Kalniņa Zane, Siliņa Karīna, Meistere Irēna, Zayakin Pawel, Rivosh Alexander, Abols Artūrs, Leja Mārcis, Minenkova Olga, Schadendorf Dirk, Linē Aija
Biomedical Research and Study Centre of Latvia, Riga, Latvia.
J Immunol Methods. 2008 May 20;334(1-2):37-50. doi: 10.1016/j.jim.2008.01.022. Epub 2008 Feb 21.
In the current study we attempted to evaluate the suitability of T7 Select 10-3b and lambdaKM8 phage display systems for the identification of antigens eliciting B cell responses in cancer patients and the production of phage-displayed antigen microarrays that could be exploited for the monitoring of autoantibody profiles. Members of 15 tumour-associated antigen (TAA) families were cloned into both phage display vectors and the TAA mini-libraries were immunoscreened with 22 melanoma patients' sera resulting in the detection of reactivity against members of 5 antigen families in both systems, yet with variable sensitivity. T7 phage display system showed greater sensitivity for the detection of antibodies against members of CTAG, MAGEA and GAGE families, both systems showed equal performance in detecting the reactivity against MAGEC and SSX2 while only lambdaKM8 allowed the detection of anti-CTAGE5 antibodies. The biological properties of both phages turned out to be equally suitable for the production of antigen microarrays however in line with the plaque assay the sensitivity for the detection of various autoantibodies differed between the vectors. However, presumably due to the higher variability of the background signals in the microarray assay, it turned out to have comparable, in some cases even slightly lower sensitivity than the plaque assay. Next, we explored the repertoire of antigens that could be identified by screening T7 phage-displayed testis cDNA library with sera from melanoma patients. From the 243 antigens identified, only 24 represented known genes translated in their natural reading frame and included known TAAs like Annexin XI-A and a novel potential CT antigen SPAG8. Another 12 were uncharacterised genes but the remaining clones contained DNA fragments in non-natural reading frames that most likely represent mimotopes, nevertheless, they may turn out to be valid biomarkers.
在本研究中，我们试图评估T7 Select 10-3b和lambdaKM8噬菌体展示系统用于鉴定引发癌症患者B细胞反应的抗原以及生产可用于监测自身抗体谱的噬菌体展示抗原微阵列的适用性。将15个肿瘤相关抗原（TAA）家族的成员克隆到两种噬菌体展示载体中，并用22例黑色素瘤患者的血清对TAA微型文库进行免疫筛选，结果在两个系统中均检测到针对5个抗原家族成员的反应性，但敏感性不同。T7噬菌体展示系统在检测针对CTAG、MAGEA和GAGE家族成员的抗体方面表现出更高的敏感性，两个系统在检测针对MAGEC和SSX2的反应性方面表现相当，而只有lambdaKM8能够检测到抗CTAGE5抗体。事实证明，两种噬菌体的生物学特性同样适合于生产抗原微阵列，然而，与噬菌斑测定法一致，载体之间检测各种自身抗体的敏感性有所不同。然而，可能由于微阵列测定中背景信号的变异性较高，结果显示其敏感性与噬菌斑测定法相当，在某些情况下甚至略低。接下来，我们探索了通过用黑色素瘤患者的血清筛选T7噬菌体展示的睾丸cDNA文库可以鉴定的抗原库。从鉴定出的243种抗原中，只有24种代表在其天然阅读框中翻译的已知基因，包括膜联蛋白XI-A等已知的TAA和一种新的潜在CT抗原SPAG8。另外12种是未表征的基因，但其余的克隆包含非天然阅读框中的DNA片段，很可能代表模拟表位，不过，它们可能会成为有效的生物标志物。
