目的 建立检测转基因兔乳腺生物反应器表达的重组蛋白药物中兔乳蛋白残留的双抗体夹心ELISA，并进行验证。方法 以非转基因兔乳免疫绵羊获得多克隆抗体，建立检测兔乳蛋白残留的双抗体夹心ELISA。对方法进行专属性、准确度、精密度、耐用性验证，并确定方法的定量限、线性及范围。同时采用建立的方法检测转基因兔乳腺生物反应器表达的重组人C1酯酶抑制剂中兔乳蛋白残留量。结果 专属性检测在450和630 nm处的吸光度均值差≤0.1；浓度范围在5~800 ng/mL时线性良好；定量限为5 ng/mL；低、中、高加标样品的回收率分别为111.0%、117.5%和110.4%，相对标准偏差（relative standard deviation, RSD）分别为5.0%、4.7%和8.9%；中间精密度RSD分别为7.9%、8.5%和7.6%；耐用性考察在3个不同实验条件下低、中、高加标样品测定值RSD分别为8.0%、4.7%和7.6%。结论 成功建立了检测转基因兔乳腺生物反应器表达产品中兔乳蛋白残留双抗体夹心ELISA，该方法专属性、准确度、精密度、耐用性、灵敏度良好。

Objective To establish and validate a double-antibody sandwich ELISA for the detection of rabbit milk protein residues in recombinant protein drugs expressed by transgenic rabbit mammary gland bioreactor.Methods Polyclonal antibodies were obtained by immunizing sheep with non-transgenic rabbit milk, and a double-antibody sandwich ELISA for detecting rabbit milk protein residues was established. The method was validated for specificity, accuracy, precision and ruggedness, and its limit of quantitation, linearity and range were determined. The established method was applied to detect the residual amount of rabbit milk protein in recombinant human C1 esterase inhibitor expressed by transgenic rabbit mammary gland bioreactor.Results The average absorbance value difference of specificity test between 450 and 630 nm was ≤0.1, with good linearity at the concentration range of 5-800 ng/mL and limit of quantitation of 5 ng/mL. The recoveries of low, medium and high spiked samples were 111.0%, 117.5% and 110.4%, with relative standard deviations (RSDs) of 5.0%, 4.7% and 8.9%, and RSDs for intermediate precision of 7.9%, 8.5% and 7.6%, respectively. For the ruggedness test, RSDs of the determined values of low, medium and high spiked samples under 3 different experimental conditions were 8.0%, 4.7% and 7.6%, respectively.Conclusion A double-antibody sandwich ELISA for the detection of rabbit milk protein residues in products expressed by the transgenic rabbit mammary gland bioreactor is successfully established, which has good specificity, accuracy, precision, ruggedness and sensitivity.