Analytical and clinical validation of a time-resolved immunofluorometric assay (TR-IFMA) for canine C-reactive protein in serum.

A time-resolved immunofluorometric assay (TR-IFMA) was developed for the determination of C-reactive protein (CRP) in canine serum. CRP was isolated from canine acute-phase serum by affinity chromatography on agarose coupled with phosphorylethanolamine. This isolated dog CRP was used as standard to calibrate the assay. Intra-assay and inter-assay coefficients of variation were in the ranges 5.3-7.1% and 4.8-13.3%, respectively. Accuracy, evaluated by adding 2 and 10 microg/ml of CRP to serum samples, provided recoveries of 99.9% and 106.8%. High correlation was found between CRP measurements by TR-IFMA and a by commercial enzyme-linked immunosorbent assay (R2 = 0.98). The limit of detection for the TR-IFMA method was 0.000067 microg/ml and the measurement of CRP in serial dilutions of acute-phase dog sera generated curves with the same slope as the one constructed with purified CRP. The TR-IFMA provides a precise, accurate and highly sensitive assay for CRP determination in dog samples. CRP levels in dogs with different diseases ranged between 10.2 and 210.7 microg/ml and were significantly higher than those observed in healthy dogs (< 7.1 microg/ml).

Production and characterisation of monoclonal antibodies against a very small hapten, 3-methylindole.

Monoclonal antibodies were produced against a very small (131.2 Da) hapten, 3-methylindole. Nine derivatives of 3-methylindole were synthesised with spacers ending in a carboxyl group, and coupled to immunogenic carriers and europium chelate labels. Almost all the antigens elicited an antihapten response, but the majority of the mAbs produced strongly recognised the spacer group and did not bind free 3-methylindole. However, specific antibodies were obtained with five immunogens. Specificity could be directed against the pyrrole ring by locating the bridging group to the aromatic moiety of the indole ring system. Any modification in the position 3 of the indole ring strongly hindered mAb binding to the compound, and the cross-reactivity of physiologically important compounds, such as tryptophan and tryptamine, was negligible for all of the mAbs. The developed hapten structures successfully focused antibody recognition to the important sub-determinants in the indole ring system. Similar constructs could also be useful in the development of antibodies against other indolic compounds.

Intake of antioxidants and risk of type 2 diabetes in a cohort of male smokers.

BACKGROUND/OBJECTIVES: Oxidative stress may induce insulin resistance in peripheral tissues and impair insulin secretion from pancreatic ß-cells. Antioxidants are suggested to decrease the risk of diabetes through reduction of oxidative stress. However, only a few studies exist on dietary antioxidants and the risk of type 2 diabetes. We investigated the association of dietary antioxidants with incident type 2 diabetes in the α-Tocopherol, ß-Carotene Cancer Prevention Study cohort. SUBJECT/METHODS: The study cohort included 29,133 male smokers aged 50-69 years. During a median follow-up of 10.2 years 660 incident cases of diabetes were observed among the 25,505 men with a completed baseline food frequency questionnaire. RESULTS: Dietary α-tocopherol, ß-tocopherol and ß-tocotrienol were positively associated with the risk of diabetes when adjusted for age and supplementation (relative risk (RR) 1.17 (95% confidence interval (CI) 0.91-1.51) P for trend 0.02; RR 1.31 (95% CI 1.02-1.68) P for trend 0.01; RR 1.28 (95% CI 1.00-1.63) P for trend 0.01, respectively), but the association disappeared after multivariate adjustment (RR 0.92 (95% CI 0.71-1.19) P for trend 0.97; RR 1.06 (95% CI 0.82-1.36) P for trend 0.48; RR 1.04 (95% CI 0.80-1.35) P for trend 0.46, respectively). Other tocopherols and tocotrienols as well as vitamin C, carotenoids, flavonols and flavones had no association with risk of diabetes. CONCLUSIONS: Dietary antioxidants were not associated with a decreased risk of incident diabetes in middle-aged male smokers.

