Jerry D J
Division of Molecular Virology, Baylor College of Medicine, Houston, TX 77030.
Biotechniques. 1993 Mar;14(3):464-9.
The amount of synthetic peptide coupled to a carrier protein is a critical parameter in production of antipeptide antibodies. In the method described, biotinylated peptide was included as a tracer to monitor the coupling of peptide to keyhole limpet hemocyanin or BSA. The extent of coupling was assessed by first fractionating free and coupled peptide by gel filtration, then slot blotting an aliquot of each fraction onto nitrocellulose. The biotinylated peptide was detected using an avidin-horseradish peroxidase conjugate. To estimate the mass of peptide incorporated, the fractions were analyzed using a competitive ELISA. The limit of detection of this assay was < 0.1 pmol and was linear to 2 pmol. The mass of biotinylated peptide in each column fraction was determined using this assay and the total mass of peptide coupled to the carrier was calculated. Using these techniques, biotin-labeling provided a sensitive and versatile means to assess the quality of peptide-carrier protein conjugates.
与载体蛋白偶联的合成肽量是抗肽抗体生产中的关键参数。在所描述的方法中，生物素化肽作为示踪剂用于监测肽与钥孔戚血蓝蛋白或牛血清白蛋白的偶联。首先通过凝胶过滤分离游离肽和偶联肽，然后将每个馏分的等分试样点样到硝酸纤维素膜上，以此评估偶联程度。使用抗生物素蛋白 - 辣根过氧化物酶缀合物检测生物素化肽。为了估计掺入的肽质量，使用竞争性酶联免疫吸附测定法分析馏分。该测定法的检测限<0.1 pmol，并且在2 pmol范围内呈线性。使用该测定法确定每个柱馏分中生物素化肽的质量，并计算与载体偶联的肽的总质量。使用这些技术，生物素标记提供了一种灵敏且通用的方法来评估肽 - 载体蛋白缀合物的质量。
