Abstract
Establishment of Enzyme-Linked Immunosorbent Assay for Sibutramine
Guangdong Provincial Key Laboratory of Food Quality and Safety/Nation-Local Joint Engineering Research Center for Precision Machining and Safety of Livestock and Poultry Products, South China Agricultural University, Guangzhou 510642, 1Tiandi No.1 Beverage INC, 2Bozhou University, 3Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, 4Licheng Detection and Certification Group Co., Ltd., Zhongshan, Guangdong 528403, 5Department of Physiology and Pathophysiology, College of Medicine, Yanbian University, Yanji, Jilin 133002, China
Correspondence Address:
Hongtao Lei, Licheng Detection and Certification Group Co., Ltd., Zhongshan, Guangdong 528403, China, E-mail: hongtao@scau.edu.cn
Polyclonal antibodies that recognize sibutramine and an indirect competition enzyme-linked immunosorbent assay were prepared to detect the content of Western butramine to be tested. Immunogen and coating progen were obtained by conjugation with bovine serum albumin and ovalbumin by active ester method with 1-(4-phenol) cyclobutamine as hapten, and the New Zealand white rabbit was immunized after the conjugate was successfully conjugated by ultraviolet scanning, and the obtained antibody serum was screened by indirect competition enzyme-linked immunosorbent assay detection to screen out the best antibody, and then the coating progen was replaced with 5-hydroxytryptamine-ovalbumin to compare the titer and inhibition rate, and the heterologous coating effect was better, and the heterologous coating was used for follow-up experiments. By changing the concentration of coating and antibody, the inhibition rate was found when the coating was 31.25 ng/ml and the antibody was diluted 8000 times. The antibody had the highest sensitivity for the two standards at the half maximal inhibitory concentration of 4.89 ng/ml, a limit of detection of 0.21 ng/ml, and a detection range of 0.68-35.21 ng/ml for the 8000 fold-dilution of the original 31.25 ng/ml and an 8000-fold dilution of the antibody. The recovery under these conditions ranged between 88 % and 105 %, which was within a reasonable range, indicating that the rapid enzyme linked immunosorbent assays was successfully established.
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