title: Identification and characterization of monoclonal antibodies against GP73 for use as a potential biomarker in liver cancer screening and diagnosis.
abstract: Golgi membrane protein 1, or GP73, is recently being evaluated as a novel cancer biomarker against prostate cancer, lung adenocarcinoma, and hepatocellular carcinoma (HCC). In the microenvironment of HCC, GP73 expression levels are significantly elevated. It is this elevation that may prove more specific and sensitive for HCC detection than that of the traditional biomarker, alpha-fetoprotein (AFP). This may be especially true if it can be measured and identified earlier in the diagnostic process. We sought to develop a testing platform to measure GP73 levels for the purposes of earlier diagnostic screening of at risk patients. We expressed recombinant GP73 protein to use as an immunogen in order to develop several monoclonal anti-GP73 antibodies. Three clones, 1D7, 2B2, and 5B4, were identified with all three having a higher than 1:5,000,000 titer. These clones were then isotyped and validated to bind the immunogen protein. Different combinations of antibody pairs were then tested in order to create a functional sandwich antibody pair. Using this pair on liver disease patient serum samples, we found that GP73 was significantly elevated when compared to healthy control patient serum (P < 0.0001). Average GP73 levels in HCC patients was 284.0 ng/mL, slightly higher than liver disease patients (265.6 ng/mL), and significantly elevated over normal serum levels is (74.86 ng/mL). The area under the receiver-operating characteristic curve (ROC) for GP73 to detect liver cancer was 0.98 (95% CI, 0.95 to 1.00; P < 0.0001), and GP73 levels had a sensitivity of 97%, a specificity of 87% for detecting liver cancer. By contrast, the sensitivity and specificity of liver disease detection was 76% and 97%, respectively. We then tested detection of 74 serum samples (n control = 46, n liver disease = 7, n liver cancer = 21) by our ELISA testing methodology and commercial kit simultaneously. The results found that our kit and the commercial kit had a good linear correlation coefficient, r(2) = 0.932. Together these clones and our ELISA pair may prove extremely useful in the detection and monitoring of GP73 in HCC and other at risk patients.
authors.affiliation: a Raybiotech, Inc ., Guangzhou , China.|c RayBiotech, Inc ., Norcross , Georgia , USA.
authors.forename: Yuming
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: b The Third Affiliated Hospital of Sun Yat-sen University , Guangzhou , China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xi
authors.affiliation: a Raybiotech, Inc ., Guangzhou , China.|c RayBiotech, Inc ., Norcross , Georgia , USA.|d South China Biochip Research Center , Guangzhou , China.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Fang
authors.affiliation: a Raybiotech, Inc ., Guangzhou , China.|c RayBiotech, Inc ., Norcross , Georgia , USA.|d South China Biochip Research Center , Guangzhou , China.
authors.forename: Shuhong
authors.initials: S
authors.lastname: Luo
authors.affiliation: c RayBiotech, Inc ., Norcross , Georgia , USA.
authors.forename: Jarad J
authors.initials: JJ
authors.lastname: Wilson
authors.affiliation: a Raybiotech, Inc ., Guangzhou , China.|c RayBiotech, Inc ., Norcross , Georgia , USA.|d South China Biochip Research Center , Guangzhou , China.
authors.forename: Ruo-Pan
authors.initials: RP
authors.lastname: Huang
mesh_terms: D000911:Antibodies, Monoclonal / Q000097:blood / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*; D008565:Membrane Proteins / Q000097:blood / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; C410824:GOLM1 protein, human; D008565:Membrane Proteins
keywords: ELISA; GP73; diagnosis; liver cancer