توسعه يك ايمونواسي جديد مبتني بر نانوبادي جهت تشخيص آنتي¬ژن اختصاصي پروستات (PSA) به عنوان يك بيوماركر سرطان پروستات در انسان

The role of immunoassay in detecting cancer biomarkers is crucial for early diagnosis and cancer prevention. Prostate cancer is the most preva‎lent cancer in men and in the clinical and biomedical arenas, there is an urgent need for accurate diagnosis and prognosis of prostate cancer. At present, the diagnosis and primary screening of prostate cancer rely on the essential biomarker known as prostate-specific antigen (PSA) that was the first cancer biomarker approved by the FDA. Nanobodies (Nbs) are the variable domain of heavy‐chain antibodies derived from the blood of camelids. They are the smallest antibody fragment with intact antigen‐binding ability. Nanobodies have unique properties that make them an ideal alternative to conventional antibodies, showing excellent prospects for various applications in disease diagnosis and therapy. The main purpose of this study was to develop a novel immunoassay for the detection of PSA based on a tri-part split-nanoluciferase system and a nanobody targeting PSA. In this approach, the nanoluciferase enzyme was splitted into three components and then two small components of the split-nanoluciferase, referred to as β9 and β10, were individually fused to two anti-PSA nanobodies, N7 and N23. When these proteins bind to PSA and in the presence of the third nanoluciferase component, called Δ11S, the split-nanoluciferase components are brought into close proximity, facilitating the reassembly of the active nanoluciferase and activation of luminescence. Following the expression and purification of the system components, this developed immunoassay could sensitively detect PSA within a linear range of 1.0 to 20.0 ng/mL with a LOD of 0.4 ng/mL. Also, the results obtained from this immunoassay were consistent with those from the ELISA test, indicating the efficiency of this new immunoassay. The results of bioinformatic studies stated that both nanobody N7 and N23 (and their fusion proteins) have high affinity to PSA and bind to different binding sites on the surface of PSA antigen within efficient distance for reconstitution of complete nanoluciferase. Additionally, the developed immunoassay is homogeneous and exhibits remarkable specificity for PSA. This immunoassay can serve as a reliable, fast, and user-friendly test for detecting PSA. The nanobody-based immunoassay may have possible applications in in vitro studies and point-of-care testing and can be used to develop commercial diagnostic kits to measure PSA levels in serum.

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