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E Usleber

Publications and source records attributed to E Usleber.

At least 19 recordsLinked to original sources
PubMed2003

Application of immunoaffinity chromatography and enzyme immunoassay in rapid detection of aflatoxin B1 in chicken liver tissues.

Existing physicochemical analytical methods for the determination of aflatoxins in animal tissues are expensive, cumbersome, and hazardous. To offer an alternative to these methods, a novel and highly sensitive immunochemical method for the rapid detection of aflatoxin B1 (AFB1) in chicken liver tissues is described in this study. Liver tissues were homogenized with cold methanol-acetone (50:50), followed by AFB1 extraction with methanol-acetone-PBS (25:25:50). The tissue extracts were, with or without further purification by immunoaffinity chromatography (IAC), applied to a highly sensitive direct ELISA for determination of AFB1. The detection limits for this assay were 15 +/- 0.77 pg/mL when standards and samples were dissolved in methanol-PBS (10:90) and 17 +/- 2.0 pg/mL when methanol-acetone-PBS (5:5:90) solution was used. The average recoveries of AFB1 were 54.3 to 65.5% in artificially contaminated tissue samples at 1 to 5 ng/g. In samples spiked with AFB1 at 1 ng/g, the method had diagnostic sensitivity and specificity of 100% for samples processed with IAC and 91.7 and 100%, respectively, for samples without IAC purification. The test was successfully applied to the detection of AFB1 in liver tissues from chickens that were experimentally dosed with AFB1. It is hoped that this test will be applicable in rapid detection of aflatoxins in poultry meats and in diagnosis of aflatoxicosis in chicken.

Aflatoxin B1↗
PubMed2001

Production of ultrasensitive antibodies against aflatoxin B1.

AIMS: To produce specific antibodies against the haptenic fungal toxin aflatoxin B1 (AFB1) and apply these antibodies in immunochemical assays for aflatoxins. METHODS AND RESULTS: Rabbits were immunized using an AFB1-bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml(-1) for AFB1, the EIA employing these antibodies is the most sensitive test for AFB1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB1, AFB2, AFG1 and AFG2. CONCLUSION: The antibodies described here are useful for the analysis of trace levels of aflatoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.

Aflatoxins↗
PubMed2001

Survey of Romanian slaughtered pigs for the occurrence of mycotoxins ochratoxins A and B, and zearalenone.

Blood serum, kidney, liver and muscle sample per animal were collected from slaughtered pigs (n = 52). The samples were analysed for ochratoxin A (OTA) and B (OTB) by HPLC methods. Zearalenone (ZEA) in serum was analysed by enzyme immunoassay. A total of 98% serum samples were OTA positive in the range of 0.05-13.4 ng/ml and 85% contained under 5 ng OTA/ml. The incidences of OTA in kidney and liver were very similar (79%, 75%) with mean levels of 0.54 ng/g and 0.16 ng/g, respectively. The lowest incidence (17%) and the lowest mean level contamination (0.15 ng/g) were in muscle samples. The mean distribution in tissues followed the pattern serum > kidney > liver > muscle (100%; 0.26%; 8.5%; 2.57%). No kidney, liver or muscle sample was found OTA positive above the maximum admitted limit in Romania (5 ng/g). No sample was found to be positive for OTB. A very similar OTA contamination (mean = 4.19 ng/ml, coefficient of variation = 34.4%) was observed in the serum samples (n = 10) collected from the same farm. A possible difference in regional distribution of OTA in Romania is suggested. Zearalenone was detected only in 17.3% of the serum samples with a maximum concentration of 0.96 ng/ml. This study shows the presence of OTA and ZEA in Romanian slaughtered pigs at levels comparable to those reported in other countries.

Animals↗
PubMed2000

Co-occurrence of ochratoxin A and citrinin in cereals from Bulgarian villages with a history of Balkan endemic nephropathy.

