Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs.

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.

Drugs for discoid lupus erythematosus.

BACKGROUND: Discoid lupus erythematosus is a chronic form of cutaneous (skin) lupus which can cause permanent scarring if treatment is inadequate. Many drugs have been used to treat this disease and some of these are potentially very toxic. OBJECTIVES: To assess the effects of drugs for discoid lupus erythematosus. SEARCH STRATEGY: We searched the Cochrane Clinical Trials Register (December 1999), MEDLINE (January 1966 to December 1999), EMBASE (January 1980 to January 2000), and the reference lists of relevant reviews. Index Medicus (1956 to 1966) was handsearched and 7 experts in the field were approached for information about unpublished trials. SELECTION CRITERIA: Randomised trials of drugs to treat people with discoid lupus erythematosus. Drugs included in the search were azathioprine, chloroquine, clofazimine, corticosteroids, (oral and topical), dapsone, gold, interferon alpha-2a, methotrexate, phenytoin, retinoids, sulphasalazine and thalidomide. DATA COLLECTION AND ANALYSIS: Two reviewers independently examined each retrieved study for eligibility. MAIN RESULTS: Two trials involving 136 participants were included. In a cross-over study of twelve weeks duration fluocinonide 0.05% cream (a potent topical corticosteroid), appeared to be markedly better than hydrocortisone 1% cream ( a mild corticosteroid). Clearing or excellent improvement was seen in 27% of people using fluocinonide and in 10% of those using hydrocortisone, giving a 17% absolute benefit in favour of fluocinonide (95% CI 4.5 to 29.5% and NNT 6). In the second trial, hydroxychloroquine was compared with acitretin in 58 people. There was marked improvement or clearing in 46% of people using acitretin and in 50% of those on hydroxychloroquine, a nonsignificant 4% absolute gain with hydroxychloroquine (95%CI -23% to 30%). The adverse effects were more frequent and more severe in the acitretin group. REVIEWER'S CONCLUSIONS: Fluocinonide cream may be more effective than hydrocortisone in treating people with discoid lupus erythematosus. Hydroxychloroquine and acitretin appear to be of equal efficacy, although adverse effects are more frequent and more severe with acitretin. There is not enough reliable evidence about other drugs used to treat discoid lupus erythematosus.

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

Identification of an antigenic peptide specific for bluetongue virus using phage display expression of NS1 sequences.

BACKGROUND: NS1 is a non-structural protein associated with the replication of bluetongue virus (BTV), an orbivirus (Reoviridae) that infects sheep and cattle. NS1 is potentially useful as a diagnostic antigen since the presence of specific antibodies is an indication that the virus has replicated in the host. It is, however, not antigenically unique and cross-reacts serologically with the analogous protein of the related epizootic haemorrhagic disease virus (EHDV). OBJECTIVES: To investigate phage display of peptides derived from the gene encoding NS1 as a way of identifying unique antigenic regions that can be mimicked by synthetic peptides. STUDY DESIGN: A cDNA clone of a large portion of the gene encoding NS1 of bluetongue virus was fragmented by partial DNase digestion. The fragments were ligated into the filamentous phage display vector, fUSE 2. Peptides expressed on the surface of the phages as part of the gene III proteins were selected from the library using antibodies affinity-purified from an antiserum to NS1. The peptides were identified by sequencing the phage DNA and alignment with the sequence of the target gene. RESULTS: Two antigenic regions were identified, one of which could be effectively mimicked by a 28 residue synthetic peptide. This peptide did not cross-react with an antiserum directed against NS1 of EHDV. CONCLUSION: The strategy of screening gene-derived phage display libraries with antibodies from an immune serum is expected to be useful in the development of highly specific peptide-based diagnostic assays.

Fine mapping of a continuous epitope on VP7 of bluetongue virus using overlapping synthetic peptides and a random epitope library.

Two complementary techniques have been used to delineate an epitope on VP7 of bluetongue virus. Two MAbs (F10 and D11), both of which bound within a region spanning amino acids 255 to 274 in the 349 amino acid protein, were used to probe overlapping synthetic peptides covering this region. A pentapeptide, QYPAL, and a hexapeptide, QY-PALT (amino acids 259-264), preferentially bound both MAbs. MAb F10 also reacted with a heptapeptide (TAEIFNV) immediately adjacent to QYPALT. The MAbs were also used to affinity-purify fusion phages from a random hexapeptide library. All phage peptides selected were similar to QYPALT. Comparison of the peptides suggested that residues Q and P at positions 1 and 3 were critical for recognition. Some affinity-purified phages displayed the hexapeptide QYPSLL, which is similar to a sequence in VP7 of another orbivirus, epizootic hemorrhagic disease virus. This finding allowed a potentially cross-reactive site to be identified.

A previously undescribed ectodermal dysplasia of the tricho-odonto-onychial subgroup in a family.

We encountered two family members with a previously undescribed pure ectodermal dysplasia. The propositus exhibited hypotrichosis, hypodontia, focal linear dermal hypoplasia on the tip of her nose, irregular hyperpigmentation on her back, bilateral amastia and athelia, and mild nerve hearing loss. Her mother displayed similar characteristics, except for present, although hypoplastic, areolae and nipples. Both mother and daughter appeared to be clinically euhidrotic. Despite a comprehensive endocrine workup, the only abnormality detected was a suboptimal cortisol response to hypoglycemia in the propositus. Five other family members seemed to be affected. The pattern of inheritance appeared to be autosomal-dominant, with variable penetrance and expressivity.