Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays.
Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays.
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与 α1-抗胰凝乳蛋白酶复合的前列腺特异性抗原 (PSA) 的纯化和表征：PSA 免疫测定国际标准化的潜在参考材料。
DOI：
10.1093/clinchem/41.9.1273
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发表时间：
1995
影响因子：
9.3
通讯作者：
T. Stamey
中科院分区：
文献类型：
--
作者：
Zuxiong Chen;A. Prestigiacomo;T. Stamey
我们描述了第一次的协议，以纯化到表观均匀性的前列腺特异性抗原（PSA）和α 1-抗胰凝乳蛋白酶（ACT）的体外制备的复合物，通过使用凝胶过滤和离子交换色谱的组合。通过凝胶电泳和Western印迹证实PSA-ACT复合物的纯度。PSA-ACT复合物在pH 6.0至7.8范围内稳定;在各种基质、温度和高浓度盐中也稳定。PSA-ACT复合物的纯化是高度可重复的。基于氨基酸分析，将280 nm处的吸光系数0.99 L x g-1 x cm-1指定给PSA-ACT复合物。由于PSA和ACT以1：1的摩尔比结合，我们将PSA-ACT复合物的分子量确定为ACT的cDNA编码的质量（加上26%碳水化合物）加上PSA的分子量（28，430 Da），其总计为89，280 Da。使用该材料，我们制备了两种常见的校准品，一种是100% PSA-ACT复合物，另一种是90% PSA-ACT复合物加上10%游离PSA（90：10校准品）。在9种商业免疫测定中用这些校准品替代制造商的校准品，显著降低了免疫测定之间的差异，特别是对于4 - 10 μ g/L的血清PSA值。推荐将90：10校准品作为PSA免疫测定国际标准化的通用校准品。
We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.