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Obtaining Specific Hybridomas for Ki-67 Protein Immunodetection

Journal: The Indonesian Biomedical Journal (Vol.13, No. 3)

Publication Date:

Authors : ; ; ; ; ;

Page : 261-70

Keywords : breast cancer; Ki-67; monoclonal antibodies; nuclear antigen; recombinant antigen; tumor cells;

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Abstract

BACKGROUND: Active proliferation is specific property of a tumor cells. However, the cost of the analysis is high due to commercial anti-Ki-67 mAbs used as the main immunoreagent for reliable identification of proliferating cells. In this study, recombinant protein was used to obtain specific mAbs for Ki-67 biomarker immunodetection. METHODS: Codon optimized fragment of ki-67 gene was cloned into the pET28c(+)vector. The recombinant protein was purified by immobilized metal affinity chromatography (IMAC) and confirmed by liquid chromatography–mass spectrometry (LC-MS)/MS. Hybridoma cells were obtained by fusing myeloma cells with mouse spleen cells immunized with recombinant antigen. The specificity and activity of mAbs was determined by enzyme-linked immunosorbent assay (ELISA), Western blot and immunocytochemistry. RESULTS: The pET-28c(+)/ki-67 plasmid, which encodes 355 amino acid protein, was obtained. Analysis by LC-MS/MS of the recombinant antigen showed that 77.5% of the amino-acid sequence belonged to Ki-67 protein. Recombinant fragment of Ki-67 protein was used to obtain specific hybridoma strains. ELISA and Western blot demonstrated high affinity and the specificity of obtained mAbs against Ki-67 protein. Newly generated anti-Ki67 mAbs detected target protein in proliferating cells of MCF-7 cell line by immunocytochemistry. CONCLUSION: Newly developed mAbs are potentially useful as an immunodiagnostic tool for assessing the proliferative activity of breast tumor cells using immunocytochemistry.

Last modified: 2022-06-08 11:40:29