Filippou Panagiota, Korbakis Dimitrios, Farkona Sofia, Soosaipillai Antoninus, Karakosta Theano, Diamandis Eleftherios P
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Department of Clinical Biochemistry, University Health Network, Toronto, Canada.
Clin Proteomics. 2017 Jan 17;14:4. doi: 10.1186/s12014-017-9140-6. eCollection 2017.
Kallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. The active form of the enzyme is predicted to have chymotryptic activity. In the present study, we produced for the first time the active recombinant protein and monoclonal antibodies, and developed novel immunoassays for the quantification of free and bound KLK9 in biological samples.
The coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. Hybridomas were further expanded and antibodies were purified. Newly-produced monoclonal antibodies were screened for reaction with the KLK9 recombinant protein by a state-of-the-art immunocapture/parallel reaction monitoring mass spectrometry-based methodology.
Anti-KLK9 antibodies were combined in pairs, resulting in the development of a highly sensitive (limit of detection: 15 pg/mL) and specific (no cross-reactivity with other KLKs) sandwich-type ELISA. Highest KLK9 protein levels were found in tonsil and sweat and lower levels in the heart, kidney and liver. Hybrid immunoassays using an anti-KLK9 antibody for antigen capture and various anti-serine protease inhibitor polyclonal antibodies, revealed the presence of an a1-antichymotrypsin-bound KLK9 isoform in biological samples.
The ELISAs for free and bound forms of KLK9 may be highly useful for the detection of KLK9 in a broad range of biological samples, thus enabling the clarification of KLK9 function and use as a potential disease biomarker.
激肽释放酶9（KLK9）是人类激肽释放酶相关肽酶家族的成员，其生理作用及在疾病过程中的影响尚不清楚。该酶的活性形式预计具有胰凝乳蛋白酶活性。在本研究中，我们首次制备了活性重组蛋白和单克隆抗体，并开发了用于定量生物样品中游离和结合形式的KLK9的新型免疫测定方法。
成熟KLK9同工型（mat-KLK9）的编码序列在Expi293F哺乳动物系统中表达，合成的多肽通过两步方案进行纯化。纯化后的蛋白用作免疫原在小鼠中制备单克隆抗体。杂交瘤进一步扩增并纯化抗体。通过基于最先进的免疫捕获/平行反应监测质谱的方法，筛选新产生的单克隆抗体与KLK9重组蛋白的反应。
抗KLK9抗体成对组合，开发出了一种高灵敏度（检测限：15 pg/mL）且特异性强（与其他激肽释放酶无交叉反应）的夹心型ELISA。在扁桃体和汗液中发现KLK9蛋白水平最高，而在心脏、肾脏和肝脏中水平较低。使用抗KLK9抗体进行抗原捕获以及各种抗丝氨酸蛋白酶抑制剂多克隆抗体的杂交免疫测定，揭示了生物样品中存在与α1-抗胰凝乳蛋白酶结合的KLK9同工型。
用于检测游离和结合形式KLK9的ELISA可能对在广泛生物样品中检测KLK9非常有用，从而有助于阐明KLK9的功能并将其用作潜在的疾病生物标志物。
