Generation of Monoclonal Antibodies against S-Adenosylmethionine and Their Application in Diagnosing Liver Diseases

Abstract

S-AdenosylMethionine (SAM) hapten-carrier conjugates were used to immunize BALB/c mice. Two strains of hybridoma cell lines that stably produced high-titer monoclonal antibodies against SAM were obtained. The biological properties of these two antibodies were characterized. A competitive ELISA (cELISA) was established using these antibodies. The sensitivity and specificity were analyzed, and human serum samples were measured using the SAM cELISA kit.Very little cross-reactivity (< 1%) was observed between the monoclonal antibodies and SAM analogues. These two antibodies are IgG2b-type antibodies. The affinity measured using the cELISA is 5.75 x 109 L/mol for the 118-6 antibody and 7.29 x 1010L/mol for the 84-3 antibody. According to the Immunohistochemistry (IHC) and flow cytometry results, normal liver cells contain much higher levels of SAM than liver cancer cells (p < 0.01). The results from 81 healthy subjects and 99 patients with liver disease showed the following sensitivity using the 240 nM cutoff: 95.65% (44/46) for hepatitis; 95% (19/20) for cirrhosis; 92.86% (13/14) for carcinoma; 100% (19/19) for liver failure; 30.86% (25/81) for healthy subjects; the detection specificity is 69.14% (56/81). Using the 120 nM cutoff, the sensitivitywas 80.43% for hepatitis; 85% for cirrhosis; 71.43% for carcinoma, 100% for liver failure; 3.70% for healthy subjects; the specificity is 96.30% (78/81). SAM, alanine transaminase and aspartate aminotransferase were compared in identifying liver diseases. Serum SAM allowed early detection of abnormal liver function and providing clinicians an important basis for a more accurate diagnosis of liver diseases than alanine transaminase and aspartate aminotransferase. Keywords: S-Adenosylmethionine (SAM); Monoclonal Antibody; Competitive ELISA; Liver Disease Diagnosis Abbreviations: Alanine Transaminase (ALT); Aspartate Aminotransferase (AST); Chronic Hepatitis B (CHB), Competitive ELISA (CELISA); Hematoxylin and Eosin (HE); Hepatocellular Carcinoma (HCC); Homocysteine (HCY); Immunohistochemistry (IHC); Keyhole Limpet Hemocyanin (KLH); L-Methionine(L-Met); Methionine Adenosine Transferase (MAT); Methylthioadenosine (MTA); Optical Density (OD); S-Adenosylhomocysteine(SAH); S-Adenosylmethionine(SAM).