| Furazolidone belongs to nitrofuran antibiotics. Because furazolidone is cheap andeffective, it is wildly used to treat animal diseases. Furazolidone can be metabolized rapidly inanimal body and the mail metabolite is3-amino-2-oxazolidinone (AOZ) which remaining inthe body. Long-term research proved that metabolite showed carcinogenic, abnormal andmutagenic characteristics. Furazolidone metabolite residue in animal food has potential healthrisks to human. Therefore, furazolidone is controlled strictly used in many countries.Countries have established analytical techniques to detect residue of drugs. HPLC,LC-MS and LC-MS/MS are the most widely used methods to detect furazolidone metabolite.But these methods need expensive equipment, extensive sample preparation, time-consuming,high cost, and highly trained individuals to operate sophisticated instruments. Therefore, theyare not suitable to be used as routing detection methods for furazolidone metabolite. The baseof ELISA is antigen-antibody reactions, which is a rapid, sensitive, convenient and low cost.ELISA has a promising application prospect. This paper aimed to develop ic-ELISA detectionmethod to measure the residue of furazolidone metabolite and a rapid detection kit.Firstly, the best synthesis condition of AOZ was discussed; using GC-MS and IRidentified the structure and purity. According to the feature of AOZ, I designed three haptensand applied in synthesis of immunogens (I, II, III and IV) and coating antigens (A, B, C and D)via active ester reaction and gelatin-glutaraldehyde (GGA) with cOVA and cBSA. Theimmunogens and coating antigens were identified by UV spectrophotometer. The antibodieswere acquired by immunizing eight New Zealand white rabbits for a cycle. Through thedetection of anti-serum titer and specificity, I chose coating antigen B and the eighth antibodyused for ELISA detection.Secondly, using selected antibody and coating antigen built ic-ELISA detection and theoptimization tests. Depending on optimization tests about ic-ELISA detection method thefurazolidone metabolite ELISA parameters were decided as followings: the dilution ratio ofthe eighth polyclonal antibody, coating antigen and HRP-GAR-IgG were1:4000,1:500and1:2000;0.05mol/L sodium bicarbonate buffer(pH9.6) was used as coating buffer; Jin Canhuaimmune-plate was used; the coating antigen was placed in the refrigerator at4℃overnight tobe enveloped;1%BSA Tween20PBST was used as blocking buffer; the best competitivetime was30min; the coloration time was15min. According to the results ofoptimization tests, calibration curve was prepared for NPAOZ (3-(2-Nitrobenzylidenamino)-2-oxazolidinone), a2-nitrobenzaldehyde derivative of AOZ, and good linearity was achieved over the concentration(0.25~10ng/mL), the obtained IC50was between0.8162ng/mL. Thelimit of detection and quantization of aquatic products tissue, livestock and poultry tissue,liver, egg, milk were0.04ng/mL,0.08ng/mL,0.1ng/mL,0.1ng/mL, and0.06ng/mL,respectively. The recoveries of shrimp tissues were80%~120%and CV<20%, they met therequirements of the analysis of veterinary drug residue. In addition, the antibody was highspecific for NPAOZ and no cross-reactivity with analog except CPAOZ and CPAHD. Hightemperature and short time derivative process can be in line with incubating at37℃overnight.The ic-ELISA rapid kits were placed at4℃and37℃for several days and they were used todetect the furazolidone metabolite. The stability test indicated that the reliability of test kitswere good at4℃within12months. |
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