[Preparation and application of melanoma-associated antigen D4 polyclonal antibody]

Objective To prepare the rabbit polyclonal antibody recognizing human melanoma-associated antigen D4 (MAGE-D4), and identify its immune characteristics for preliminary application. Methods MBP/MAGE-D4 fusion protein was expressed from pMAL-C2/MAGE-D4 recombinant plasmid constructed in the previous work. Then the purified MBP/MAGE-D4 protein was used to immunize the New Zealand white rabbits for generating polyclonal antibody. Subsequently, the anti-MAGE-D4 antibody was purified with protein A affinity chromatograph and identified by SDS-PAGE. The titer and specificity of the antibody were further detected by indirect ELISA and Western blotting, respectively. MAGE-D4 expression and localization of lung cancer tissues were analyzed by immunohistochemical staining. Results MAGE-D4 polyclonal antibody with high purity was obtained. Its titer was about 1:256 000. Western blot analysis demonstrated that the antibody could specifically react to the recombinant MAGE-D4 protein. Immunohistochemical staining showed that the antibody could recognize the endogenous MAGE-D4 protein in lung cancer tissues, and its positive rate was 69.6% (17/23). Conclusion The MAGE-D4 polyclonal antibody with high specificity and sensitivity has been successfully prepared.