A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients

Abstract

BackgroundWe report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals.ResultsFive cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera.ConclusionThis protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.

Autoantibodies against tumor-associated antigens for detection of hepatocellular carcinoma

“…In contrast, there are no data available, to our knowledge that associates autoantibodies against p53 with patients' clinical characteristics. In patients with colorectal cancer (CRC), there is an increase in the prevalence of anti-p53 autoantibodies in carcinoma in-situ (6%) compared with adenomas (1%), indicating that the level of anti-p53 autoantibody increases with CRC [36] hnRNP L Akada et al [42] HSP70, SOD2, and PRDX6 Shao et al [41] Glucose-regulated protein 78 Nomura et al [39] Ku86 Liu et al [40] CENPF, DDX3, HSPA4, HSPA5, VIM, LMNB1, and p53 Pekáriková et al [21] CRT Chen et al [28] Sui1, RalA Wang et al [43] KRT23, AHSG and FTL Wang et al [44] RalA Looi et al [45] HSP60, HSP70 Li et al [35] DDX3, eEF2, AIF, hnRNP A2, PBP, and TIM Chen et al [46] EIF3SI, LDHA, RFC2, and MCART1 Zhang et al [27] IMP1, IMP2, IMP3, p53, c-myc, cyclin B1, survivin and p16 Akere et al [13] p53 Zhou et al [47] HCC-22-5 Takashima et al [48] HSP70, GAPDH, PRX, Mn-SOD Looi et al [49] p16 Yagihashi et al [25] Survivin Su et al [17] IMP2 Himoto et al [19] IMPs Himoto et al [18] IMPs, p53, c-myc, and survivin Zhang et al [24] c-myc, cyclin B1, IMP1, Koc, p53, IMP2, and survivin Soo Hoo et al [50] p53, IMP2, Koc, CENP-F, p90 Le Naour et al [51] CRT, CK8, NDK-A, and ATP5B Zhang et al [23] IMP2, CENPF Zhang et al [20] IMP2 Raedle et al [52] p53 Covini et al [22] Cyclin B1 Imai et al [53] HCC1 TAAs: Tumor-associated antigens; HCC: Hepatocellular carcinoma; IMP: Insulin-like growth factor mRNA-binding; CENPF: Centromere protein F. 9…”

Relationship between Autoimmunity and Cancer or Metabolic Abnormality in Liver Diseases

“…Interestingly, Akada and colleagues isolated antibodies to heat shock protein (HSP) 70 from the sera of patients with HCV-related HCC alone, implying that persistent HCV infection may induce a tumor-associated antigen during malignant transformation [44]. Recently, glucose-regulated protein 78 (GRP78), which is a chaperone protein belonging to the HSP70 family, was captured as a novel TAA in patients with HCC [32].…”

“…This approach is mainly based on a classical proteomics workflow combining effective separation on 2-dimensional gel electrophoresis and identification by mass spectrometry. More recently, proteosome-covered array chips have been introduced as a novel technique for the detection of TAAs [44]. Cytoplasm Golgi processing, transporting and sorting 6% [38] of intracellular proteins IMP1 regulation of IGF-II mRNA 4-17% [22,39] p62/IMP2 regulation of IGF-II mRNA 1-14% [22,39] Koc/IMP3 regulation of IGF-II mRNA 5-10% [22,39] fatty acid synthase oncogenic protein 97% [40] heat shock protein 70 stress protein 47% [41] peroxiredoxin cellular redox regulation 33% [41] Mn-SOD scavenger of oxidative stress 33% [41] N/A,not available…”

Two novel protein chips for the detection of antibodies against porcine parvovirus

“…Additionally, chemical amplifications using Au nanoclusters or nanoparticles as catalytic centers have primarily been utilized for the silver enhancement/amplification reaction [19]. Cyanine dye 3(Cy3) was also applied to detect antigenantibody reaction products specifically, which is a procedure that was applied relatively early to the field of protein chips; furthermore, Cy3, Cy5, and IR800 can also be used to label protein chips [20,21]. Both of these methods were applied to evaluate the seroprevalence of PPV in serum samples collected from pig herds in China.…”