OBJECTIVETo prepare immunoaffinity column of zearalenone.

METHODSThe zearalenone immunoaffinity column (IAC) was prepared by coupling CNBr-activated Sepharose 4 Fast Flow (4FF) with the anti-zearalenone monoclonal antibody which was purified by caprylic acid-ammonium sulfate method. The coupling reaction was identified by UV-absorbance measurements, and the IAC prepared was evaluated by indirect-competition ELISA and HPLC.

RESULTSThe column capacity was determined to be 0.40 microg when using 0.5 ml of CNBr activated Sepharose 4FF and 350 microg of purified anti-zearalenone monoclonal antibody. The mean true recoveries were in the range 76.33% - 90.10% and RSD was 6.68% - 10.93% at levels of 60 microg/kg - 300 microg/kg. 30 samples of wheat and maize were detected by the anti-ZEN IAC produced by the laboratory, 17 samples were observed to be contaminated in a comparable range from 31.33 microg/kg - 377.84 microg/kg. Detection limit based on a signal-to-noise ratio 3:1 was 10. 00 microg/kg for ZEN in wheat and maize.

CONCLUSIONIAC, a simple separating method which is used in ZEN extraction from cereals, is able to purify and condense ZEN in one step. The cost of detection can be lowered down because the IAC developed is hopefully to substitute the imported IAC.

Antibodies, Monoclonal ; isolation & purification ; Chromatography, Affinity ; methods ; Chromatography, High Pressure Liquid ; Zearalenone ; antagonists & inhibitors ; immunology

A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were from 25 to 750 ppb and from 12.5 to 100 ppb, respectively. The detection limit of both methods as measured with standard solutions was 10 ppb. The intra-plate and inter-well variation of both ELISAs were less than 10%. The IC50 values for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol compared to ZEN were 108.1, 119.3, 114.1, and 130.3% for the ZEN-coated ELISA. These values were 100.7, 120.7, 121.6, and 151.6% for the anti-ZEN antibody-coated ELISA. According to the anti-ZEN antibody-coated ELISA, the average recovery rates of ZEN from spiked animal feed containing 150 to 600 ng/mL of ZEN ranged from 106.07 to 123.00% with 0.93 to 2.28% coefficients of variation. Our results demonstrate that the mAb developed in this study could be used to simultaneously screen for ZEN and its metabolites in feed.
Aminooxyacetic Acid/*chemistry ; Animals ; Antibodies, Monoclonal/*immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Female ; Inhibitory Concentration 50 ; Mice ; Mice, Inbred BALB C ; Reproducibility of Results ; Serum Albumin, Bovine/chemistry ; Zearalenone/*immunology

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, is known to cause testicular toxicity in animals. In the present study, the effects of ZEA on spermatogenesis and possible mechanisms involved in germ cell injury were examined in rats. Ten-week-old Sprague-Dawley rats were treated with 5 mg/kg i.p. of ZEA and euthanized 3, 6, 12, 24 or 48 h after treatment. Histopathologically, spermatogonia and spermatocytes were found to be affected selectively. They were TUNEL-positive and found to be primarily in spermatogenic stages I-VI tubules from 6 h after dosing, increasing gradually until 12 h and then gradually decreasing. Western blot analysis revealed an increase in Fas and Fas ligand (Fas-L) protein levels in the ZEA-treated rats. However, the estrogen receptor (ER)alpha expression was not changed during the study. Collectively, our data suggest that acute exposure of ZEA induces apoptosis in germ cells of male rats and that this toxicity of ZEA is partially mediated through modulation of Fas and Fas-L systems, though ERalpha may not play a significant role.
Animals ; Antigens, CD95/*immunology ; Apoptosis/*drug effects/immunology ; Estrogens, Non-Steroidal/*toxicity ; Fas Ligand Protein/*immunology ; Histocytochemistry ; Immunoblotting ; In Situ Nick-End Labeling ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatocytes/cytology/*drug effects/immunology ; Spermatogenesis/drug effects/immunology ; Spermatogonia/drug effects/immunology ; Testis/cytology/*drug effects/immunology ; Zearalenone/*toxicity