【摘要】 为建立犬免疫球蛋白G(Ig G)的检测方法,本研究以兔抗犬Ig G Fc段多克隆抗体为包被抗体,过氧化酶标记的兔抗犬Ig G(全分子)多克隆抗体为检测抗体,建立了双抗体夹心ELISA定量检测方法,并采用Protein A方法从犬血清中纯化犬Ig G作为ELISA检测标准品。该检测方法与小鼠、兔、马、牛、鸡及羊常见动物血清均无交叉反应,特异性好;最低检测下限可以达到4.8 ng/m L,敏感性较高。以本研究建立的ELISA方法检测犬细小阳性血清和阴性血清,阳性血清Ig G含量明显高于阴性血清;检测转染犬源化抗体序列重组质粒的细胞培养液上清,重组质粒在96 h表达量达到峰值。本研究为进一步建立稳定表达犬源化抗体的细胞系提供了特异性的定量检测方法。更多还原

【Abstract】 To develop the quantitative detection method of canine immunoglobulin G(Ig G),a double-antibody sandwich ELISA was established in this study,using the rabbit anti-dog Ig G Fc fragment antibody as the coating antibody and rabbit anti-dog Ig G-HRP as the detecting antibody,The Ig G from canine serum purified using Protein A was used as standards for ELISA detection.The established assay was specificity for detection of canine Ig G,and had no cross-reaction with other animal sera such as mouse,rabbit,horse,cow,chicken and sheep;the detection limit of the assay was around 4.8 ng/m L of serum Ig G.Using the assay to test the canine parvovirus positive and negative serum,the data showed that the amount of Ig G in positive serum was much higher than that in negative serum.The detection results of culture medium from the cells transfected with the recombinant plasmid expressing canine humanized antibody sequences showed the peak expression of the antibody occurred at 96 hrs post the transfection.This study provided a high specificity quantification detection method for further research on establishing cell line with stable expression of canine humanized antibody.更多还原