Biomedical subjects

E Usleber

Publications and source records attributed to E Usleber.

26 records · Page 2Linked to original sources

PubMed1992

Studies on the application of enzyme immunoassays for the Fusarium mycotoxins deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone.

Polyclonal antisera against zearalenone (ZEA) were produced in rabbits after immunization with ZEA-oxime coupled to human serum albumin. Using these antibodies and a ZEA-oxime-horseradish peroxidase conjugate in a competitive direct enzyme immunoassay (EIA), the detection limit for ZEA was 70 pg/ml. The relative cross-reactivities of the assay with ZEA, alpha-zearalenol, beta-zearalenol, zearalanone, alpha-zearalanol, and beta-zearalanol, respectively, were 100%, 37.3%, 7.2%, 59.2%, 5.3%, and 3.9%, respectively. This EIA and two EIAs for deoxynivalenol (DON) and 3-acetyldeoxynivalenol(3-AcDON) (Usleber et al., 1991) were used to analyze wheat samples. The limits of determination for DON, 3-AcDON, and ZEA in wheat were 200 ppb, 50 ppb, and 20 ppb, respectively. The analysis of reference materials (wheat flour) containing DON by EIA showed good agreement with the nominal values. The EIA for ZEA was in addition used to analyze biological fluids, obtained during a feeding trial. Two lactating cows were administered 25 mg and 100 mg ZEA per day, respectively, over a period of 6 days. Serum, milk, urine, and feces were assayed in the ZEA-EIA with and without sample treatment with beta-glucuronidase prior to the analysis. Maximum toxin levels (ZEA-equivalents) found in milk were 0.4 and 1.2 ppb (glucuronides). The toxin concentration in milk decreased rapidly after the last toxin administration. In the urine, maximum levels of toxin-glucuronide conjugates were 23 ppb and 24 ppb, respectively. The serum toxin levels corresponded to those found in milk. In the feces, mean values were 150 ppb and 500 ppb, respectively, no conjugated toxins were found in feces.

Animals↗

PubMedDate not supplied

Do saxitoxin-like substances have a role in scombrotoxicosis?

Evidence is presented which establishes that mackerel fed in captivity can, by relay from contaminated shellfish via sand eels, accumulate paralytic shellfish poisons (PSP) in the edible flesh at a level (250 micrograms saxitoxin equivalents per kg) similar to that in the contaminated shellfish. Data from ELISAs performed independently in two laboratories show that commercial mackerel fillets which have been associated with incidents of scombrotoxicosis contained 0.02-1.30 micrograms saxitoxin equivalents per kg, concentrations some two to four orders of magnitude below that normally detectable by the mouse bioassay. The doses, expressed as saxitoxin equivalents, administered inadvertently during volunteer testing of such fillets ranged up to 0.5 ng/kg bw, at least four orders of magnitude less than the fatal oral dose for an adult. The doses associated with the rapid induction of nausea/vomiting and/or diarrhoea, 0.11-1.0 ng/kg bw, could not be distinguished from the doses which failed to produce such symptoms in susceptible volunteers (up to 0.5 ng/kg bw). Factors that might explain this lack of correlation between dose (saxitoxin equivalents) and volunteer response are discussed along with previously published reports of PSP relay through the food web. It is suggested that the relay of algal toxins, particularly PSP, but possibly in combination with diarrheic shellfish poisons, may be responsible for scombrotoxicosis.

Animals↗

PubMedDate not supplied

Multimycotoxin dipstick enzyme immunoassay applied to wheat.

A membrane-based visual dipstick enzyme immunoassay for the simultaneous detection of up to five mycotoxins was developed. Multiple dots of the respective antibodies against aflatoxin B1 (AFB1), T-2 toxin (T-2), 3-acetyldeoxynivalenol (3-AcDON), roridin A (RA), and zearalenone (ZEA) were applied onto a dipstick membrane. The competitive immunoreactions were performed by incubation of the dipstick in a test tube containing sample solution and a mixture of the respective toxin-horseradish peroxidase conjugates. After a second incubation in enzyme substrate/chromogen solution, a complete suppression of the (blue) colour development of the respective dot was scored positive. Visual detection limits for AFB1, T-2, 3-AcDON, RA, and ZEA in buffer solution were 2 ng/ml, 5 ng/ml, 50 ng/ml, 30 ng/ml, and 5 ng/ml respectively. Using a simple extraction procedure, the detection limits of the multimycotoxin assay in artificially contaminated wheat samples were 30 ng/g, 100 ng/g, 600 ng/g, 500 ng/g, and 60 ng/g, respectively.

Food Contamination↗

PubMedDate not supplied

Two formats of enzyme immunoassay for the detection of saxitoxin and other paralytic shellfish poisoning toxins.

