Monoclonal Anti-gremlin 1 And Identification Of Antibodies

Objective:To prepare the monoclonal antibody against gremlin1and identify their affinity and specificity. Methods:Prepare monoclonal antibodies using hybridoma technique. Measure the concentrations of antibodies by ultraviolet spectrophotometry. Determine their titers in ascitic fluid with indirect ELISA. Identify their affinity and specificity with immunofluorometric assay of HEECs and Western blotting. Results:This study successfully prepared seven anti-gremlin1monoclonal antibodies. Their concentrations in ascitic fluid were from13.7to37.7mg/ml. The titers in ascitic fluid of1D4、1H5、1C9、1A4and1C4were all above1:3.2×104by indirect ELISA assay. By immunofluorometric assay of HEECs, all showed positive fluorescence except the blank control and the fluorescence was universe distributed in cytoplasms. For the positive staining, the antibody1A4exhibited the brightest signal of all. The expected band of about47KDa was obtained with the each antibody during western blotting procedure, especially1A4, but another unexpected band also appeared. Conclusions:We successfully prepared seven monoclonal antibodies. The titers in the ascitic fluid all met the needs for affinity chromatography and ELISA. Identified by immunofluorometric assay of HEECs and Western blotting, the seven monoclonal antibodies exhibited affinity to gremlin1, especially1A4, but some unspecified band also emerged.