Study On Immunochromatographic Assays For Detecting Aflatoxin B1 In Semen Cassia

Aflatoxin B1(AFB1)is the most toxic mycotoxin among all the known mycotoxins.It has strong carcinogenicity,mutagenicity,and genotoxicity.Humans or animals will suffer from liver damage if taking in excess AFB1.Crops are likely to be contaminated by mycotoxins such as AFB1 during growth,transportation,and storage,which has caused widespread concern.In recent years,much emphasis has been put on the situation that medicinal and food homologous seed crops such as Semen cassia are contaminated by AFB1.In this paper,two methods of immunochromatographic assay(ICA),which were colloidal gold(CG)and fluorescent microsphere(FM),were used to qualitatively and quantitatively detect AFB1 in Semen cassia.In the experiment,ultra-high performance liquid chromatography(UPLC)was used to detect AFB1 for getting the negative samples as the matrix for the ICA method of AFB1 in Semen cassia.Based on the chromatographic conditions used in this paper,three negative samples were successfully screened from the four Semen cassia samples purchased locally.The content of AFB1 in positive sample was 0.129?g/kg.Then,the pH values,the concentration of antibody,and the concentration of AFB1-BSA on T line were optimized in the systems of the colloidal gold immunechromatographicassay(CG-ICA)andfluorescentmicrospheres immunechromataography assay(FM-ICA),respectively.The effective way of pretreatment was selected as well.The CG-ICA used in this paper was a kind of qualitative method based on different color bewteen T and C line without test strip reader.It was quick,convenient,and suitable for detection of a lot of Semen cassia samples.The detection of limit of the method by naked eye in extracting solution and real sample were 0.4?g/L and 5?g/kg,respectively.The sensitive FM-ICA was studied in the paper.The T/C values were read using a fluorescent microsphere immunochromatographic reader and a competitive inhibition curve in Semen cassia was developed for quantitative detection.The sensitivity of the method was 0.034?g/L,and two linear independent regression equations were expressed with y=-0.5854 log(x)+0.09948(R~2=0.9846)for low AFB1concentrations between 0.1?g/L and 0.6?g/L,and y=-0.2234 log(x)+0.1654(R~2=0.9888)in high AFB1 concentrations in the range from 0.6?g/L to 2.0?g/L.Compared with the UPLC method,the result of FM-ICA had a good correlation.Therefore,FM-ICA was accurate,reliable,and suitable for the quantitative detection of AFB1 in Semen cassia.The FM-ICA could be used in the quantitative detection for positive samples after screening by CG-ICA.Two methods of immunochromatography(CG-ICA and FM-ICA)could be used in combination and meet the need for on-site screening of a mass of samples.To further evaluate the performances of CG-ICA and FM-ICA,the specificity,stability,precision,false-negative rate,and false-positive rate were studied in the paper.The results showed that the two immunochromatographic test strips had good specificity,stability,low CV of inter-assay and intra-assay(less than 15%),low false-negative rate(0%),and low false-positive rate(no more than 5%).