Cereal samples were collected in 1998 from Bulgarian villages without [control village (C), n = 20] or with [endemic villages (E); E1, n = 21; E2, n = 30; E3, n = 23] a history of Balkan endemic nephropathy (BEN). Sampling included foods (wheat, corn) and feeds (barley, oats, wheat bran). Analysis of ochratoxin A and citrinin was done by enzyme immunoassays (EIA), with detection limits of 0.5 and 5 ng/g, respectively. Ochratoxin A-positive results were confirmed by HPLC after immunoaffinity chromatography. Highest toxin levels were found in wheat, wheat bran, and oats. For ochratoxin A, the percentages of positives were 35% (C), 29% (E1), 30% (E2), and 47% (E3), the mean/median values of positives were 1.5/1.3 ng/g (C), 11/1.6 ng/g (E1), 18/1.6 ng/g (E2), and 3.5/1.5 ng/g (E3). For citrinin, 5.0% (C), 14% (E1), 3.3% (E2), and 13% (E3) were positive, and the mean/median values were 6.1/6.1 ng/g (C), 180/83 ng/g (E1), 10/10 ng/g (E2), and 84/20 ng/g (E3). Highest concentrations of ochratoxin (maximum = 140 ng/g) and citrinin (maximum = 420 ng/g) were found in samples from endemic villages. Co-contamination with ochratoxin A and citrinin was found for one sample (14% of positives) from village C and for six samples (22% of positives) from villages E1-E3. Citrinin levels in these samples were 2-200 times higher than those of ochratoxin A.

Animal Feed↗
PubMed2000

[Occurrence of zearalenone, alpha- and beta-zearalenol in bile of breeding sows in relation to reproductive performance].

Although zearalenone-induced reproductive disorders and the clinical appearance of hyperestrogenism were reproduced and documented quite often the role of zearalenone-contaminated fodder as a cause for fertility problems in sow breeding is still discussed controversial. Therefore the correlation of zearalenone and zearalenone-derivatives in bile (n = 794) and feed (n = 158) with fertility problems of unknown origin was investigated in this study. For the analysis of zearalenone and its derivatives in bile a HPLC/EIA combination was used. On the one hand, this procedure guaranteed the quantitatively reproducible detection, on the other hand, the investigation expenditure could be kept small with regard to a later effort in the routine diagnostics. The detection limits for zearalenone, alpha- and beta-zearalenol in bile were at 1.0 ng/ml, 1.0 ng/ml, and 3.0 ng/ml, respectively. Results were confirmed by GC-MS. Zearalenone and zearalenone-derivatives were detected in almost every bile analysed. The contamination rate was 96.2%. In opposition to recent investigations beta-zearalenol was perceived as a relevant metabolite in swine. The contamination rate of feeding stuffs was 25.9%. Incubation of samples with beta-glucosidase did not elevate the detected amounts of zearalenone. As the measurable concentrations in bile and fodder were only slightly correlated the analysis of bile represents a reasonable alternative for fodder investigation. However, a correlation between the occurrence of zearalenone, alpha- and beta-zearalenol in bile of sows and non-infectious reproductive disorders could not be established at the loading level found. These results are in line with those statements obtained in feeding experiments regarding the risk evaluation of zearalenone in sow reproduction.

Animal Feed↗
PubMed1999

Comparison of ELISA and HPLC for the determination of histamine in cheese.

A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for histamine in cheese was compared with a reversed-phase liquid chromatography (RP-HPLC) method. Cheese was homogenized with phosphate-buffered saline (PBS), centrifuged, and filtered, and the supernatant was diluted with PBS for CD-ELISA. For RP-HPLC, biogenic amines (histamine, tyramine, putrescine, and cadaverine) were derivatized with 9-fluorenylmethylchloroformate, followed by reversed-phase chromatography and fluorescence detection. Detection limits and mean recoveries (10-1000 mg/kg) were 2 mg/kg and 93% for CD-ELISA and 1 mg/kg and 99% for RP-HPLC, respectively. Analysis of 50 commercial cheeses according to both methods showed good agreement for histamine (r = 0.979; concentration range = 2-1800 mg/kg). At a threshold level of 10 mg/kg, the ELISA gave no false-negative and three false-positive results. The results show that the ELISA is suitable for the determination of histamine in cheese.

Biogenic Amines↗
PubMed1998

Immunochemical screening for antimicrobial drug residues in commercial honey.

