抗苏丹红Ⅰ号单抗的制备及用于检测食品中苏丹红含量的酶联免疫方法建立

Analysis on the competitive ELISA assay to detect SudanⅠand para-red dyes in foods

将羧基苏丹红Ⅰ号分别与BSA和OVA偶联,制得免疫抗原和包被抗原。免疫BALB/c小鼠,取脾脏细胞与骨髓瘤细胞SP2/0融合,筛选获得能分泌抗羧基苏丹红抗体的杂交瘤细胞株。将该细胞株接种BALB/c小鼠腹腔,产生的腹水经辛酸-饱和硫酸铵法纯化获得腹水型苏丹红单克隆抗体,效价为1:128 000。以该腹水型单抗建立的对苏丹红Ⅰ号和对位红的竞争ELISA检测法,IC_(50)值分别为0.8μg/L和4μg/L,与苏丹红Ⅱ、Ⅲ、Ⅳ号和其他染料的交叉反应率均小于2.2%。用乙腈法提取番茄酱中的苏丹红Ⅰ号或者对位红,回收率为69.8%～118%。该方法灵敏特异,可作为快速检测食品中添加苏丹红Ⅰ号和对位红的初筛方法。

Immunogen and coating antigen were prepared by coupling SudanⅠand BSA or OVA.After the BALB/c mice were immunized,the splenocytes were taken out from mice and fused with Myeloma cells SP2/0,and the McAb hybridoma cells secreting antibodies specifically against SudanⅠwere obtained through several filtrations.The cells were then injected into the abdominal cavities of other normal BALB/c mice to induce ascites.The ascites was purified by caprylic ammonium sulfate precipitation to obtain the antibody titer of 1：128 000.An competitive ELISA assay to detect SudanⅠor para-red dye by using ascitic fluid was thus established.and its IC50 for SudanⅠand para-red dyes were found to be 0.8μ/L and 4μg/L,respectively.The cross-reactivity rates within SudanⅡ,Ⅲ,Ⅳand other four dyes were all less than 2.2%,and the recovery rate of the detection of SudanⅠor para-red dye in tomato sauce by the acetonitrile was 69.8%～118%.It is evident that this sensitive and specific method can be used as a quick and primary immunoassay device to detect SudanⅠor para-red dye in the foods.