Studies On Immunochemistry For Residue Analysis Of Chlorsulfuron

Chlorsulfuron is a sulphonylurea herbicide. It is effective in controlling a variety of weeds common to cereal grain. The sensitive crops could be damaged when the chlorsulfuron residue in soil is higher than 0.5ng/g.With the developed polyclonal anti-chlorsulfuron antibodies, the immunoaffinity chromatography (IAC) for chlorsulfuron was established. Anti-chlorsulfuron immunosorbent was prepared by coupling purified antibodies (IgG) to GDI-activated Sepharose 4B. The optimized conditions of IAC were pH7.2 phosphate buffer (PB) as equilibrium medium, and 80% methanol as eluent. The dynamic column capacity was up to 3.5μ g chlorsulfuron ml-1 gel. The efficiency of enrichment by IAC was higher than 250 times when the concentration of chlorsulfuron in sample solution was lower than 2ng/ml. The blank sample extract (extracted by dichloromethane route) was cleaned up by IAC and the eluate was used as diluent of chlorsulfuron and the working curve prepared by E-H direct competitive ELISA. The half-maximal inhibition (IC50) was 10.14ng/ml, the limit of detection was 0.41431ng/ml and the average recovery was 94.09% at the spiked level of 0.1 μg/g with coefficient of variation (c.v.) of 10.11%. The working curve was well correspond to standard curve.The conditions (including pH values, ionic strengths, time of competitive reaction and so on) of E-H direct competitive ELISA were optimized. When the extracts (extracted by carbonate buffer route) were used as diluent of chlorsulfuron without IAC clean up step, the average IC50 from working curve was 0.1095ug/ml and the limit of detection was 1.53×10~3μg/ml. By E-H direct competitive ELISA and bioassay, the chlorsulfuron residue in soil samples from different farm land were determined in which the chlorsulfuron had been used for different years. The results showed that the chlorsulfuron residue were 0.35ng/g, 0.295ng/g, 0.234ng/g when the chlorsulfuron had been used for five years, three years and one years respectively. The average recovery at spiked level of 0.1μg/g was 84.69%. The results of ELISA for chlorsulfuron residue were well verified by by bioassay.PFIA is a competition method based on the difference of polarization fluorescencebetween the fluorescent-labeled hapten (tracer) and its immuno-complex with specific antibody. The optimized conditions for PFIA were 2×10-7mol/L antibody (150mP) and 6 ×10-8mol/L tracer. Under the optimized conditions, the standard solution of chlorsulfuron was added to the mixed solution of tracer and antibody to prepare PFIA standard curves by the change of polarization fluorescence. The resluts showed that the regression equation was I=70.87+19.40LogC, IC50 and IC20were 0.08397 @g/mL, 2.39 ×10-3μg/mL.