Broad-spectrum detection of zeranol and its analogues by a colloidal gold-based lateral flow immunochromatographic assay in milk.

Based on colloidal gold and broad-spectrum monoclonal antibody that binds to zeranol and its five analogues with high sensitivity, a lateral flow immunochromatographic assay (LFIA) in a competitive format was developed to specifically determine residues of zeranol, an illegal growth promoter in livestock. In this study, the assay had high sensitivity and was broad-spectrum only for zeranol and its five analogues, and the results were obtained within 10 min without needing sophisticated procedures. The cutoff values for zeranol and its five analogues were 10 ng/mL, and the IC50 values for zeranol, ß-zearalanol, zearalanone, α-zearalenol, ß-zearalenol and zearalenone were 1.250, 1.800, 1.775, 1.225, 1.709 and 1.319 ng/mL, respectively. The recovery rates were ranged from 85.6 to 93.9%, with the coefficient of variations less than 12.4%. The results demonstrated that the LFIA could be used for rapid, simultaneous, semi-quantitative and quantitative detection of residues of zeranol and its five analogous in milk.

Preparation of a broad-spectrum anti-zearalenone and its primary analogues antibody and its application in an indirect competitive enzyme-linked immunosorbent assay.

A broad-spectrum monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed for rapidly screening zearalenone (ZEN) and its primary analogues in various samples using an easy sample preparation procedure. Primarily, a group-specific mAb, 6C2, was produced, which had IC50 values for ZEN, α-zearalenol, ß-zearalenol, α-zearalanol, ß-zearalanol and zearalanone of 114.0, 127.4, 290.4, 114.9, 205.6 and 257.1 ng L-1, respectively. The limit of detection and limit of quantitation of this method for ZEN and its five primary analogues in various matrix samples ranged from 114.2 to 812.3 ng L-1 and 237.1 to 1653.9 ng L-1, respectively. The recoveries of the above samples spiked with ZEN and its five primary analogues were in the range of 62.9-113.6%. The CVs were less than 13.2%. A good correlation (R2 = 0.995) between the ic-ELISA results and the HPLC-MS/MS results for swine feeds supported the reliability of the developed ic-ELISA.

Preparation of highly specific anti-zearalenone antibodies by using the cationic protein conjugate and development of an indirect competitive enzyme-linked immunosorbent assay.

Although anti-zearalenone (ZEN) antibodies have been widely prepared, these antibodies cross-react with α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and ß-zearalanol (ß-ZAL). To overcome this problem and improve the specificity of immunoassays, we produced anti-ZEN antibodies based on a ZEN-cationic protein conjugate. In this study, ZEN was coupled with cationic bovine serum albumin (cBSA) via a Mannich reaction. After BALB/c mice were immunized with ZEN-cBSA, an immunological response was rapidly induced. The titers of the polyclonal antisera and monoclonal antibody were 30,000 and 20,000, respectively. Cross-reactivity (CR) values of the anti-ZEN polyclonal antisera and monoclonal antibody with the 5 analogs were <7% and <2%, respectively. An indirect competitive enzyme-linked immunosorbent assay based on the monoclonal anti-ZEN antibody was established. The recovery rates of ZEN in spiked cereal and feed were in the range of 80%-120% with coefficients of variation <15%. The intra-assay variation and inter-assay variation in assay buffer were both <5%. Therefore, the results demonstrated a feasible approach for preparing highly specific, higher titer and more rapidly induced antibodies against ZEN by using a ZEN-cBSA conjugate as the immunogen instead of currently used immunogens.

[Preparation and preliminary application of monoclonal antibody against alpha-zearalanol].

AIM: To prepare monoclonal antibody (mAb) against alpha-zearalanol (alpha-ZER) and develop an immunoassay for the detection of alpha-ZER and its analogues residues in food derived from animal tissues. METHODS: alpha-ZER was conjugated to BSA as immunogen to immunize BALB/c mice and mAb were prepared by hybridoma technique. mAb's characteristics (titer, Ig subclass, specificity and relative affinity) were identified by ELISA. Standard inhibitive cure was made and sensitivity of the mAb was identified. 37 samples derived from animal liver were detected for alpha-ZER residues by competitive ELISA established in the study. RESULTS: 8 hybridoma cell lines stably secreting anti-alpha-ZER mAb were obtained. The titer of one of them (4E5) was 5.142x10(7). The Ig subclass was IgG1. The mAb was specific for alpha-ZER and its analogues and had no cross-reactivity with other compounds.8 positive results were found from the 37 samples derived from animal liver which were negative detected by HPLC. CONCLUSION: Anti-alpha-ZER mAb has been prepared successfully. A rapid method using the mAb for detecting alpha-ZER and its analogues residues in animal tissues has been established.

Development and evaluation of immunoassay for zeranol in bovine urine.

A high affinity polyclonal antibody-based enzyme linked immunosorbent assay (ELISA) was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1:5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 microg/ml, and the detection limit was 0.02 microg/ml for the assay. The overall recoveries and the coefficients of variation (CVs) were in the range of 82% to approximately 127% and 3.5% to approximately 8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol (ranging from 0.2 to 10 microg/ml) were detected by the ELISA and liquid chromatography (LC) method, and good correlations were obtained between the two methods (R(2)=0.9643). We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an alternative for the conventional LC method for zeranol in bovine urine.

A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle.

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp. toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n = 12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n = 20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n = 18) and the within-assay variation ranged between 4.5 and 9.0%.

Free and conjugated zeranol residues determined by radio-immunoassay in urine and plasma of calves treated with Forplix.

Anti-zeranol 7-hemisuccinate-bovine serum albumin was raised in rabbits. This antiserum was used in a radio-immunoassay of zeranol residues in urine and plasma of calves implanted with Forplix. The antibody was specific for zeranol but cross-reacted with its metabolite zearalenone and the mycotoxin zearalenone. Detection limits in plasma and in urine were 100 pg/ml and 1 ng/ml respectively. In veal calves treated with zeranol containing implant, no residues were detected in plasma even if plasma proteins were hydrolysed with pronase before the radio-immunoassay. Free and conjugated residues in urine were easily measured. The urine concentration of conjugated residues increased markedly after the 20th day of treatment and was still high (19 ppb) at day 40.

Preparation and properties of monoclonal antibodies to the anabolic agent zeranol.

Monoclonal antibodies have been produced using two haptens, zeranol-7-hemisuccinate coupled to bovine serum albumin and zeranol-16-carboxypropyl ether coupled to human serum albumin. An assessment of cross-reactivity demonstrated that the monoclonal antibody raised against the 7-hemisuccinate derivative reacted with zeranol (100%), talernol (12%), zearalenone (17%), zearalanone (100%) and alpha- and beta-zearalenol (17% and less than 0.01%, respectively). In contrast the antibodies to the 16-carboxypropyl ether derivative reacted only with zeranol (100%) and also with a alpha-zearalenol (13-16%). All monoclonal antibodies were more specific than the polyclonal antibodies raised to the same haptens by conventional methods using sheep.

Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies.

To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6'-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The porcine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.