We newly cloned the gene encoding the human aspartyl (asparaginyl) beta-hydroxylase (HAAH) from the surgical tissue of a patient with hepatocellular carcinoma. This study was designed to generate HAAH-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were co-immunized with naked plasmid DNA containing N-terminal domain of encoding HAAH gene and recombinant HAAH polypeptide. Hybridomas were developed by the electrofusion of the splenocytes from mice immunized with plasmid DNA to Sp2/0 myeloma cells in vitro. Three hybridoma cell lines (designated G3, G9, and F11, respectively) stably secreting HAAH-specific MAbs were obtained. The specificity and sensitivity of MAbs were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Results showed that the three MAbs belong to IgG1 kappa isotype, the titer of MAbs reached was 5 x 10(4) - 1 x 10(5), and the affinity constant (k(aff)) of MAbs ranged between 2.5 x 10(8) - 1.1 x 10(9). MAb G3 was preliminarily applied to detection expression of HAAH for seven tumor tissues, including hepatocellular carcinoma, lung cancer, kidney cancer, cholangiocarcinoma, prostate cancer, breast cancer, and glioblastoma by immunohistochemical stain. Our studies demonstrated that co-immunization of naked DNA containing encoding gene of target antigen and recombinant target protein, and combined with in vitro electrofusion, is an effective and simple method to raise MAbs.