Effect of alpha-tocopherol and beta-carotene supplementation on the incidence of type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is associated with reduced antioxidant defence. Only a few human studies have investigated the role of antioxidants in the pathogenesis of diabetes. This study aimed to examine whether alpha-tocopherol or beta-carotene affected the occurrence of type 2 diabetes. METHODS: In the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, a double-blind, controlled trial, 29,133 male smokers aged 50-69 years were randomised to receive either alpha-tocopherol (50 mg/day) or beta-carotene (20 mg/day) or both agents or placebo daily for 5-8 years (median 6.1 years). Baseline serum samples were analysed for alpha-tocopherol and beta-carotene using HPLC. Cases of diabetes were identified from a nationwide Finnish registry of patients receiving drug reimbursement for diabetes. Of 27,379 men without diabetes at baseline, 705 men were diagnosed with diabetes during the follow-up of up to 12.5 years. RESULTS: Baseline serum levels of alpha-tocopherol and beta-carotene were not associated with the risk of diabetes in the placebo group: the relative risk (RR) between the highest and lowest quintiles of alpha-tocopherol was 1.59 (95% CI 0.89-2.84) and that for beta-carotene was 0.66 (95% CI 0.40-1.10). Neither supplementation significantly affected the incidence of diabetes: the RR was 0.92 (95% CI 0.79-1.07) for participants receiving alpha-tocopherol compared with non-recipients and 0.99 (95% CI 0.85-1.15) for participants receiving beta-carotene compared with non-recipients. CONCLUSIONS/INTERPRETATION: Neither alpha-tocopherol nor beta-carotene supplementation prevented type 2 diabetes in male smokers. Serum levels of alpha-tocopherol and beta-carotene were not associated with the risk of type 2 diabetes.

Determination of androstenone in pig fat using packed column supercritical fluid chromatography-mass spectrometry.

Packed column supercritical fluid chromatography (SFC) in combination with atmospheric pressure chemical ionisation mass spectrometry was applied to the analysis of androstenone in pig fat samples. Liquefied fat samples were dissolved in dichloromethane and analysed directly by SFC without any sample purification. Chromatographic separation was achieved with a density/pressure gradient using pure carbon dioxide as the mobile phase and the analysis resulted in a quantitation limit of 0.25 microg/g with 1 microl injection volume. Good agreement was found between the SFC method and time-resolved fluoroimmunoassay by the analysis of 15 boar back fat samples.

Development and validation of dry reagent time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine.

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 96/23/EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin alpha-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TRFIAs, the results being available within 1h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits.

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1). The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

Prevalence of zeranol, taleranol and Fusarium spp. toxins in urine: implications for the control of zeranol abuse in the European Union.

There is currently little information concerning the prevalence of zeranol and taleranol in animal urine following metabolism of the naturally occurring Fusarium spp. toxins. An epidemiological study is described which involves four European Union control laboratories in which 8008 urine samples were screened for the presence of zeranol using a time-resolved fluoroimmunoassay (TR-FIA). Of these samples, 93.6% screened negative for zeranol. All samples testing positive for zeranol were then analysed with a confirmatory method. Based on the confirmatory results, the TR-FIA-positive samples were then categorized as false-positive, true-positive or 'equivocal' (zeranol/taleranol and the Fusarium spp. toxins detected). The true-positive samples represented only 0.05% of the total number of samples (n = 4). After statistical analysis, 170 of 174 equivocal samples proved to belong to a 'normal' population in which the amount of zeranol/taleranol could be related to the total amount of Fusarium spp. toxins through a linear regression with a 99% prediction interval. This suggested that the presence of zeranol in these samples might be due to in vivo metabolism of the Fusarium spp. toxins. The presence of zeranol in the four remaining 'outliers' might be attributable to zeranol abuse rather than to natural contamination. The results are of interest for control laboratories as they might provide an analytical tool to help distinguish between abuse and natural contamination in zeranol testing.