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Biomedical subjects

A J Radford

Publications and source records attributed to A J Radford.

At least 19 recordsLinked to original sources
PubMed1992

Removal of false-positive reactions from plasma in an enzyme immunoassay for bovine interferon-gamma.

A monoclonal antibody-based sandwich enzyme immunoassay (EIA) for bovine interferon-gamma (IFN-gamma) has been developed and can be used in conjunction with a whole blood culture system to diagnose tuberculosis in cattle. During its development, normal bovine plasma samples were tested to establish background levels of circulatory IFN-gamma. Of 191 samples tested, 81 (42.4%) were positive (OD > 0.1) when tested undiluted in intact monoclonal antibody (IgG1)-coated wells compared to only 8 (4.2%) in F(ab')2-coated wells, which suggested non-specific interference in the EIA rather than circulatory IFN-gamma. Reactivity of all remaining samples was removed by diluting plasmas 1/2 with 1% casein-PBS-0.05% Tween 20 supplemented with an optimum amount (5%) of normal mouse serum (NMS). Serum pools derived from BALB/c, DBA/2, C3H/HeJ, CBA/CaH and Swiss, but not C57BL/6J, mice were found to inhibit equally the reactions of five strong false-positive bovine plasma samples but had no effect on the titre of IFN-gamma in the sample. Sera from other species tested were less effective. This suggests that the interfering factors possess a high degree of specificity, since the immunoglobulin heavy chain of IgG1 produced by all these five strains of mice are allotypically identical and different to IgG1 produced by C57BL/6J mice. The use of F(ab')2 antibody fragments to coat plate wells and sample diluent containing 5% NMS has resulted in an EIA for bovine IFN-gamma that is virtually free from false-positive reactions, has a high degree of reproducibility and a sample detection limit equivalent to approximately 80 pg/ml recombinant bovine IFN-gamma.

Animals↗
PubMed1992

Characterisation of a novel repetitive DNA sequence from Mycobacterium bovis.

We report characterisation of three copies of a novel repeat sequence isolated from a Mycobacterium bovis genomic library. The repeat occurs within open reading frames, potentially encoding a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-X-Gly. The tandem array is present up to five times in M. bovis and it is proposed that they may occur in a family of genes expressing functionally related proteins. We postulate that these proteins may play a role in binding of M. bovis to host cell receptors.

Amino Acid Sequence↗
PubMed1992

Rational attenuation of Corynebacterium pseudotuberculosis: potential cheesy gland vaccine and live delivery vehicle.

The phospholipase D (PLD) gene (pld) has been deleted from the Corynebacterium pseudotuberculosis chromosome by using site-specific mutagenesis. Sheep infection trials indicate that the PLD-negative C. pseudotuberculosis strain (Toxminus) is incapable of inducing caseous lymphadentis (cheesy gland) even at doses two logs higher than that at which the wild-type strain produces the disease. This clearly establishes PLD as a major C. pseudotuberculosis virulence factor. Vaccination of sheep with live Toxminus C. pseudotuberculosis elicits strong humoral and cell-mediated immune responses and protects the animals from wild-type challenge.

Animals↗
PubMed1991

Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector.

We have constructed a set of novel Mycobacterium-Escherichia coli shuttle vectors using either a kanamycin- or hygromycin-resistance gene and the replication region from a Corynebacterium plasmid. Important features of these new vectors (pEP2 and pEP3) are that they are small, contain multiple cloning sites, and replicate to high copy number in various Mycobacterium species and E. coli. These vectors are unusual in that plasmid replication in gram-negative and gram-positive bacteria appears to be controlled from a single region. These plasmids will be useful for the genetic analysis of Mycobacterium and gene expression in this genus, particularly Mycobacterium bovis BCG.

Cloning, Molecular↗
PubMed1991

A randomised study of outcomes in a defined group of acutely ill elderly patients managed in a geriatric assessment unit or a general medical unit.

The aim of this study was to identify differences in the medical management and clinical outcome in a group of elderly patients admitted to a designated geriatric assessment unit (GAU) or to two general medical units (GMUs). A prospective randomised controlled trial was undertaken in 267 patients aged 70 years and over (mean age = 78.3 years). Following discharge from hospital, patients were followed up at three monthly intervals for a total of 12 months. At the time of discharge, no significant differences were found in inpatient management, length of stay, mortality rates, discharge rates to institutional care or utilisation of community services in patients admitted to the GAU and the GMUs. Similarly, no significant differences were found at three, six, nine, and 12 month follow up in case fatality, activities of daily living indices, mental health status, rates of institutional referral and the level of community service support in patients admitted to the GAU and the GMUs studied. These findings do not show any advantage for the unselected 70+ acutely ill elderly patient who is admitted to a designated geriatric assessment unit rather than to a general medical unit. Therefore, an admission policy to GAU, based solely on age 70+ is medically inappropriate and cost-inefficient. Evidence from other sources suggests that an age cohort of acutely admitted patients beyond 80 years may well have returned more optimistic findings for the GAU. In future, GAUs will require a more selective admission policy to maximise the benefits of their rehabilitative and interdisciplinary approach.

Acute Disease↗
PubMed1991

Cloning and sequencing of the ovine gamma-interferon gene.

