Comparison of homologous and heterologous formats in nanocolloidal gold-based immunoassays for parathion residue determination.

The effectiveness of homologous and heterologous formats in a nanocolloidal gold-based immunoassay for pesticide residue determination was investigated. Parathion, one of the most toxic organophosphorus pesticides, was used as the target analyte. One-step homologous and heterologous test strips based on a nanocolloidal gold-labeled monoclonal antibody were developed for the rapid detection of parathion residues. The results showed that the heterologous format was more effective than the homologous format, being more sensitive, more specific to parathion and more tolerant of matrix interferences. The best competitive hapten was found to have a moderate heterology and the opposite electronic distribution to the immunizing hapten. The detection limits for parathion using the preferred heterologous strip were 1 µg/L in water samples and 5 µg/kg in soil and food samples.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural samples.

A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC(50) and IC(10) of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries in six agricultural products were between 79.5% and 118.0%, with the intra-assay coefficient of variation being less than 8 %. The limit of detection for all tested products was 30 ng/g. To the best of our knowledge, this assay has the best specificity among all the published research on ELISAs for chlorpyrifos.

Homology modeling of anti-parathion antibody and its interaction with organophosphorous pesticides and analogues.

The mechanism of specific recognition in pesticide immunochemistry was investigated by computer-based strategy, and a rapid method for the identification of antibody specificity was developed. Based on the previously produced anti-parathion monoclonal antibody (mAb), the DNA sequence was analyzed by polymerase chain reaction (PCR). From the translated amino acid sequences, a three-dimensional structure of the antibody was constructed by homology modeling method, and then it was coordinated by 1 ns molecular dynamics under the explicit solvent condition. The stereochemical property and folding quality were further assessed by Procheck and Profile-3D. The self-compatibility score for the antibody model was 98.7, which was greater than the low score 46.2 and close to the top score 102.6. In addition, parathion and several structural analogues were docked to the constructed antibody structure. The docking results showed that the interaction energy (-40.54 kcal/mol) of antibody-parathion complex was the lowest among all the tested pesticides, which accounted for the high specificity of the antibody to parathion and perfectly matched with the experimental data. Moreover, three residues, Phe165, Asp107 and Thr100 were recognized as the most important residues for antibody reacting with parathion. The interaction energy negatively correlated with the antibody specificity.

Specific antibody for pesticide residue determination produced by antibody-pesticide complex.

A new method for specific antibody production was developed using antibody (Ab)-pesticide complex as a unique immunogen. Parathion (PA) was the targeted pesticide, and rabbit polyclonal antibody (Pab) and mouse monoclonal antibody (Mab) were used as carrier proteins. The Ab-PA complexes were generated by conjugating the two antibodies with an excessive dosage of PA. It was shown that the sensitivity of homologous enzyme-linked immunosorbent assay (ELISA) using the new antibodies was similar to that using original antibodies. However, the new mouse Pab had not only similar positive recognizing spectrum as the original Mab, but also a significantly improved sensitivity in heterologous ELISA when some recognizable competitors were applied. IC(50) value of ELISA based on a combination of the new mouse Pab and hapten 9 was 0.24 ng/mL, which was 445.54 times as that of the homologous ELISA. An Ab-pesticide complex may be a suitable alternative immunogen for producing highly specific antibody to improve the immunoassay sensitivity.

Monoclonal antibody produced by heterologous indirect enzyme-linked immunosorbent assay and its application for parathion residue determination in agricultural and environmental samples.

