Rapid Detection For Illegal Additives Using Immunoassays In Slimming Tea And Coffee

Health drinks with nutritional or physiological effects is suitable for certain people,and it can be used to regulate body functions but not for curing diseases.Therefore,substances with therapeutic functions cannot be included in drinks,as they are classified as special drinks and are specifically regulated.The use of health drink has increased worldwide in the last few decades.However,illegal activities such as the manufacture and marketing of fake commodities,false advertising,and fraudulent sales have restricted the sustainable development of the health drink industry.In particular,there is much concern regarding health drink illegally adulterated with pharmaceuticals and their analogs because of the notable risk to public health.Therefore,there is urgent need to develop accurate,sensitive,and rapid detection methods for analysis of such compounds.In this study,sibutramine(SB),phenolphthalein(PPH),sildenafil(SD),and tadalafil(TDL)were used as the research objects.The aim of this study was to establish a rapid and sensitive assay to detect illegal additives using immunoassay methods.In the first we designed haptens of SB,SD,and TDL based on their chemical structures.The antigens were prepared and conjugation of haptens with carrier protein(BSA,KLH,or OVA).After the procedure of mouse immunization,cell fusion,cell culture and screening,ascites preparation and purification of the monoclonal antibody(m Ab)were successfully prepared.We selected the m Ab of 3D7,3C9,1A2,and 5A1 for SB,PPH,SD,and TDL,respectively.Under optimal conditions,the half-inhibitory concentration(IC₅₀)of monoclonal antibodies against SB,PPH,SD,and TDL using ELISA method were 4.52 ng m L^-1,18.14 ng m L^-1,0.48 ng m L^-1,and 12.3 ng m L^-1,respectively.Furthermore,colloidal gold and fluorescence immunochromatographic test strip were established.Under optimal conditions,the cut off limits using gold labelled antibody(GLM)was found to be 250 ng m L^-1(TDL)and 500 ng m L^-1(SB,SD,and PPH)while using fluorescent labelled antibody(FLM)was found to be 100 ng m L^-1(TDL)and 250 ng m L^-1(SB,SD,and PPH)in 0.01 M PBS(p H 7.4).In this study slimming tea and coffee was spiked with the SB,PPH,SD,and TDL with the cut off was 250 ng m L^-1 for TDL and 500 ng m L^-1 for SB,SD,and PPH using GLM,meanwhile for using FLM was found to be 100 ng m L^-1 for TDL and 250 ng m L^-1 for SB,SD,and PPH.The recovery rate was 98.2-102.7%(GLM)and with FLM were 98.4-102.6%that spiked4 different concentration of TDL in slimming tea and coffee,meanwhile spiked with SD has recovery rate was 98.0 to 105.3%for the GLM strip and 96.0 to 102.7%for the FLM strip in slimming tea and coffee.The recovery rate was 96.2 to 104.7%for the GLM and 94.7 to 104.7%for the FLM strip that spiked with PPH in slimming tea and coffee,in the other hand,the recovery rate that spiked with SB was 99.5 to 102.7%for the GLM strip,and 90.7 to 103.0%for the FLM strip in slimming tea and coffee.All results of using lateral flow immunoassay(LFIA)could be obtained by 10 min.These showed using LFIA in GLM and FLM to detect 4illegal additives is a high reliability and sensitive assay to analysis the real sample.