Detection and Quantification of Tp53 and p53-Anti-p53 Autoantibody Immune Complex: Promising Biomarkers in Early Stage Lung Cancer Diagnosis

Abstract

Lung cancer is a leading cause of death worldwide, claiming nearly 1.80 million lives in 2020. Screening with low-dose computed tomography (LDCT) reduces lung cancer mortality by about 20% compared to standard chest x-rays among current or heavy smokers. However, several reports indicate that LDCT has a high false-positive rate. In this regard, methods based on biomarker detection offer excellent potential for developing noninvasive cancer diagnostic tests to complement LDCT for detecting stage 0∼IV lung cancers. Herein, we have developed a method for detecting and quantifying a p53-anti-p53 autoantibody complex and the total p53 antigen (wild and mutant). The LOD for detecting Tp53 and PIC were 7.41 pg/mL and 5.74 pg/mL, respectively. The detection ranges for both biomarkers were 0–7500 pg/mL. The known interfering agents in immunoassays such as biotin, bilirubin, intra-lipid, and hemoglobin did not detect Tp53 and PIC, even at levels that were several folds higher levels than their normal levels. Furthermore, the present study provides a unique report on this preliminary investigation using the PIC/Tp53 ratio to detect stage I–IV lung cancers. The presented method detects lung cancers with 81.6% sensitivity and 93.3% specificity. These results indicate that the presented method has high applicability for the identification of lung cancer patients from the healthy population.

The Usefulness of the Ratio of Antigen–Autoantibody Immune Complexes to Their Free Antigens in the Diagnosis of Non-Small Cell Lung Cancer

“…A recent method using a 9-guanine DNA chip with high affinity to AIC and its free antigen proved to have good clinical applicability [ 24 , 25 , 26 ]. This study aimed to explore biomarkers used in the diagnosis of NSCLC using a combination of the ratios of AIC to its free antigen for four well-known lung cancer-associated protein markers, including CYFRA21-1, which is one of the most effective discriminators of NSCLC [ 28 , 29 , 30 ].…”

“…Based on previous studies, bioconjugates were prepared using six types of syntheses—marker protein-capture antibody (cAb)-DNA, anti-mouse IgG-Cy5, anti-human IgG-Cy5, marker protein-detection Ab (dAb)-fluorescent beads (FB), anti-human-IgG-FB, and anti-human-IgG-FB—and Cy5-DNA for four protein markers, including CYFRA21-1, ProGRP, NGAL, and NSE [ 24 , 26 ]. The lateral flow strip membranes (LFSM) were also prepared to detect the eight markers (CIC, CYFRA21-1, PrGIC, ProGRP, NGIC, NGAL, NSIC, and NSE).…”

“…After washing, the bound materials of each marker were detected and quantified by scanning the LFSM using a BMT Membrane Reader TM (Biometrix Technology Inc., Chuncheon, Republic of Korea). The fluorescence intensity was expressed in arbitrary units according to validated standard curves [ 24 , 26 ].…”

“…However, the amount and type of tumour-specific proteins that increase in each individual may differ during cancer development. Therefore, if a specific cut-off is used to discriminate between patients with cancer and healthy individuals, the overlap with healthy individuals will result in a lower specificity, which was also observed for AICs and free antigens separately measured in a previous study using the same method [ 24 , 25 , 26 ]. Irrespective of the number of tumour-specific proteins present in each individual, the ratio of AIC to its free antigen confirmed its potential as a marker to discriminate between the healthy controls (HCs) and patients with early-stage lung cancer [ 24 , 25 ].…”

“…A new method that uses a 9-guanine DNA chip to measure the antigen–autoantibody immune complex (AIC) and its antigen based on the ratio of AIC to its free antigen was verified for its efficiency in detecting lung cancer [ 24 , 25 , 26 ]. Previously, tumour marker-specific autoantibodies were overexpressed in patients with cancer [ 27 ].…”