Rickard E C, Sittampalam G S, Clodfelter D K
Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, IN 46285.
Biotechniques. 1988 Nov-Dec;6(10):982-92.
Several techniques were evaluated for the quantitation of the total protein content of an IgG2a monoclonal antibody, KS1/4, and its deacetylvinblastine (DAVLB) conjugate. The UV assay is rapid, but it requires an extensive calibration of the response factor, and impurities may cause a high bias. Amino acid analysis (AAA) is an absolute method that has few interferences, but it requires evaluation of hydrolysis recovery factors. Kjeldahl nitrogen is very sensitive to minor impurities, and it requires a conversion factor to calculate percent protein. The Kjeldahl assay also is less precise (observed RSD of 3-4%) than the UV and AAA assays (observed RSD of 2-3% for both). The bicinchoninic acid assay, a representative of colorimetric assays, inherently requires comparison to a calibrated standard of the same material and tends to be less precise than the other assays. Thus, the UV and AAA assays are the techniques of choice for measurement of the total protein content of KS1/4 and its DAVLB conjugate.
对几种用于定量IgG2a单克隆抗体KS1/4及其去乙酰长春碱（DAVLB）缀合物总蛋白含量的技术进行了评估。紫外测定法速度快，但需要对响应因子进行广泛校准，且杂质可能导致高偏差。氨基酸分析（AAA）是一种干扰较少的绝对方法，但需要评估水解回收率。凯氏定氮法对微量杂质非常敏感，需要一个换算因子来计算蛋白质百分比。凯氏定氮法也不如紫外和AAA测定法精确（观察到的相对标准偏差为3-4%，而紫外和AAA测定法的观察到的相对标准偏差均为2-3%）。比色测定法的代表——二辛可宁酸测定法，本质上需要与相同物质的校准标准进行比较，且往往不如其他测定法精确。因此，紫外和AAA测定法是测量KS1/4及其DAVLB缀合物总蛋白含量的首选技术。
