title: [Preparation and application of melanoma-associated antigen D4 polyclonal antibody].
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Guangxi Colleges and Universities Key Laboratory of Biological Molecular Medicine Research, Guangxi Medical University, Nanning 530021, China.
authors.forename: Shujia
authors.initials: S
authors.lastname: He
authors.affiliation: First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
authors.forename: Yongyao
authors.initials: Y
authors.lastname: Gu
authors.affiliation: First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
authors.forename: Shaowen
authors.initials: S
authors.lastname: Xiao
authors.affiliation: Department of Histology and Embryology, Guangxi Colleges and Universities Key Laboratory of Preclinical Medicine Research, Guangxi Medical University, Nanning 530021, China.
authors.forename: Qingmei
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Chen
authors.affiliation: Department of Histology and Embryology, Guangxi Colleges and Universities Key Laboratory of Preclinical Medicine Research, Guangxi Medical University, Nanning 530021, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Luo
authors.affiliation: Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.affiliation: Department of Histology and Embryology, Guangxi Colleges and Universities Key Laboratory of Preclinical Medicine Research, Guangxi Medical University, Nanning 530021, China. *Corresponding author, E-mail: xiaoxunxie@gmail.com.
authors.forename: Xiaoxun
authors.initials: X
authors.lastname: Xie
mesh_terms: D000328:Adult; D000368:Aged; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology*; D005260:Female; D006801:Humans; D008297:Male; D008545:Melanoma / Q000276:immunology*; D008875:Middle Aged; D011993:Recombinant Fusion Proteins / Q000276:immunology
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; C430404:MAGE-E1 antigen, human; D011993:Recombinant Fusion Proteins