Development of a Competitive Chemiluminescent Assay for Quantitative Determination of TP53 Fusion Protein Using Reagent Strips.

ABSTRACT

Recent studies have shown that almost half of all cancers occur due to DNA damage. For the early diagnosis of cancer, a highly sensitized and swift identification for TP53 is needed since the corresponding TP53 protein is effectively recognized as "the guardian of the genome." To improve the detection sensitivity, numerous analytical methods were previously used for the determination of the TP53 protein, including denaturing high-performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). Currently, immunochromatographic tests (ICTs) that are simple to use, stable over time, and show low interference are regarded as valuable tools for the quick screening of food and environmental monitoring along with clinical diagnosis. ICTs often have limited sensitivity even if a variety of novel reporters possessing optimum photostability and improved brightness are used as signal-intensity reporters. Compared with N-(4-aminobutyl)-N-(ethylisoluminol) or luminol, a novel luminescent probe, 2',6'-diMethyl-4'-(N-succiniMidyloxycarbonyl) phenyl-10-sulfopropylacridiniuM-9-carboxylate (NSP-DMAE-NHS) has achieved a much higher efficiency, improvement in the biosensor's performance, and amplification of the signal without causing any damage to the biomolecule in terms of its biochemical activity. In this study, the reagent strip method was initially used to detect TP53 fusion protein by combining the advantages of NSP-DMAE-NHS and immunochromatography. In our experiment, the control and study lines on the strips were immobilized through HRP-conjugated goat anti-rabbit IgG and TP53 antigen, respectively. The optimized concentration of the anti-TP53 antibody-NSP-DMAE-NHS immunoconjugates was then added to the TP53 antigen samples. After, the test strips were inserted and left in the aforementioned buffer solution for an additional 20 min. Finally, a lab-made luminous measurement device was used to analyze the corresponding control and study lines on the strips. Under optimized conditions, this method was found to be ultrasensitive, with a wide range of linear responses from 0.0008 ng mL-1 to 1 µg mL-1 and a limit of detection of 0.0008 ng mL-1 (0.013 pM). Thus, a novel competitive chemiluminescent assay based on reagent strips was established for the determination of the TP53 fusion proteins. The strategy has potential applications for ultrasensitive detection in the early diagnosis of cancer.