Preparation And Application Of Anti-GP73 Monoclonal Antibody And Development Of Immunoassays

Objection Hepatocellular carcinoma(HCC) is one of the most frequent malignant tumors and is the fifth most common cause of cancer death in China. The incidence and mortality of HCC is found increasing year by year. The worldwide new cases of HCC are approximately 560,000 each year, of which nearly 50% of cases occur in China. HCC conventional screening and diagnosis methods include physical exa minations, imaging technologies and serum Alpha fetoprotein(AFP) examination. Nowadays, serum AFP is the most commonly used biomarker for HCC, but the clinical diagnostic accuracy to detect early HCC has been questioned as its sensitivity and specificity are not satisfactory. Recent studies revealed that the sensitivity and specificity of GP73 in serum are superior to AFP in the diagnosis of HCC. GP73 is expected to be a potential biomarker for early diagnosis of HCC. So, the aim of this research is to prepare the monoclonal antibody(m Ab) against GP73 to develop a Sandwich ELISA method and magnetic enzyme-linked immunoassay(MEIA) for quantitative measurement of serum GP73(s GP73).Methods 1. His-GP73 fusion protein and a pair of anti-GP73 m Ab were prepared purified. The fusion protein was identification by Western blot. The titer of the m Ab was detected by ELISA, the subclasses of m Ab were identified by the kit for Mouse Monoclonal subclasses and its specificity was evaluated by Western blotting and immunohistochemistry.2. anti-GP73 m Ab 8E7 was designed as coating antibody to coat the 96-well plate and the anti-GP73 m Ab 5A10 was designed as detecting antibody, which optimal concentrations were defined by titration. Standard curve was performed using purified GP73 protein to develop Sandwich ELISA method, then 119 HCC and 103 normal serum samples were detected by this newly designed method.3. anti-GP73 m Ab 8E7 was designed as coating antibody to coat immunomagnetic beads and the anti-GP73 m Ab 5A10 was designed as detecting antibody, which optimal concentrations were defined by titration. Standard curve was performed using purified GP73 protein to develop MEIA, and this newly designed method was performed to measure the s GP73 level in healthy people and HCC patients.Results 1. The GP73 fusion protein was successfully expressed, purified and then validated by SDS-PAGE, the purity of the protein is more than 90%. Two hybridoma cell lines against GP73 were obtained, which named 8E7 and 5A10, the heavy chain of immunoglobulin subclasses was Ig G2 a and Ig G1, respectively, and the titer is above 1:243,000. Western blot and immunohistochemistry showed that anti-GP73 m Ab can identify the GP73 protein.2. A sandwich ELISA assay for human s GP73 quantitative measurement was successfully established and the sensitivity of this assay was 1.56ng/m L. Clinical s GP73 samples in 103 healthy individuals and 119 HCC patients were detected by this method. The results suggested that the expression concentration of s GP73 in HCC patients were much higher than that of healthy individuals(P< 0.05).3. MEIA for s GP73 quantitative measurement was successfully established, and the sensitivity of this assay was 0.78 ng/m L. The GP73 level of sera in 30 healthy people and 30 HCC patients were quantitative measured by MEIA, the detecting results was the same as that of ELISA, but compared with ELISA, MEIA was more and convenient and timesaving.Conclusions In our research, GP73 fusion protein was used as antigen and we successfully prepare a-GP73 m Ab. The s GP73 level in healthy peoplev and HCC patients were quantitative measured by using the established sandwich ELISA assay and MEIA. Those two detection methods were compared, and the results indicated that MEIA was more efficient, convenient and tamesaving in detecting s GP73 than ELISA. The level of GP73 in HCC patients was higher than that in healthy individuals, which indicated that GP73 may be a potential biomarker for the diagnosis of HCC, and those immunoassay methods based on anti-GP73 m Ab is expected to promote their application in HCC clinical detection and disease screening in the future.