In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative IHC analysis depends on both the specificity and affinity of the binding reagent, which are inherently difficult to quantify in situ. Here we describe a label-free method that allows for the direct and real-time assessment of molecular binding kinetics in situ on FFPE tissue specimens using quartz crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumour specimens in situ. rVAR2 interacted with FFPE human placenta and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody (anti-AR). As the KD value produced by this method is independent of the number of epitopes available, this readout offers a quantitative and unbiased readout for in situ binding-avidity and amount of binding epitopes. In summary, this method adds a new and important dimension to classical IHC-based molecular pathology by adding information about the binding characteristics in biologically relevant conditions. This can potentially be used to select optimal biologics for diagnostic and for therapeutic applications as well as guide the development of novel high affinity binding drugs.

中文翻译：

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实时，无标记地测定与原发癌组织标本的配体结合动力学；一种用于评估生物标记物靶向性的新颖工具。

在临床肿瘤学中，最佳治疗策略的诊断和评估主要基于组织病理学检查，结合福尔马林固定石蜡包埋（FFPE）组织活检组织中癌相关抗原的免疫组织化学（IHC）表达分析。但是，信息丰富的IHC分析取决于结合试剂的特异性和亲和力，这固有地难以原位定量。在这里，我们描述了一种无标记方法，该方法允许使用启用了石英晶体微天平（QCM）的生物传感器技术直接和实时评估FFPE组织标本上的分子结合动力学。我们分析了rVAR2蛋白与其原胎盘组织以及乳腺和前列腺肿瘤标本中的胎盘样硫酸软骨素（pl-CS）受体之间的相互作用。rVAR2以纳摩尔级的亲和力与FFPE人胎盘和癌组织相互作用，并且与p1-CS阴性正常组织无相互作用。我们通过包含雄激素受体N-20抗体（抗AR）的分析进一步验证了该方法。由于通过此方法产生的KD值与可用的表位数量无关，因此该读数可提供定量且无偏见的原位结合亲和力和结合表位数量。总之，该方法通过添加有关生物学相关条件下结合特性的信息，为基于IHC的经典分子病理学增加了新的重要意义。这可以潜在地用于选择用于诊断和治疗应用的最佳生物制剂，并指导新型高亲和力结合药物的开发。