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目的:建立一种灵敏度高、特异性好且快速的ELISA方法,用以检测食蟹猴体内的帕妥珠单抗含量。方法:采用双抗夹心的ELISA方法对帕妥珠单抗进行定量。以Erb B2单抗作为一抗,将其包被至96孔板上,加入待检样品,之后加入稀释好的二抗(猴血清吸附的羊抗人Ig G-HRP),令结合到一抗上的帕妥珠单抗与其特异性结合,再加入底物进行显色,在酶标仪上用双波长读取D450/560nm值。结果:建立了室温稳定性、冻融稳定性、稀释效应稳定性良好的检测帕妥珠单抗的ELISA方法并得到确证,其定量下限为0.78 nm/m L,线性范围为0.78~50 nm/m L,板间及板内的准确度、精密度均在±15%之内。结论:该ELISA方法学的确证表明用该方法测定帕妥珠单抗浓度具有特异性好、灵敏度高、速度快的特点,能够满足新生物制药临床前药代动力学的指导要求,可应用于帕妥珠单抗的检测。
Objective: To establish a sensitive, specific and rapid ELISA method for the determination of pertuzumab in cynomolgus monkeys. METHODS: Pertuzumab was quantified by double-antibody sandwich ELISA. Erb B2 monoclonal antibody was used as a primary antibody, which was coated on a 96-well plate and the sample to be tested was added. After that, the diluted secondary antibody (goat anti-human Ig G-HRP adsorbed by the monkey serum) was added to bind to the primary antibody Pertuzumab was specifically bound to it, and then the substrate was added for color development. The D450 / 560 nm value was read on a microplate reader using a dual wavelength. Results: The ELISA method for the determination of pertuzumab was established, which was stable at room temperature, freeze-thaw stability and good dilution stability. The lower limit of quantitation was 0.78 nm / m L and the linear range was 0.78-50 nm / m L, the accuracy between plate and board, precision within ± 15%. Conclusion: The confirmatory test of this ELISA method shows that the determination of pertuzumab with this method has the characteristics of good specificity, high sensitivity and fast speed, which can meet the guidance requirements of preclinical pharmacokinetics of new biopharmaceuticals, and can be applied to Pertuzumab test.