Cytokines are major modulators of the immune system of all animals. The cloning and expression of recombinant cytokine genes have permitted the analysis of their immune function and role in the control of the immune response to disease and vaccination. While human, murine, and bovine genes have been cloned and sequenced, the cloning of ovine cytokine genes has not yet been reported. As sheep are of dominant economic importance to the Australian farming industry, it is of significance to clone and express these genes to facilitate the development of new and better vaccines and pharmaceuticals. We have initially selected ovine gamma-interferon (gamma-IFN) as a target cytokine gene. By the use of the polymerase chain reaction (PCR), using primers based on the bovine gamma-interferon sequence, we have amplified the ovine gamma-interferon gene from crude messenger RNA extracted from lymphocytes. After cloning and DNA sequencing the gene, we found that ovine gamma-IFN is 93% identical to bovine gamma-IFN in amino acid sequence. This result indicates that the PCR method will be a rapid and efficient means for cloning other ovine cytokine genes.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of major antigens from a Mycobacterium bovis culture filtrate.

Ten major antigens from Mycobacterium bovis culture filtrate of 39, 32, 30, 25, 24, 22 (a and b forms), 19, 15, and 12 kDa have been purified and characterized by classical physicochemical methods. With monoclonal antibodies and/or N-terminal amino acid sequencing data, it was found that the antigens of 32, 30, 24, 22 (a), 19, and 12 kDa are related to M. bovis or M. tuberculosis antigens P32, MPB59, MPB64, MPB70, 19 kDa, and 12 kDa, respectively. The 39-, 25-, 22 (b)-, and 19-kDa antigens showed concanavalin A-binding properties and were positive in a glycan detection test, suggesting that they are glycoproteins. The 25- and 22 (b)-kDa proteins were found to be glycosylated forms of MPB70.

Amino Acid Sequence↗
PubMed1990

Mapping of the T and B cell epitopes of the Mycobacterium bovis protein, MPB70.

A clone coding for the entire gene for the Mycobacterium bovis protein antigen MPB70 was used to produce a series of overlapping subclones by making a series of deletions from the 3' end of the gene. The subclones expressed incomplete MPB70 proteins as fusions with glutathione-S-transferase. The insert DNA was sequenced to determine the extent of the deletion and the proteins expressed by the clones were examined for the presence of T cell and B cell epitopes. T cell epitopes were mapped by measuring the ability of recombinant antigens to stimulate gamma interferon (gamma-IFN) production in a whole blood culture system. gamma-IFN production was measured using a sandwich enzyme immunoassay specific for bovine gamma-IFN. B cell epitopes were mapped with a series of anti-MPB70 monoclonal antibodies using an indirect enzyme immunoassay.

Amino Acid Sequence↗
PubMed1990

Epitope mapping of the Mycobacterium bovis secretory protein MPB70 using overlapping peptide analysis.

The gene encoding the major Mycobacterium bovis secreted protein MPB70 was cloned and isolated from a DNA library in lambda EMBL 3, and the restriction map of the area of chromosome containing the gene ascertained. After sub-cloning, the complete DNA sequence and predicted amino acid sequence were determined, and from this information a series of overlapping octapeptides encoding all possible linear epitopes of eight or less amino acids were synthesized. These peptides were probed with monoclonal antibodies specific for M. bovis and with sera from M. bovis-infected cattle. Epitopes defined by this technique were then examined using a substitution analysis that allowed the significance of each amino acid in the putative epitope to be quantified, and the exact specificity of the antibody response for the epitope determined.

Amino Acid Sequence↗
PubMed1990

The community care of terminally ill patients.

The authors outline the results of a survey, through interviews or posted questionnaires, of 122 Adelaide general practitioners. General practitioners shared or handed over most aspects of terminal care, particularly the spiritual and social aspects. Comprehensive care for these patients is directly related to the general experience of the general practitioners. The need for improved undergraduate and postgraduate education in terminal illness is stressed.

Australia↗
PubMed1989

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis.

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigen in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K. A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.

Animals↗
PubMed1989

Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes.

Two lambda gt11 recombinant clones, JKL2 and JKL15, each containing an insert coding for part of the highly immunogenic 70-kilodalton (kDa) protein antigen, were isolated from a Mycobacterium leprae genomic library by immunoscreening with the monoclonal antibody L7. Clone JKL2 contained the largest insert, 2.3 kilobase pairs. Nonoverlapping fragments of this insert were used as probes and showed strong hybridization to a number of Mycobacterium tuberculosis-lambda gt11 recombinants producing proteins recognized by an anti-M. tuberculosis 71-kDa monoclonal antibody, IT11. One clone from a recombinant Mycobacterium bovis library was also characterized by using L7, and the insert from this clone, B5bt, hybridized strongly to the M. leprae probes as well. The nucleotide sequence of the 1,037-base-pair coding region of the JKL2 M. leprae clone which encodes the carboxy-terminal half of the 70-kDa protein had extensive homology with genes from a number of species. In all cases, these genes, including the recently described Ag63 and Ag361 of Plasmodium falciparum, were found to be members of the heat shock protein 70 (hsp 70) family of genes. At the amino acid level, homology was maximal between amino acids 83 through 107 and 159 through 184, which showed extreme conservation (92 and 85% identity) with Escherichia coli DnaK amino acids 386 through 409 and 460 through 485, respectively, and was 51% homologous over the entire coding region (amino acids 1 through 344 of JKL2). In contrast, amino acids 129 through 158 had maximal homology with the phylogenetically more distant Xenopus laevis hsp70. Homology declined substantially in the carboxy-terminal 34 amino acids. The predicted ATP-binding functional activity of the 70-kDa antigen from M. bovis was confirmed with affinity purification of the antigen by binding to ATP-agarose and elution with ATP. In view of the conservation of sequences between these mycobacterial antigens and mammalian endogenous cellular enzymes, further evaluation of these molecules in vivo may aid in understanding tolerance to self-antigens as well as provide potentially useful immunodiagnostic reagents.

Adenosine Triphosphate↗
PubMed1988

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis.

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.

Animals↗