Detection of Circulating Antibodies to p16 Protein-Derived Peptides in Hepatocellular Carcinoma

Yangchun Xu, MM; Litong Gu, MM; Jiaxin Wang, MD; Zhenqi Wang, MD; Ping Zhang, MD, PhD; Xuan Zhang, MD, PhD


Lab Med. 2020;51(6):574-578. 

In This Article

Materials and Methods


A total of 122 patients were recruited for this study by the First and Second Hospitals of Jilin University, Changchun, China, ranging from March 2010 to November 2015. Their HCC diagnosis was performed via CT scans and circulating AFP levels, and their median age was 54.8 years ± 9.7 years. HCC staging was confirmed as per the Barcelona Clinic Liver Cancer staging system.[23] Table 1 contains the patient demographics and clinical characteristics. To explore the correlation between clinical stages and plasma anti-p16 IgG levels, all 122 patients were classified into 3 subgroups, as follows: group 1 (stages 0 + A), group 2 (stage B), and group 3 (stages C + D). Plasma specimens were collected prior to anticancer therapy. Also, 134 healthy control subjects (107 males and 27 females) aged 55.2 years ± 9.2 years were recruited from local communities for this study. The controls, with no malignancy or severe autoimmune disease history, were included based on a clinical interview and image examination.

The recruited patients and controls were all of Chinese Han origin, and all provided informed written consent. This work was approved by the ethics committee of the Second Hospital of Jilin University, Changchun, China, and conformed to the requirements of the Declaration of Helsinki.

Plasma Anti-p16 IgG Level Detection

According to B-cell and human leukocyte antigen-II restricted epitopes, we designed a human p16-derived peptide antigen (CGFLDTLVVLHRAGARLDVRDAWGRLPVD) via computational prediction ( and synthesized this peptide antigen using solid-phase chemistry to ensure >95% purity. It was then applied to the development of an in-house ELISA assay based on the plasma anti-p16 IgG, as the method described in previous study.[24] Briefly, the peptide antigen was dissolved in 67% acetic acid to obtain a 5 mg/mL stock solution, and a 20 μg/mL working solution was then obtained by diluting with coating buffer (0.1 M phosphate buffer containing 0.15 M NaCl and 10 mM ethylenediaminetetraacetic acid, pH 7.2). Next, antigen coating of the maleimide-activated plates (Thermo Scientific) was completed as per the corresponding specifications and then washed twice using 200 μL wash buffer (phosphate-buffered saline [PBS] containing 0.05% Tween 20). Fifty microliters of specimens and 50 μL of assay buffer (PBS containing 0.5% bovine serum albumin) were added to each specimen well (positive control [PC]) and negative control (NC) well incubating for 1.5 h at room temperature, respectively, in which plasma specimens were diluted (1:200) in assay buffer in advance. After washing the plates thrice, 50 μL of peroxidase-conjugated goat anti-human IgG antibody (1:50,000, ab98567; Abcam) was added and incubated for an hour at room temperature, followed by color development using 50 μL of stabilized chromogen (SB02; Life Technologies, Beijing, China). Twenty minutes later, 25 μL of stop solution (SS04; Life Technologies) was added to terminate the reaction. A microplate reader was used to determine the optical density (OD) within 10 min at 450 nm, and 620 nm was selected as the reference wavelength.

Two repeats were set up for all specimens. The relative levels of plasma IgG antibodies were displayed with specific binding ratio (SBR), calculated as (OD specimen − OD NC)/(OD PC − OD NC). Additionally, to minimize intra-assay deviation, the accuracy of the in-house ELISA antibody detection method was evaluated using the ratio of the repeated OD value of each specimen to its sum. Specimens with ratios of >10% were excluded from the analysis. Unrelated healthy subjects (>100) were randomly selected as quality control (QC) specimens for calculating interassay deviation, and ELISA repeatability was represented by the coefficient of variation.

Data Analyses

Data are expressed as the mean ± standard deviation (SD) in SBR. The Mann-Whitney U test (2-tailed; IBM SPSS Statistics 21.0) was used to compare the difference in plasma anti-p16 IgG levels between patients and controls. Receiver operating characteristic (ROC) curve analysis was performed to obtain the area under the ROC curve (AUC) with the 95% confidence interval (CI), as well as the sensitivity of the anti-p16 IgG assay against a specificity of 95%.