Honey samples (n = 100; origin: various countries from Eurasia, Oceania, and the Americas) were analysed by enzyme immunoassays (EIA) for tetracyclines, streptomycin, and sulfathiazole. Considering antibody specificity, these EIAs are either quantitative (streptomycin) or qualitative (tetracyclines, sulfathiazole) tests. Honey extract purification was achieved by liquid-liquid partition (tetracyclines), and by solid phase extraction-immunoaffinity chromatography (streptomycin, sulfathiazole). Detection limits were 20 micrograms kg-1 (tetracycline equivalents), 10 micrograms kg-1 (streptomycin), and 50 micrograms kg-1 (sulfathiazole equivalents), with mean recoveries of 100-117%. A total of 42% of the samples was found positive by EIA; 25% were positive in one assay, 13% in two, and 3% were positive in all three tests. In the EIA for tetracyclines, 26% were positive, with 12 samples exceeding a level of 50 micrograms kg-1 (tetracycline equivalents). In the EIA for streptomycin, 19% were positive, with a mean concentration of 19 +/- 12 micrograms kg-1. In the sulfathiazole EIA, 16% of the samples were positive, with 13 samples exceeding a level of 100 micrograms kg-1 (sulfathiazole equivalents). However, when samples which were positive in the sulfathiazole EIA were reanalysed for sulfonamides by HPLC, no sulfa drugs could be detected. Experimental heating (40 degrees C) of honey spiked with sulfathiazole indicated that the sulfa drug(s) responsible for positive EIA results could be present a sugar derivatives.

Animals↗
PubMed1998

The potential of monoclonal antibodies against ampicillin for the preparation of a multi-immunoaffinity chromatography for penicillins.

Monoclonal antibodies (Mab) against ampicillin were prepared by immunization of mice with an ampicillin-keyhole limpet hemocyanin conjugate coupled by a glutaraldehyde method. Sensitivity and specificity of these antibodies were tested in a direct competitive enzyme immunoassay, in which an ampicillin-horseradish peroxidase conjugate prepared by a carbodiimide method served as the labelled antigen. According to their cross-reactivities with the other beta-lactam antibiotics, the Mabs could be divided into two groups, which are represented by the clones designated 1D1 and 3B5. While Mab 3B5 (IgG1) showed no major cross-reactions with the other penicillins frequently used in veterinary medicine except for amoxicillin (108%), Mab 1D1 (IgG2a) had marked cross-reactivities with most of the 17 tested beta-lactam antibiotics (e.g., amoxicillin 187%, penicillin G 31%, cloxacillin 30%, dicloxacillin 44%, and oxacillin 14%). The detection limits for ampicillin, calculated from the antibiotic concentration giving 30% binding inhibition, were 11.7 (Mab 3B5) and 16.6 ng ml-1 (Mab 1D1). To prepare multi-immunoaffinity chromatography columns, Mab 1D1 and a previously described antibody against cloxacillin (Mab 1F7) were each coupled to CNBr activated sepharose. The capacity of the resulting immunosorbents was approximately 6.6 and 5.4 micrograms ml-1 gel for ampicillin and cloxacillin, respectively. Recoveries of amoxicillin, ampicillin, cloxacillin, dicloxacillin, penicillin G and oxacillin (in buffer solutions) from the produced immunoaffinity columns were in the range from 67 to 100%.

Ampicillin↗
PubMed1996

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat.

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 micrograms/kg (DON) and 17 micrograms/kg (ZEA), maximum concentrations were 1800 micrograms kg-1 and 120 micrograms kg-1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1% and 0.7% of the samples, at at maximum level of about 100 micrograms kg-1. Only one sample was positive for T-2 (55 micrograms/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.

Bulgaria↗
PubMed1996

Immobilization of small molecules on solid matrices: a novel approach to enzyme-linked immunosorbent assay screening for saxitoxin and evaluation of anti-saxitoxin antibodies.

