农药的免疫化学，免疫亲和色谱及其在环境检测中的应用

Chlorsulfuron is a highly effective sulphonylurea herbicide in controlling a variety of weeds common to cereal grain. The sensitive crops could be damaged when the chlorsulfuron residue in soil is more than 0.5ng g-1.With the highly specific polyclonal anti-chlorsulfuron antibody that we developed first in the world, the antibody coated (enzyme labeled-hapten, E-H) direct competitive ELISA was established and the working curve was formed by using the blank soil extract as the diluent of chlorsulfuron. The half-maximal inhibition (IC50) of chlorsulfuron to antigen-antibody reaction was 0.1095μg mL-1 and the detection limit was 1.53 ng mL-1 under the optimized conditions. The average recovery was 84.69% at spiked level of 0.1μg chlorsulfuron per gram soil. The experiments showed thet the limit of quantification of chlorsulfuron in soil was 0.16 ng g-1. By E-H direct competitive ELISA and bioassay, the chlorsulfuron residue in soil samples from different farmlands were monitored. The results showed that the chlorsulfuron residua were 0.35ng g-1, 0.295ng g-1 and 0.234ng g-1 when the chlorsulfuron had been used for five years, three years and one year respectively. The results from ELISA were in correspondence with that from bioassay.Anti-chlorsulfuron immunosorbent was prepared by coupling purified antibodies to CDI-activated sepharose CL 4B and the immunoaffinity chromatography (IAC) for chlorsulfuron was established. Under the optimized conditions, the dynamic column capacity was up to 3.5μg chlorsulfuron mL-1 gel. The efficiency of enrichment by IAC was higher than 500 times when the concentration of chlorsulfuron in sample solution was lower than 1ng mL-1. The spiked sample at the level of 0.1μg chlorsulfuron per gram soil was separated by IAC and determined by E-H direct competitive ELISA (verified by HPLC), the average recovery was 94.09% with coefficient of variation (c.v.) of 10.11%, the limit of. quantification of chlorsulfuron in soil was 0.03 ng g-1..Polarization fluorescence immunoassay (PFIA) is a homophase competition Immunoassay based on the difference of polarization fluorescence between the fluorescent-labeled hapten (tracer) and its complex with specific antibody. To develop PFIA of chlorsulfuron, the fluorescent-labeled hapten was synthesized and the working concentration of tracer and antibody was optimized. Then, the standard solution of chlorsulfuron was added to the mixed solution of tracer and antibody to prepare PFIA standard curves by the changes of polarization fluorescence. The results showed that the regression equation was I=70.87+19.40LogC with the correlation factor of 0.9906, the IC50 was 83.97 ng mL-1 and the detection limit was 2.39 ng mL-1.The antibody coated (enzyme labeled-hapten, E-H) direct competitive ELISA for residue determination of carbaryl was established. This method was used to detect the carbaryl residue in cabbage and apple.

以对环境有影响的氯黄隆或甲黄隆为对象，应用免疫学原理，开展免疫化学研究，本项目在酶免疫化学研究的基础上，进行发光免疫化学研究。同时还利用抗体抗原的特异性和可逆性结合反应，研究免疫亲和色谱（IAC），将特异性抗体固相化作为免疫亲和色谱的基质，对痉掷牒痛炕＝ⅲ↖AC）和HPLC结合的方法，可以简化样本前处理，防止复杂样本中基质的干扰。