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23 April 2012

Establishment of a monoclonal antibody against a peptide of the novel zinc finger protein ZNF32 proved to be specific and sensitive for immunological measurements

Yuyan WeiBCDEF, Jie ZhangCD, Kai LiBF, Lugang HuangBD, Jun LiBF, Xiujie WangF, Ping LinACDEFG, Yuquan WeiG

DOI: 10.12659/MSM.882725

Med Sci Monit 2012; 18(5): BR167-173

Abstract

Background: ZNF32 has been predicted to be a zinc finger protein and is involved in cell differentiation and tumor development, but its precise function is unknown. Specific monoclonal antibodies (mAbs) have been widely used in research and clinical diagnosis and treatments. Therefore, we established an anti-ZNF32 mAb to characterize this protein’s function.
Material/Methods: Peptide49–63, a specific small peptide of ZNF32, was chosen and the synthetic keyhole limpet hemocyanin (KLH)-peptide49–63 was used as an antigen to immunize mice. A mAb against peptide49–63 was generated by hybridoma technology, and hybridoma cells were screened by limiting dilution. The isoform of mAb-pZNF32-8D9 was identified by double agar diffusion. The sensitivity and specificity of the mAb and expressed levels of ZNF32 in various cells and tissues were identified by enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, immunohistochemistry, and Western blotting.
Results: A stable anti-pZNF32-8D9 hybridoma secreting the anti-peptide49–63 mAb was established and the clone positive to the peptide49–63 in supernatant was 92% in ELISA. The mAb-pZNF32-8D9 is an immunoglobulin-1 that can be used for detecting the ZNF32 protein by immunocytochemistry, immunohistochemistry, and Western blotting and is highly sensitive and specific. We also found ZNF32 expressed at high levels in Jurkat and pulmonary squamous carcinoma cells, but it was not expressed in squamous epidermis cells.
Conclusions: mAb-pZNF32-8D9 can be used for the identification and expression of ZNF32. It might also provide a new tool for diagnostics or therapy for ZNF32-related diseases.

Keywords: Limit of Detection, Kruppel-Like Transcription Factors - immunology, Hybridomas, Chromosomes, Cell Line, Blotting, Western, Antibodies, Monoclonal - immunology, Amino Acid Sequence, Molecular Sequence Data, Peptide Fragments - immunology

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750