspension by rabbit IgG. This made it possible to develop an scFv based inhibition ELISA for BTV antibodies using recombinant VP7 without prior purification. The resulting immunoassay detected antibodies to 24 BTV serotypes, but not those directed against three serotypes of the related epizootic haemorrhagic disease virus. A phage library displaying fusion peptides expressed by fragments of the BTV genome segment 7 cDNA was constructed and screened using F10. Comparing selected peptides with the amino acid sequence of VP7 showed that recognition by the scFv required at least 131 residues representing the protein's upper domain. By providing well-characterised immunological reagents, recombinant antibody technology can contribute to the development of improved immunoassays for BTV diagnosis.
Assuntos
Anticorpos Antivirais/análise , Antígenos Virais/análise , Vírus Bluetongue/classificação , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Vírus Bluetongue/genética , Galinhas , Sorotipagem , Proteínas do Core Viral/imunologiaRESUMO
BACKGROUND: Epitopes can be mapped by comparing immunoaffinity-selected peptides from fragmented-gene display libraries with the target gene. With larger libraries derived from unsequenced genomes, this is not possible. Spurious epitope mimics may be created by expressing DNA in a variety of meaningless reading frames and orientations. OBJECTIVES: To determine empirically whether panning a large fragmented-genome phage display library with antibodies to MAP1, the major antigenic protein of the rickettsial parasite Cowdria ruminantium, would result in the selection of irrelevant, cross-reactive mimotopes. STUDY DESIGN: A gene III phage library displaying peptides derived from C. ruminantium was constructed using cloned DNA from a bacteriophage lambda genomic library. After in vivo excision, plasmids were cleaved with PvuII followed by PCR. Genes with a PvuII site, including MAP1 were therefore not amplified. DNA was sonicated, partially digested with DNase and cloned into the display vector fUSE2. Affinity-purified MAP1 antibodies were used for panning. Peptides expressed by panned phages were tested for recognition in Western blot and ELISA. Oligonucleotides representing antigenic sequences were used to locate their encoding DNA sequences in the original lambda library. The phage display library was also panned with two monoclonal antibodies (Mabs) against bluetongue virus (BTV). RESULTS: Five different peptide sequences were selected from the MAP1-deficient phage display library. None was identical to MAP1, but four peptides had regions that were similar, both to each other, and to the parasite protein. They produced strong signals in ELISA and Western blot. None could be located to any C. ruminantium open reading frame. Two BTV Mabs recognised a sequence similar to their authentic epitope. CONCLUSION: Large genome-targeted phage display libraries may be sufficiently diverse to allow the selection of peptides that mimic actual antigenic determinants. This diversity may be exploited in the search for useful epitopes.
Assuntos
Antígenos de Bactérias/imunologia , Ehrlichia ruminantium/imunologia , Epitopos Imunodominantes/imunologia , Mimetismo Molecular , Biblioteca de Peptídeos , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Bacteriófagos/genética , Western Blotting , Reações Cruzadas , Ehrlichia ruminantium/genética , Ensaio de Imunoadsorção Enzimática , Genoma Bacteriano , Dados de Sequência Molecular , Peptídeos/genéticaRESUMO
The numbers of patients treated for seven types of carcinoma during 1977 at 10 hospitals in South Africa have been reviewed. The total number of patients admitted to the 10 hospitals in 1977 was 286 373. Slightly more than 1%, namely 3 409, of these patients suffered from carcinoma of the cervix, oesophagus, breast, lung, liver, stomach or colon. Carcinoma of the cervix was commonest among Black patients and carcinoma of the colon among Whites. The relative incidence of the different types of carcinoma among Whites was almost the opposite of the sequence among Blacks.