乳铁蛋白双抗体夹心ELISA检测方法的建立

Establishment of Double Antibody Sandwich ELISA for Detecting Lactoferrin

乳铁蛋白(Lactoferrin, LF)是一种重要的铁离子结合蛋白,因其在抗菌杀菌、抗病毒及调节机体免疫方面效果显著,近年来被广泛应用于食品领域。试验旨在建立LF双抗体夹心法检测方法。试验以乳铁蛋白为抗原免疫BALB/c小鼠,应用常规的细胞融合技术获得18株杂交瘤细胞株,进而获得腹水抗体18个,效价测定结果显示13株单克隆抗体的效价均在1︰2.56×10^(5)以上;用效价测定较高的13株抗体作为包被抗体,用辣根过氧化物酶标记过的多克隆抗体作为酶标抗体,经过配对筛选, 8号单克隆抗体作为包被抗体,命名为FL8,建立牛乳铁蛋白的双抗体夹心ELISA检测方法,最低检测限为0.05 ng/mL,最适检测范围为0.05~3.2 ng/mL。

Lactoferrin, an important iron-binding protein, had been widely used in food areas due to its antibacterial and sterilization, antiviral and immune regulation. Aim to establish double antibody sandwich ELISA method for detection of lactoferrin(LF). About 18 strains of hybridoma cells were obtained by lymphocyte hybridoma technique using lactoferrin as antigen to immunize BALB/c mice. About 18 monoclonal antibodies were obtained and measured for the antibody titer. The titer degree of ascites fluid of 13 monoclonal antibodies were above 1︰2.56×10^(5). Therefore, with these 13 monoclonal antibodies as coated antibodies, and rabbit HRP-labeled polyclonal antibody as a second detection antibody, the double antibody sandwich ELISA was established for detection of LF with the monoclonal antibody obtained from the strain named FL8. The lowest detectable limit of ELISA was achieved 0.05 ng/mL, with the range of 0.05-3.2 ng/mL.