Goodell Vivian, McNeel Douglas, Disis Mary L
Center for Translational Medicine in Women's Health, University of Washington, Seattle, WA 98109-8050, USA.
BMC Immunol. 2008 May 29;9:23. doi: 10.1186/1471-2172-9-23.
The application of high throughput molecular techniques such as SEREX are resulting in the identification of a multitude of tumor associated antigens. As newly identified antigens are incorporated into a variety of clinical trials, standardization of immunologic monitoring methods becomes increasingly important. We questioned whether mammalian cell expression of a histadine-linked human protein could be used to produce antigen suitable for detecting tumor-specific humoral immunity and whether such an assay could be amenable to standardization for clinical use.
We designed a his-tagged capture ELISA based on lysate from genetically engineered CHO cells for detection of antibodies to insulin-like growth factor binding protein 2, a novel tumor antigen. We performed technical and preliminary clinical validation studies, including comparison to a standard indirect ELISA based on commercially prepared recombinant antigen.
The his-tagged capture ELISA could be standardized. Precision experiments resulted in CVs < 15%. Linearity and calibration experiments demonstrated r2 values of 0.99. In comparison to Western blot analysis, his-tag and indirect ELISA accurately identified 88% and 93% of samples, respectively. Sample concordance between capture and indirect assays was highly significant (p = 0.003). Furthermore, significantly greater levels of IGFBP-2 antibody immunity were found in cancer patients compared to normal controls (p = 0.008).
A genetically engineered cell lysate based ELISA can be amenable to standardization and can detect increased levels of antibody immunity to tumor-associated antigen in cancer patients compared to non tumor-bearing healthy controls.
诸如SEREX等高通量分子技术的应用正在促使大量肿瘤相关抗原得以鉴定。随着新鉴定出的抗原被纳入各种临床试验，免疫监测方法的标准化变得愈发重要。我们质疑与组氨酸相连的人类蛋白的哺乳动物细胞表达是否可用于生产适合检测肿瘤特异性体液免疫的抗原，以及这样一种检测方法是否适合临床应用的标准化。
我们基于基因工程CHO细胞的裂解物设计了一种用于检测胰岛素样生长因子结合蛋白2（一种新型肿瘤抗原）抗体的组氨酸标签捕获ELISA。我们进行了技术和初步临床验证研究，包括与基于商业制备的重组抗原的标准间接ELISA进行比较。
组氨酸标签捕获ELISA可以标准化。精密度实验得出的变异系数<15%。线性和校准实验显示r2值为0.99。与蛋白质印迹分析相比，组氨酸标签ELISA和间接ELISA分别准确鉴定出88%和93%的样本。捕获法和间接法之间的样本一致性非常显著（p = 0.003）。此外，与正常对照组相比，癌症患者中发现的胰岛素样生长因子结合蛋白-2抗体免疫水平显著更高（p = 0.008）。
基于基因工程细胞裂解物的ELISA适合标准化，并且与非肿瘤健康对照相比，能够检测癌症患者中针对肿瘤相关抗原的抗体免疫水平升高。
