Antibody Microarray Technology For The Identification Of Potential Serum Biomarkers For Liver Cancer

Background:Liver cancer is one of the most common malignant tumors, earlier diagnosis of liver cancer is critical to improve patient survival. Current diagnostics include a blood test for the level of a-fetoprotein (AFP) and ultrasound imaging of liver, but it is difficult to realize the early diagnosis of liver cancer for lacking enough diagnostic sensitivity. Therefore, discovering new serum protein biomarkers for liver cancer would have great clinical diagnostic value. Many potential protein biomarkers have been discoveried by analyzing the relative protein abundances in samples from liver cancer tissue and controls, but most of them haven't been identified systemly in human serum samples.Objective:Developing an antibody microarray technology for the identification of potential liver cancer serum biomarkers.Methods:An antibody microarray targeting for 14 potential liver cancer biomarkers (AFP, HSP27, HSP 70, CK-19, VEGF, GGT, CA125, GP73, AFU, PTMA, HBxAg, clusterin, GPC3, HCCR) was prepared on aldehyde coated glass slides. Clinical serum samples were collected from 44 patients with liver cancer and 46 healthy individuals according to standard operating procedures. The antibody microarrays were incubated with NHS-biotin labeled serum samples, then visualized with streptavidin-Cy3. Potential liver cancer biomarkers were identified by the comparative analysis of protein expression levels based on antibody microarray.Results:Optimum conditions of antibody microarray technology for the analysis of directly labeled serum samples were developed as follows:5% nonfat milk powder as the blocker and blocking the slides 1 hour at room temperature, the optimal mass ratio between NHS-biotin reagents and serum proteins was 1:40, the final dilution ratio of labeled serum samples for antibody microarray analysis was 1:200. The results of antibody microarray tested for 90 serum samples showed that the protein expression levels of HSP70,CK19,GPC3,PTMA,AFU,GP73,HBXAg,CA125 were statistical significance difference in serum between liver cancer and healthy, the positive predictive value and specificity of liver cancer diagnosis based on the joint detection of these six kinds of proteins were 88.6% and 97.8% respectively.Conclusions:As an effective high-throughput analytical technology, antibody microarray can be used to identify potential serum biomarkers of liver cancer or other diseases on a large scale.