Roy Jackman

Antibodies to the quinolones and fluoroquinolones for the development of generic and specific immunoassays for detection of these residues in animal products

Several quinolone and fluoroquinolone haptens have been used to raise polyclonal antibodies exhibiting both specific and generic properties for these classes of antimicrobial compounds. The antisera have been assessed in rapid enzyme immunoassays (ELISAs) designed to exploit the specificities obtained. A direct generic ELISA for both the quinolones and fluoroquinolones has been developed that uses the cross-reactivity of an antibody raised against norfloxacin (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid) linked to ovalbumin via a secondary amine group on the piperazinyl moiety to detect nine different drugs in these classes. Specific ELISAs to ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid), enrofloxacin (1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carboxylic acid), flumequin (9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo(ij)quinolizine-2-carboxylic acid) and nalidixic acid (1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid) have also been developed with a high degree of specificity to the individual compounds. The assays measure drug residues in bovine milk and ovine kidney with an interassay relative standard deviation (sr) of 10.5% or less and intra-assay Sr of 11.2% or less. Sensitivity is less than 4 μg kg-1 for both the generic and specific assays for all but one of the compounds tested. (Pipemidic acid (8-ethyl-5,8-dihydro-5-oxo-2-(1-piperazinyl)pyrido(2,3-d)pyrimidine-6-carboxylic acid) is detectable at 6 μg kg -1 in kidney.)

Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion

Abstract A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrPC, the pathological isoform PrPSc forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage.

Molecular Profiling of Ovine Prion Diseases by Using Thermolysin-Resistant PrPSc and Endogenous C2 PrP Fragments

ABSTRACT Disease-associated PrP fragments produced upon in vitro or in vivo proteolysis can provide significant insight into the causal strain of prion disease. Here we describe a novel molecular strain typing assay that used thermolysin digestion of caudal medulla samples to produce PrPres signatures on Western blots that readily distinguished experimental sheep bovine spongiform encephalopathy (BSE) from classical scrapie. Furthermore, the accumulation of such PrPres species within the cerebellum also appeared to be dependent upon the transmissible spongiform encephalopathy (TSE) strain, allowing discrimination between two experimental strains of scrapie and grouping of natural scrapie isolates into two profiles. The occurrence of endogenously produced PrP fragments, namely, glycosylated and unglycosylated C2, within different central nervous system (CNS) regions is also described; this is the first detailed description of such scrapie-associated fragments within a natural host. The advent of C2 fragments within defined CNS regions, compared between BSE and scrapie cases and also between two experimental scrapie strains, appeared to be largely dependent upon the TSE strain. The combined analyses of C2 fragments and thermolysin-resistant PrP species within caudal medulla, cerebellum, and spinal cord samples allowed natural scrapie isolates to be separated into four distinct molecular profiles: most isolates produced C2 and PrPres in all CNS regions, a second group lacked detectable cerebellar C2 fragments, one isolate lacked both cerebellar PrPres and C2, and a further isolate lacked detectable C2 within all three CNS regions and also lacked cerebellar PrPres. This CNS region-specific deposition of disease-associated PrP species may reflect the natural heterogeneity of scrapie strains in the sheep population in the United Kingdom.

Development of a polarization fluoroimmunoassay for sulfamethazine using an automated analyser

A rapid, non-isotopic, polarization fluoroimmunoassay for the antibiotic sulfamethazine has been developed. The influence of the structure of fluorescein-labelled tracers on the sensitivity of the assay was investigated. Reagents were adapted for use with the Abbott TDx Analyzer and existing software was adapted for automated analyses. Total time for the assay of 1 and 20 samples was 14 and 22 min, respectively. All analytical criteria for the assay were satisfied. The detection limit for sulfamethazine in 20 µl of aqueous sample was 10 ng ml–1(200 pg), which compares well with the sensitivity of enzyme linked immunosorbent assay methods.

Enzyme immunoassay for the detection of levamisole in meat and milk

Ovine polyclonal antisera were raised to aminolevamisole to develop an enzyme immunoassay for levamisole. The assay has been validated in milk and in 100 g l-1 muscle homogenate. The limit of detection is 1 microgram l-1. In milk the inter-assay relative standard deviation (Sr) is 6% at 10 micrograms kg-1 and the intra-assay Sr value 7.2%. In tissue homogenate the inter-assay Sr value is 12.7% at 5 micrograms kg-1 and the intra-assay Sr value is 3.8%. Total incubation time is 1 h.

Improved spectrophotometric assay for β-lactam residues in kidney tissue

This paper describes a detection system for β-lactams using a commercially prepared carboxypeptidase enzyme (CPase) and a substrate system in which lactic acid is cleaved from a synthetic peptide, Nα-NIµ-diacetyl-L-lysyl-d-alanyl-d-lactic acid. The lactate is itself oxidized by lactate dehydrogenase to form NADH. Oxidized NAD+ is regenerated by diaphorase with the simultaneous reduction of the colourless 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride hydrate (INT) indicator substrate to produce a red–mauve colour that is proportional to CPase activity. The presence of β-lactams decreases the intensity of colour produced. The lower limit of detection for benzyl penicillin (Pen G) by this system is 20 ng g–1 compared with 50 ng g–1 by the same assay but using an R-d-ala-d-ala substrate from a commercial kit.