A heterologous indirect enzyme-linked immunosorbent assay (ELISA) was developed, which was based on monoclonal antibody (Mab) to determine parathion residue in agricultural and environmental samples. Eight Mabs were produced by introducing the heterologous indirect ELISA into the screening procedure. It was shown that these Mabs had more broad-reactivity with twenty competitors than that of 5H7 (Mab produced by homologous screening). So it became much easier using these new Mabs to develop heterologous immunoassays. In addition, all the developed heterologous ELISAs could be used to determine parathion residue in semiquantitative or quantitative level, and their detection limitation (LOD) was around 2 ng/mL. Compared with the previous heterologous ELISA (hapten 11/5H7) with IC(50) of 13.3 ng/mL, a better heterologous ELISA (hapten 11/1E1) was obtained with IC(50) of 3.8 ng/mL, which improved the sensitivity 3.5 times. Finally, the latter was applied to parathion residue determination in spiked agricultural and environmental samples without extraction or cleanup. The average recoveries of parathion added to water, soil, cucumber, Chinese cabbage and carrot were between 80.4 % and 111.8 %. The LOD for water and soil samples was 5 ng/mL, and the LOD for cucumber, rice and corn samples was 10 ng/mL.

Pubertal exposure to saisentong: effects on thyroid and hepatic enzyme activity in juvenile female rats.

Saisentong, as a thiadiazole fungicide, is widely used in China. The structure of saisentong shows a closer relationship to N, N-methylene-bis (2-amino-1, 3, 4-thiadiazole) (Bis-A-TDA), which is a teratogen. A few studies have shown that some of the thiadiazole fungicides act as endocrine disruptors via disturbance in thyroid hormone homeostasis. Little is known about the effect of pubertal exposure to saisentong on the development in pubertal female rats. Based on the protocol of the 20-Day Pubertal Female Assay, we attempt to estimate the possible effect of exposure to saisentong on thyroid hormone and hepatic enzyme activity in female rats. Postnatal days (PND) 22 old SD rats were administered with saisentong daily by oral gavage at doses of 0, 5, 10 or 15mg/kg/day for 20 days. After treatment, the rats were sacrificed for blood collection; the reproductive organs, liver, pituitary, adrenal and thyroid gland were harvested. The results indicated that saisentong administration increased thyroid weight and thyroid stimulating hormone (TSH) concentrations, and induced hepatic uridine diphosphate glucuronyl transferase (UDPGT) activities in the highest-dose group, although not statistically significant. The high dose caused a decrease in weight at vaginal opening (VO), but the age at VO was unaffected by saisentong in all treatment groups. No histological changes were observed in uterus and ovaries. These data and changes demonstrate that saisentong is a potential thyroid disrupter in female rat following exposure during development, but does not affect the development of pubertal female rats.

Pubertal exposure to bismerthlazol inhibits thyroid function in juvenile female rats.

Bismerthiazol, as a thiadiazole fungicide, is widely used in China. The structure of bismerthiazol shows a close relationship to N,N-methylene-bis (2-amino-1,3,4-thiadiazole) (Bis-A-TDA), which is a teratogen. A few studies have shown that this fungicide acts as an endocrine disruptor in mature rats via disturbance in thyroid hormone homeostasis at a certain dose. Little is known about the effect of pubertal exposure to bismerthiazol on the development in pubertal female rats. Based on the protocol of the 20-Day Pubertal Female Assay, postnatal days (PND) 22 old SD rats were administered with bismerthiazol daily by oral gavage at doses of 0, 10, 20 or 40 mg/kg/day for 20 days. After treatment, the rats were sacrificed for blood collection; the reproductive organs, liver, pituitary, adrenal and thyroid gland were harvested. The results indicated that changes in thyroid endpoints following bismerthiazol administration were decreased serum triiodothyronine (T(3)) and thyroxine (T(4)) concentrations, increased thyroid stimulating hormone (TSH) concentrations and the ratios of thyroid gland weights, and produced thyroid gland hyperplasia. No histological changes were observed in the uterus and ovaries; moreover, the age and weight at vaginal opening (VO) were unaffected by bismerthiazol in all treatment groups. These data and changes demonstrate that bismerthiazol is likely a thyroid disrupter in female rat following exposure during development, but does not affect the development of pubertal female rats. Further studies using environmentally relevant doses are needed for hazard identification.