The Establishment And Preliminary Application Of ELISA Method Of Fc-ApoH

[Objects]Primary liver cancer(PLC) is one of the most deadly types of cancer in the world, ranking third in all cancers. Early diagnosis is the effective mean to improve the treatment of liver cancer. Now, the serum AFP and AFP-L3 are commonly used as the diagnosis markers of liver cancer in China. Because about 40% of clinical liver cancers do not have elevated AFP levels, the application of AFP and AFP-L3 is limited greatly. Therefore, we urgently try to find other diagnostic markers for early diagnosis of hepatocellular carcinoma. AFP-L3, which is also named as AFP heterogeneity, is highly fucosylated AFP and can specifically bind to lentil lectin(LCA). In previous study, we found that many glycoproteins in human serum are fucosylated, which have the potential to become candidate markers for the diagnosis of liver cancer. The expression levels of fucosylated glycoproteins in serum are significantly different between HCC and non-HCC patients. Based on lentil lectin affinity, we try to establish an ELISA method to detect Fc-APOH in serum. Further, we try to examine the correlation between the serum level of Fc-APOH and the development and process of liver cancer. Further we explore the possibility of Fc-APOH as a diagnostic marker for liver cancer, and attempt to provide a new method for early diagnosis of hepatocellular carcinoma.[Methods]HCC, 57 patients with benign liver disease and 52 healthy donors. 2. By using apolipoprotein H antibody as coating antibody and peroxidase labeled lentil lectin-horseradish as object, ELISA method was established to detect Fc-APOH in human serum, followed by process optimization and performance analysis.3. The patient’s serum levels of Fc-APOH were detected by as-established ELISA method. The cutoff value of Fc-APOH for HCC diagnosing was calculated by ROC curves. Its sensitivity and specificity for the diagnosis of HCC was analyzed and compared with AFP.[Results]1. The reaction conditions of ELISA were as follows:1:800 dilution of coating antibody, 1:200 dilution of HRP lentil lectin; optimal coating conditions is 4℃ for the night; the most suitable sealing liquid is 1% BSA PBST; the optimum conditions for the closure is 37℃ for 60 min; the optimum reaction conditions of ELISA lentil lectin and samples is at room temperature for 30 min; the optimum reaction condition of coating antibody and samples in serum is 37 ℃ for 60 min; The optimum substrate reaction conditions is 37℃ for 15 min. 2. For as-established ELISA method, the linear range is 78.1 ng/ml-1250 ng/ml, the limit of detection was 60.02ng/ml, the lower limit of normal is 348.1ug/ml and the variance coefficient of intra and inter is less than 10%. 3. The serum level of Fc-APOH is significantly lower in liver cancer group than in benign liver disease group and healthy control group. When cutoff value was 327.5μg/ml, the sensitivity and specificity of Fc-APOH for HCC diagnosing were 77.5% and 84.4%. The area under the ROC curve was 0.887. 4. In 80 HCC patients, the sensitivity of Fc-APOH(77.5%) was significantly higher than AFP(62.5%) for diagnosing liver cancer.[Conclusions]Fc-APOH, which may provide an effective tool for studying the expression of Fc-APOH in hepatocellular carcinoma, and provide important reference for setting up a method to detect fucosylated proteins in human serum. 2. The expression levels of Fc-APOH successively decreased in healthy control group, benign liver disease group and liver cancer group. It suggests that the serum Fc-APOH levels closely related with the occurrence and development of HCC. And this result is different from those published previously.3. The method established in this study can be used for the diagnosis of liver cancer with high sensitivity and specificity. It is suggested that Fc-APOH be a noval candidate marker for liver cancers, especially for liver cancers with low AFP.