A competitive direct enzyme-linked immunofiltration assay for the detection of saxitoxin was developed, using polyclonal antibodies against saxitoxin and a saxitoxin-horseradish peroxidase conjugate. The test was performed in an eight-well plastic test device, in which antibody-coated nylon membranes were pressed tightly to an absorbent cellulose layer. Saxitoxin standard or sample extract solution, saxitoxin-conjugate, and enzyme substrate/chromogen solution were sequentially added on to the membrane. The test was evaluated visually by comparing the intensity of the resulting coloured (blue) dot with that of a negative control. The detection limits for saxitoxin in buffer solution and in shellfish tissue were 4 ng/ml and 80 ng/g respectively, with an assay time of less than 15 min. Under the conditions of the immunofiltration assay, decarbamoyl-saxitoxin, gonyautoxin 2/3, and neosaxitoxin standards (in buffer) gave a positive response at concentrations of about 10 ng/ml, 40 ng/ml, and 80 ng/ml, respectively. The relative cross-reactivity of the antibody to these PSPs was similar when determined using both direct and indirect (using a saxitoxin-bovine serum albumin conjugate) competitive enzyme immunoassays in microtitre plate format. In competitive direct microtitre plate assays, the 50% binding values found for saxitoxin, decarbamoyl-saxitoxin, gonyautoxin 2/3 and neosaxitoxin were 15 pg/ml, 47.5 pg/ml, 163.5 pg/ml, and 510 pg/ml respectively. In competitive indirect microtitre assay, the respective values were 138 pg/ml, 404 pg/ml, 1582 pg/ml, and 6982 pg/ml.

Animals↗

PubMedDate not supplied

Comparison of enzyme immunoassay and mouse bioassay for determining paralytic shellfish poisoning toxins in shellfish.

Sixty shellfish samples (mussel [Mytilus edulis], queen scallops [Chlamys opercularis] and king scallops [Pecten maximus]), collected during routine surveillance for paralytic shellfish poisoning (PSP) toxins conducted by the UK Ministry of Agriculture, Fisheries and Food and the Scottish Office Agriculture, Environmental Fisheries Department, were analysed for contamination with PSP toxins by mouse bioassay (MBA), and by a competitive direct enzyme immunoassay (Ridascreen Saxitoxin ELISA). Using the MBA as the reference method (detection limit: 350 micrograms kg-1), no false negative results out of a total of 45 MBA-positive samples and one false positive result out of a total of 15 MBA-negative samples were obtained by ELISA. The correlation coefficient for MBA-positive samples (n = 45) was 0.78. At the regulatory level for PSP toxins (800 micrograms kg-1), 39 of the samples were correctly determined by ELISA to be above or below this level, toxicity was overestimated in two samples, and underestimated in four samples.

Animals↗

PubMedDate not supplied

Rapid determination of citrinin in corn by fluorescence liquid chromatography and enzyme immunoassay.

A rapid assay procedure was developed for mycotoxin citrinin in corn using liquid-liquid extraction (LLE) cartridges. Ground corn was extracted with methylene chloride and 0.5 N phosphoric acid. The extract was added to an LLE cartridge containing a diatomaceous-earth adsorbant, previously impregnated with sodium bicarbonate solution. After aspiration to dryness, the cartridge was eluted with methanol-water (4 + 1), and aliquots were taken for quantitation by reversed-phase liquid chromatography with fluorescence detection. Recoveries of citrinin added to ground corn at 200-1600 ng/g ranged from 71.2 to 86.3%, with coefficients of variation between 4.1 and 10.6%. An indirect enzyme immunoassay was also evaluated, using sodium carbonate solution for extraction. Recoveries of citrinin added to ground corn at 200-2000 ng/g ranged from 53.2 to 67.2%, but the coefficients of variation varied between 18.4 and 51.5%. The LLE cartridge procedure offers the advantages of low solvent consumption and speed, and is amenable to automation.

Adsorption↗

PubMedDate not supplied

Immunoassay methods for paralytic shellfish poisoning toxins.

The current status of immunochemical techniques for analysis of paralytic shellfish poisoning (PSP) toxins is summarized. Important aspects regarding production of the biological reagents necessary for immunochemical methods, the characteristics of polyclonal and monoclonal antibodies against saxitoxin and neosaxitoxin, and the importance of test sensitivity and specificity are discussed. Applications of immunochemical techniques for PSP toxins include microtiter plate enzyme immunoasays and enzyme-linked immunofiltration assays for toxin detection, and immunoaffinity chromatography (IAC) for sample extract cleanup. A major advantage of enzyme immunoassay (EIA) is simplicity and rapidity of the test procedure, and higher sensitivity than other methods. However, quantitative agreement between EIA and mouse bioassay is dependent on antibody specificity and the toxin profile in the shellfish; thus, both over- and underestimation of total toxicity may occur. For screening purposes, however, EIAs offer major advantages over the mouse bioassay, which is criticized in Europe because of animal welfare. A major application of antibodies against PSP toxins is their use for extract cleanup by IAC, which gives highly purified extracts, thereby enhancing determination of PSP toxins by conventional physicochemical methods such as liquid chromatography. IAC can also be used to isolate PSP toxins for preparation of analytical standard solutions.

Animals↗

PubMedDate not supplied

[Demonstration of zearalenone metabolites in bile of breeding sows with fertility disturbances].

Bile of 52 gilts with reproductive problems have been analyzed for zearalenone and alpha-zearalenol. The applied methods, HPLC in combination with a fluorescence detector and with ELISA, were suitable for survey of biological materials with a complex matrix such as bile. The detection limit and recovery rate were 1 ng/ml and 75%, respectively. Zearalenone and alpha-zearalenol, which were present as glucuronide conjugates, were detected up to 40.0 ng/ml and 66.1 ng/ml in all samples analyzed. This suggests that zearalenone occurs more frequently in feed for pigs than it was recognized by feed analysis. Further investigation have to show, if the detection of zearalenone and alpha-zearalenol in samples of bile indicates a participation of these mycotoxins in reproductive problems.

Animals↗