便潜血单克隆一步法胶体金检测试卡的研制

Preparation of Colloid Gold Kit by One-Step Chromatography Immunoassay for Fecal Occult Blood Detection

采用鼠抗人血红蛋白(Hb)单克隆抗体1和胶体金标记鼠抗人血红蛋白(Hb)单克隆抗体2及对照抗体羊抗鼠IgG,利用双抗体免疫夹心反应原理特异性地结合人血红蛋白抗原,在5 min内可得到胶体金显色结果的原理研制便潜血单克隆一步法胶体金检测试卡,用于诊断因各种原因引起的消化道出血疾病。应用杂交瘤技术制备两株鼠抗人血红蛋白(Hb)抗体(McAb);ELISA方法检测McAb效价;免疫胶体金技术制备胶体金检测试卡。ELISA法检测McAb效价的结果显示,鼠抗人血红蛋白(Hb)单克隆抗体1效价为1∶3.2×103;鼠抗人血红蛋白(Hb)单克隆抗体2效价为1∶1.6×103。试卡的灵敏度检测结果显示,检测粪便中血红蛋白的最低浓度为0.1μg/mL;试卡的特异性检测结果显示,特异性地结合人血红蛋白抗原,而不结合其他物种的血红蛋白(如动物血红蛋白),本法测便潜血不受维生素C、含过氧化物酶的绿叶蔬菜及铁剂等干扰。大便潜血一步检测法是一种快速、灵敏、简便的免疫层析检测法,由于具有高度敏感性和特异性,以及快速直观等优势,对于诊断各种原因引起的上下消化道出血疾病具有重要的定性判别意义。

It was to prepare a colloid gold kit by one-step chromatography immunoassay for the qualitative detection of fecal occult blood based on the principle of the highly specific immunochemical reactions of antigens and antibodies which are used for the analysis of specific human hemoglobin in human stool at ideal cut-off concentrations.To obtain monoclonal antibodies（McAb）of mouse against Hb from hybridoma through limiting dilution,then construct the kit.The titers of McAb were tested by enzyme linked immunosorbent assay.Results showed that the titers of the first McAb of mouse anti-human hemoglobin were 1∶ 3.2×103,the second McAb of mouse anti-human hemoglobin were 1∶ 1.6×103;the colloid gold kit with the following cut-off concentrations of 0.1 μg/mL;the colloid gold kit showed good specificity without cross reaction with Vitamin C,HRP,Fe.It proved that the colloid gold kit is a rapid,specific,sensitive immunoassay suitable for the detection of possible human hemoglobin in the stool,and it＇s important to diagnose alimentary tract bleeding caused by any reasons.