Monoclonal antibody-based enzyme-linked immunosorbent assays for the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN).

This study aimed at developing competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) using a monoclonal antibody (mAb). Of the five EPN derivatives (haptens) prepared for use as an immunogen or as a competitor, two of them were used as the immunogen for the production of the mAbs. By using the antibody with the highest specificity and a coating antigen (hapten-OVA conjugate), a competitive indirect ELISA was developed, which showed an IC(50) of 2.9 ng/mL with a detection limit of 0.3 ng/mL. A competitive direct ELISA using a different antibody and an enzyme tracer was also developed, which showed an IC(50) of 0.6 ng/mL with a detection limit of 0.09 ng/mL. The mAbs in both assays showed negligible cross-reactivity with other organophosphorus pesticides. The recoveries of EPN from spiked samples determined by the developed ELISA ranged from 59 to 143%. Dilution of the samples improved the recovery. The assay performance of the present ELISAs based on the mAb was compared with that of the EPN ELISAs based on polyclonal antibodies (pAbs) that had been developed previously and was found to be better in dynamic response.

Development of enzyme-linked immunosorbent assays for the organophosphorus insecticide EPN.

Competitive enzyme-linked immunosorbent assays (ELISAs) in indirect and direct format were developed for the quantitative detection of the organophosphorus insecticide EPN. Five EPN derivatives (haptens) were synthesized and coupled to carrier proteins to use as an immunogen or as a competitor. Rabbits were immunized with two of the five haptens coupled to KLH for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin (OVA). Using the serum with the highest specificity and a coating antigen (hapten-OVA conjugate), an indirect (antigen-coated) ELISA was developed, which showed an IC(50) of 5.6 ng/mL with a detection limit of 0.2 ng/mL (20% inhibition). A direct (antibody-coated) ELISA using an enzyme tracer (hapten-enzyme conjugate) was also developed, which showed an IC(50) of 8.4 ng/mL with a detection limit of 0.9 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticide parathion-ethyl only in the direct ELISA. The recoveries of EPN from spiked samples determined by the indirect ELISAs were between 37 and 164%.

Effects of soybean isoflavone extract on the plasma lipid profiles and antioxidant enzyme activity in streptozotocin-induced diabetic rats.

The present study evaluated the effects of various dosages of soybean isoflavone extract on lipid profiles, lipid peroxidation and antioxidant activities in streptozotocin-induced diabetic rats. The one normal control group was fed an AIN-76-based experimental diet and four diabetic groups were fed the same diet, supplemented with four different levels of soybean isoflavone extract for seven weeks. The daily dosages of pure isoflavone for four diabetic groups were set to be 0 mg (diabetic control), 0.5 mg (ISO-I), 3.0 mg (ISO-II) and 30.0 mg (ISO-III) per kilogram of body weight, respectively. The plasma total cholesterol levels and the TBA-reactive substances contents in the liver and kidney were significantly lowered in ISO-II and ISO-III groups compared to those in the diabetic control group. The levels of plasma HDL-cholesterol, plasma vitamin A and hepatic superoxide dismutase were significantly increased in those two groups compared with the diabetic control group. The present study demonstrated the possibility that the diets supplemented with 3.0 mg and 30.0 mg of soybean isoflavone extract may have beneficial effects on the plasma lipids, tissue lipid peroxidation and partly on antioxidant system in diabetic animals and there were no significant differences between the ISO-II and ISO-III groups. The results suggest that the effective daily dosage level of isoflavone for improving lipid metabolism in diabetic rats may be above 3.0 mg per kilogram body weight.

Soybean isoflavone extract improves glucose tolerance and raises the survival rate in streptozotocin-induced diabetic rats.

The present study evaluated the effect of various dosages of soybean isoflavone extract on body weight changes, glucose tolerance and liver function in streptozotocin-induced diabetic rats. One group of normal rats (normal control) was fed an AIN-76-based experimental diet and four groups of diabetic rats were fed the same diet supplemented with four different levels of soybean isoflavone extract for seven weeks. The daily dosages of pure isoflavone for four diabetic groups were set to be 0 mg (diabetic control), 0.5 mg (ISO-I), 3.0 mg (ISO-II) and 30.0 mg (ISO-III) per kilogram of body weight, respectively. The daily consumption of isoflavone at the level of 3.0mg per kilogram of body weight resulted in the suppression of body weight loss and increased the survival rate of diabetic animals one and half times compared to that of the diabetic control group. Blood glucose levels in a fasting state and after the oral administration of glucose were significantly lower in the ISO-II group during the oral glucose tolerance test. The ISO-II group showed a tendency to elongate the gastrointestinal transit time. The activity of serum aminotransferases, indicator of liver function, was not negatively affected by any intake level of isoflavone. The present study demonstrated that the soybean isoflavone extract may be beneficial to diabetic animals by improving their glucose tolerance and suppressing weight loss without incurring hepatotoxicity at the daily dosage of 3.0 mg per kg of body weight.