Diagnostic Value Of Antibodies Against A Panel Of Multiple Tumor-associated Antigens In Diagnosis Of Primary Hepatocellular Carcinoma

Background and ObjectiveHepatocellular carcinoma (HCC) is one of the most common tumors worldwide, particularly prevalent in Africa and Asia. There are 600,000 people died of HCC worldwide, and 320,000 hundred thousand in China. They are both in the second place of the incidence rate and the mortality worldwide. The majority of people with HCC will die within 1 year after its detection. This high case-fatality rate can in part be attributed to lack of diagnostic methods that allow early detection. How to establish a methodology to identify the high-risk individuals for HCC remains to be investigated.Many studies demonstrated that cancer sera contain antibodies which react with a unique group of autologous cellular antigens called tumor-associated antigens (TAAs), the detection of TAA will be useful for the diagnosis of HCC. A feature of HCC is that antecedent liver cirrhosis and chronic hepatitis are common precursor conditions and during transition to malignancy some patients develop autoantibodies which were not present during the preceding chronic liver disease phase, suggesting a new way of diagnosis of HCC. The major concern is that sensitivity is usually low when an individual antigen is used. In order to overcome this problem, some researchers propose to use a panel of selected TAAs to enhance anti-TAA antibody detection for immunodiagnosis of HCC.Methods1. Sample preparation: in this study, all patients were divided into two groups: primary hepatocellular cancer group consisted by 153 cases, all cases were the first visit to hospital and had been proved by pathologic diagnosis, and with no chemotherapy or radiotherapy; All health comparison group had health examination and confirmed have no tumour-associated disease.2. The antibodies against fourteen TAAs in sera from patients with primary hepatocellular cancer and normal human sera were detected by enzyme-linked immunosorbent assay (ELISA). The validity of testing results for each anti-TAA antibody was evaluated by epidemiological method.3. AHSG gene which was found to be different in HCC,hepatitisand and normal individuals xenogenic sera screening was cloned.ResultsThe results of single anti-TAA antibody was judged respectively1. When anti-TAA antibody was judged respectively, the sensitivity was low, the highest was 32.2%. But the positive rate for HCC was higher than that for normal human sera.2. When anti-TAA antibody was judged respectively, the sensitivities of the HBV-related HCC was higher than that of no- hepatitis HCC or normal.The results of TAA array dectected1. With the successive addition of TAAs to a final total of fourteen antigens, there was a stepwise increase of positive antibody.The sensitivity didn't increase until when there was eight TAAs, so eight TAA array was optimal in the present study. The positive antibody reaction of a array of eight TAAs reaching a sensitivity of 72.2% and a specificity of 91.2%. Positive and negative predictive values were 86.5% and 81.4%, respectively, which indicated that parallel assay of eight TAAs raised the diagnostic value greatly. Positive likelihood ratio and negative likelihood ratio were 6.25 and 0.14, which showed that the clinical diagnostic value of parallel assay of TAAs was high. Kappa value was 0.65, which indicated the observed value of this assay had midrange coincidence with actual value.2. When eight-TAA array was judged respectively, the sensitivity was no difference between male and female.the sensitivitie of people between 50 years and 60 years was higher than that of people less than 50years or older than 60 years.3. There was no difference between AFP dectection and TAAs detection.Ten AFP-negative HCC patients with anti-TAAs. When AFP detection and TAAs detection were connected in parallel, The sensitivitie was 91.0% (32/35) much higher than that of AFP detection 62.99%(22/35) or TAAs detection 65.7% (23/35) .Result of AHSG clone1. The recombinant plasmid of AHSG was digested into two fragments which are clone vector fragment (2.7kb) and ahsg gene fragment (1104bp). 1104bp ahsg gene fragment could be amplified by specific PCR. It was demonstrated that recombinant plasmid of AHSG gene (pTA) was constructed successfully.Gene sequencing results: AHSG gene fragment was 1104bp long. It encoded the polypeptide of 367 amino acid residues, corresponding to calculated molecular masses of 39 kDa. and the analysis of the AHSG cDNA sequences was performed by using Genbank database and BLAST program to search for homologs. The result showed that there was 7 nucleotide diffenence, and the homology was 99%.Conclusions1. This study shows that a panel of eight TAAs can enhance antibody detection for diagnosis of hepatocellular carcinoma, the approach may be used as a screening tool for population at high risk in hepatocellular carcinoma, and also a routine test in clinical practice.2. There was some sense of TAA detection to the AFP-negative HCC patients, We can consider it as a adjunctive tool for AFP detection. 3. The recombinant plasmid of AHSG gene (pTA) was constructed successfully. It was base of expression of protein AHSG.