| Background:The morbidity and mortality of Hepatocellular carcinoma(HCC) and hepatitis B is very high in China, especially in Guang Xi province. At present, the major ways to diagnose HCC are clinical examination, imaging, pathological diagnosis, but since the early clinical symptoms and signs of HCC is not obvious, once found is too late. Therefore, looking for early detection of serum markers of HCC and to carry on the simple and effective detection is the main content of this research. Alpha fetoprotein (AFP) is the only serum marker for HCC diagnosis at present, but the sensitivity is not good enough. A number of experiment showed that several indexes could increase the detection rate.Our research group have found that the expression level of anti-SMP30 antibody and anti-CNN2 antibody in the serum of HCC was remarkable increased than healthy people and other patients, but these wasn’t any report about the expression level of SMP30 and CNN2 protein level in serum of HCC. In the meantime, GP73 can increase the detection rate of HCC and can complement each other with AFP was reported.Objective:To explore the diagnostic value of serum SMP30, CNN2, GP73 in the patient of HCC, discuss the diagnostic value of the joint roles of serum SMP30, CNN2, GP73 and AFP in the diagnosis of HCC, and provide a better choice for the diagnosis of HCC.Methods:Using genetically engineered bacterium BL21-pET30a-SMP30、 BL21-pGold III-CNN2, which be structured and saved by our research group at-80 ℃, to express and purify human senescence marker protein-30 (SMP30) and H2-calponin (CNN2); To develop and analytically validate an enzyme-linked immunosorbent assay (ELISA) for the quantification of SMP30 protein and CNN2 protein in human serum from HCC, chronic HBV, HBV carrier, cirrhosis and health, respectively; To buy commercial Golgi protein 73 ELISA assay kit for the quantification of GP73 protein in serum from HCC, chronic HBV, HBV carrier, cirrhosis and health.Results:1. The concentration and purity of SMP30 were 0.6mg/mL and 92.82%, respectively. The optimum concentration of coated antibody(rabbit anti-SMP30 antibody), detect antibody(mouse anti-SMP30 antibody), enzyme marked antibody(goat anti-mouse 1g G-HRP) and serum were diluted to 1:200,1:400,1:5000,1:5, respectively. The standard protein curve regression equation was:y=1.673x+0.025, R2=0.996, P<0.001. The detection limit of the assay was 3.6 ng/mL, its linear detection was 3.6 to 464ng/mL. Coefficients of variation for intra and inter-assay variability 8% and 12%, respectively. The ELISA protocol produced reproducible standard curves, all of the R2>0.94. The concentration and positive rate of SMP30 in HCC patients were both significantly higher than viral hepatitis and healthy control, and there was no difference between viral hepatitis and healthy control. Diagnostic sensitivity and specificity of SMP30 were 8.4% and 98.86%, respectively. No significant correlation between the concentration of SMP30 in HCC and clinical data.2. The optimum concentration of coated antibody(goat anti-CNN2 antibody),detect antibody(mouse anti-CNN2 antibody), enzyme marked antibody(goat anti-mouse 1g G-HRP) and serum were diluted to 1:200,1:2000, 1:5000,1:10, respectively. The positive rate of HCC, healthy and hepatitis B were 2.1% (3/143),2.8%(6/217) and 2.4% (4/170), respectively. No statistical significance between the three groups. Diagnostic sensitivity and specificity of CNN2 were 2.4% and 97.2%, respectively. No relationship between the concentration of CNN2 in HCC and clinical data.3. The level of serum GP73 (ng/mL) in the group of HCC, cirrhosis, hepatitis B, hepatitis B carriers and healthy were 88.09±64.13、184.20±88.70、 149.53±89.76、39.32±8.99 and 25.27±12.221. The level of serum GP73 had no difference between male and female in healthy group (P=0.001); the level of serum GP73 of male in the group of HCC was significantly higher than that of female, with cirrhosis was significantly higher than that without cirrhosis, tumor diameter> 3cm was significantly higher than that≤3cm. The critical value for HCC was determined to be 61.93ng/mL, the sensitivity and specificity of the GP73 was 56.93% and 98.56%.4. Multiple HCC associated antigen detection at the same time can significantly increase the detection rate of HCC. The best combination was AFP+GP73, positive rate was 82.35%.Conclusions:1. Successfully established a sandwich ELISA assay for SMP30 protein with good repeatability. Serum SMP30 level in HCC patients was significantly increased compared to healthy and hepatitis B,indicate that SMP30 may become a new diagnostic biomarker for HCC.2. Serum CNN2 level in HCC patient had no difference compared to healthy and hepatitis B, So CNN2 might not be a diagnostic biomarker for HCC yet.3. The GP73 was as good as AFP with high sensitivity and specificity to be a diagnostic biomarker for HCC.4. The joint detection could reduce effectively the missed diagnosis of HCC whose AFP was negative, it could improve the detection rate of HCC. |
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