A novel enzyme-linked immunosorbent assay (ELISA) technology was developed for detecting saxitoxin or evaluation of anti-saxitoxin antibodies, which is based on non-covalent immobilization "free' saxitoxin to Maxisorp microtitre plates. The effect of pH on immobilization was studied in media with wide-range buffering capacities (piperazine-glycylglycine and barbiturate buffers). Increasing pH resulted in better responses, although this was mainly due to non-specific interactions. At pH 10.0, however, saxitoxin immobilization was quite effective and specific. The same pattern was found under four different conditions; absence vs presence of bovine serum albumin precoating and absence vs presence of 150 mM NaCl. The best results (high specific response) were achieved with bovine serum albumin precoating in the presence of 150 mM NaCl. The method of choice involved precoating Maxisorp with 5 micrograms/ml albumin followed by addition of 5 microM saxitoxin in 0.01 M piperazine-glycylglycine buffer, pH 10.0. The efficacy of this technology was demonstrated on a polyclonal rabbit anti-saxitoxin antibody and compared with a conventional ELISA of saxitoxin using saxitoxin-bovine serum albumin conjugate as the coating antigen. In the experiments investigating cross-reactivities of various saxitoxin derivatives based on a competitive assay, significantly greater sensitivity was achieved with the novel approach, e.g. 35 pM saxitoxin could be detected (3 x 10(4) times lower concentrations than using the conjugate). The assay works well with mussel tissue homogenates, and because it does not require the use of the covalent saxitoxin-carrier conjugates it offers a simpler alternative to the traditional ELISA for saxitoxin.

Antibodies↗
PubMed1995

Use of monoclonal antibodies for the analysis of mycotoxins.

Hybridomas producing monoclonal antibodies against aflatoxin M1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, 3-acetyl-deoxynivalenol, fusarenon X, and roridin A were developed after immunization of BALB/c mice and fusion of the splenocytes with myeloma cells. The antibodies were characterized in terms of immunoglobulin subclass, sensitivity, and specificity. The use of these antibodies in competitive enzyme immunoassays, either as microtiter plate assays or membrane-based quick tests, as well as for the production of immunoaffinity columns is described. The advantages and disadvantages of monoclonal antibodies compared to polyclonal antisera for the improvement of mycotoxin analysis are discussed.

Animals↗
PubMed1995

An indirect enzyme immunoassay for the mycotoxin citrinin.

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%.

Animals↗
PubMed1994

Immunochemical detection of antibiotics and sulfonamides.

To control the maximum residue limits (MRLs) for residues of veterinary drugs in food of animal origin, according to EU regulations, a broad spectrum of sensitive analytical methods is required. One effective approach is the development of immunoassays, particularly for screening purposes. Strategies for the production of specific polyclonal and monoclonal antibodies against beta-lactams, tetracyclines, streptomycin, chloramphenicol, sulfonamides and trimethoprim, are outlined, as well as methods for the synthesis of the respective enzyme-labelled antigens. The sensitivity and the specificity of the antibodies were characterized, and the immunochemical test systems were designed as quantitative routine tests (microtitre plate format) and as rapid qualitative tests (membrane-based assay formats). The detection limits of the assays were found to be well below the regulatory limits. The range of recovery, for the analysis of artificially contaminated samples, was between 68 and 104%. In principle, the enzyme immunoassays for antimicrobial drugs showed the advantage of sensitivity and speed together with the simplicity of manipulations involved in the procedure. However, because of the results of the specificity studies, as well as the possibility of false positive results owing to unspecific inhibition of the assay, confirmation of immunoassay results is still required for all legal and statutory purposes.

Animals↗
PubMed1994

Enzyme immunoassay for the detection of isoxazolyl penicillin antibiotics in milk.

Polyclonal antibodies were raised against isoxazolyl penicillins in rabbits after immunization with a cloxacillin-human serum albumin conjugate. The antisera were tested in direct and indirect competitive enzyme immunoassays (EIAs), using glucose oxidase or horseradish peroxidase conjugates of oxacillin, cloxacillin, or dicloxacillin, respectively, as the labelled antigen. The relative cross-reactivities of each test system with oxacillin, cloxacillin, and dicloxacillin, determined from the amount of antibiotic required for 50% inhibition of labelled antigen binding, were dependent on the antibiotic used as the labelled antigen. Other beta-lactam antibiotics did not cross-react in these test systems. In a direct EIA using a cloxacillin-horseradish peroxidase conjugate, cloxacillin and dicloxacillin in milk were detected at levels of 10 and 30 ng ml-1; the average recoveries at these levels were 102 and 84%, respectively.

Animals↗
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↗