Chemiluminescence Enzyme Immunoassay For Determination Of SD-methoxy-pyrimidine

This paper was about chemiluminescent technology combined with immunological techniques, namely, chemiluminescence enzyme immunoassay for quantitative determination of antibiotic-methoxy-pyrimidine sulfonamide:residues in foodstuffs. An anti-SMD lgG was passively adsorbed onto the wells of polypropylene plates. The labeled conjugates were horseradish peroxidase (HRP) or alkaline phosphatase (ALP) conjugate of SMD. The unbound antigen was washed, then a substrate was put into well, chemiluminescent intensity was determined and the content of antigen could be calculated by regression equation. According to the basic theory, Luminol was used as the substrate of HRP, AMPPD was used as the substrate of ALP, the food residues of SMD was detected rapidly.This issue focused on the following aspects of research:1 HRP-luminol-H2O2 system1.1 First, an ideal method to enzyme markers was chosen in the small molecule drugs, the diazotization-coupling method could be used to combine horseradish peroxidase with SMD and the following three methods were used to identify marked complex:firstly, the conjugate turned into a purple-red color; Secondly, from the UV curve, the characteristic absorption peak was seen at 233nm and there was displacement; Finally, from the infrared spectra of conjugates, the results were below: 3500～4000cm-1 region,2250～2500 cm-1 region and 1250～1750 cm-1 region of symbolize the characteristic peaks of amino acids of horseradish peroxidase,900～1160 cm-1 region had shows SMD characteristic peak marks.1.2 Then the optimal conditions:antibody-coated solid-phase concentration was 2.5μg/ml, the HRP-SMD was diluted to 1:4. Every well was added by standards or samples (50μl) and HRP-SMD (50μl), then plates were placed at 37℃for 1h. After washed, each well was added by 100μl Luminol substrate. The result was obtained by luminometer after adding substrate for 1.5min.The limit of detection was 3.2pg/ml, the relative standard deviation of intra-assay and inter-assay of ten samples were below 13%, below 18%, respectively. The regression equation was y=-0.0063x+6.6846(x was the logarithm of standard sample concentration, y was the logarithm of light intensity), the linear range for the method was from 10 pg/ml to 2000pg/ml, the correlation coefficient was 0.9952.2 ALP-AMPPD System2.1 First, an ideal method to enzyme markers was chosen in the small molecule drugs, the diazotization-coupling method could be used to combine horseradish peroxidase with SMD and the following three methods were used to identify marked complex:firstly, the conjugate turned into a purple-red color; Secondly, from the UV curve, the characteristic absorption peak was seen at 238nm and there was displacement; Finally, from the infrared spectra of conjugates, the results were below: 3500～4000cm-1 region,2250～2500 cm-1 region and 1250～1750 cm-1 region of symbolize the characteristic peaks of amino acids of alkaline phosphatase,800～1200 cm-1 region had shows SMD characteristic peak marks.2.2 Then the optimal conditions:antibody-coated solid-phase concentration was 5.0μg/ml, the ALP-SMD was diluted to 1:10. Every well was added by standards or samples (50μl) and ALP-SMD (50μl), then plates were placed at 37℃for 1h. After washed, each well was added by 100μl AMPPD. The result was obtained by luminometer after adding substrate for 25min.The limit of detection was 28.0pg/ml, the relative standard deviation of intra-assay and inter-assay of ten samples were below 15%, below 18%, respectively. The regression equation was y=-0.0215x+5.1726 (x was the logarithm of standard sample concentration, y was the logarithm of light intensity), the linear range for the method was from 50 pg/ml to 10000pg/ml, the correlation coefficient was 0.9961.3 Tested Biological Samples3.1 HRP-Luminol-H2O2 system was used to test biological samples, such as milk, eggs, honey and serum, the recoveries were:91.0%-104.0%,84.0%-92.0%, 90:0%-113.0%,110.0%～119.0%.3.2 The CLEIA was compared with ELISA and HPLC, the limit of detection of CLEIA was the lowest and its operation was more convenient. Although the stability of HPLC was good, pre-treatment was very troublesome, the cost was great and time-consuming. So it was showed that the CLEIA was rapid, convenient and sensitive.