| 1. Development of ELISA method for detecting tartrazine in urine and analyzation of exposure level on random samplesPeople attach great importance to food safety with the improvement of material life. Tartrazine is a kind of widely used food colorants with bright yellow color. It can improve sensory quality of food. According to the standard of edible pigment in China, it's safe to use tartrazine within the limitation, because most of tartrazine eliminates from body in prototype. However, some businesses use tartrazine illegally and excessively in order to increase sales. It dues to the increase of tartrazine internal exposure and causes adverse effect to human body. Therefore, we need to monitor the exposure level of tartrazine in human body. Through the preparation of monoclonal antibody, we established a highly sensitive ELISA method to detect tartrazine and monitor the internal exposure level in people. We collected and analyzed the data of tartrazine exposure level in people through the analyzation of 576 human urine samples.Objective:Analyze exposure level of tartrazine on random samples. Methods:TAR-BSA and Freud's immune adjuvant were used to immunize the BALB/c mice. The monoclonal antibody was produced by hybridoma which was screened through cell fusion technique. Finally, we developed the indirect competitive ELISA method to analyze 576 human urine samples from Zhejiang Province, China. Results:The concentration of purified monoclonal antibody was 2.72mg/mL. There was no cross-reactive with other food colorings, such as sunset yellow, allura red, indigo, carmine, erioglaucine and amaranth. The LOD and IC50 of this method were 0.31ng/mL and 2.96ng/mL. The coefficient of variation of intra-assay was from 3.69% to 4.89% while the inter-assay was from 1.17% to 4.54%. The recovery rate was from 99.14% to 108.40%. Compared with traditional HPLC, these two detection methods showed good correlation (R2=0.99, n=6).576 urine samples were analyzed by the indirect competitive ELISA method, and the detection rate was 100%. Concentration was from 0.36ng/mL to 38.31ng/mL. The mean concentration and geometric mean were 5.15ng/mL and 3.70ng/mL. The mean concentration of male (5.36ng/mL) was a little higher than female (4.86ng/mL), but there was no significant difference. The concentration of tartrazine in people under 50 years old was significantly higher than people over 50 years old (P<0.05). Among them, people in 41 to 50 years old had significantly higher concentration of tartrazine than people in 50 to 60 (P<0.05). Conclusions:The indirect competitive ELISA method based on monoclonal antibody was sensitive and reliable for the detection of tartrazine in human urine samples. We collected exposure data of tartrazine in random samples and analyzed characteristics of exposure level.2. Development of ELISA method for detecting diethylstilbestrol in urine and analyzation of exposure level on random samplesThe demand of meat and aquatic products is increasing with the improvement of living standard. As a result, aquaculture is developing rapidly. In order to raise output and shorten the growth cycle, growth hormones, such as diethylstilbestrol, are added into fodder illegally by feeders or manufacturers. Finally, it causes the pollution of water and soil. At the same time, people expose to diethylstilbestrol through the food chain. However, as a kind of estrogen, diethylstilbestrol can affect growth and development of teenagers. Therefore, through the preparation of monoclonal antibody, we established a highly sensitive ELISA method to detect diethylstilbestrol and monitor the internal exposure level in people. We collected and analyzed the data of diethylstilbestrol exposure level in people through the analyzation of 576 human urine samples.Objective:Analyze exposure level of diethylstilbestrol on random samples. Methods: DES-BSA with Freud's immune adjuvant and Quick-antibody immunologic adjuvant were used to immunize the BALB/c mice, separately. The monoclonal antibody was produced by hybridoma which was screened through cell fusion technique. Finally, we developed the indirect competitive ELISA method to analyze 576 human urine samples from Zhejiang Province, China. Results:The concentration of purified monoclonal antibody was 8.32mg/mL. There was no cross-reactive with two kinds of estrogens and two structural analogues with diethylstilbestrol. The LOD and IC50 of this method were 0.29ng/mL and 3.33ng/mL. The coefficient of variation of intra-assay was from 8.79% to 15.74% while the inter-assay was from 3.25% to 10.23%. The recovery rate was from 91.20% to 118.07%. Compared with traditional HPLC, these two detection methods showed good correlation (R2=0.99, n=6).576 urine samples were analyzed by the indirect competitive ELISA method, and the detection rate was 98.78%. Concentration was from LOD to 42.85ng/mL. The mean concentration and geometric mean were 4.70ng/mL and 3.50ng/mL. The mean concentration of male was 5.07ng/mL, higher than female (P<0.05). People in 41 to 50 years old had the highest mean concentration of diethylstilbestrol (5.60ng/mL). While people over 50 years old had lower concentration. Conclusions:The indirect competitive ELISA method based on monoclonal antibody was sensitive and reliable for the detection of diethylstilbestrol in human urine samples. We collected exposure data of diethylstilbestrol in random samples and analyzed characteristics of internal exposure level. |
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