Obtaining Specific Hybridomas for Ki-67 Protein Immunodetection
Turgimbayeva A. Abeldenov S. Sarina N. Khassenov B. Eskendirova S.
2021Prodia Education and Research Institute
Indonesian Biomedical Journal
2021#13Issue 3260 - 270 pp.
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 proliferating cells. In this study, recombinant protein was used to obtain specific mAbs for Ki-67 biomarker immunodetection. 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 enzymelinked immunosorbent assay (ELISA), Western blot and immunocytochemistry. RESULTS: The pET-28c(+)/ki-67 plasmid, which encodes 355 amino acid protein, was obtained. Analysis by LCMS/ 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 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
breast cancer , Ki-67 , monoclonal antibodies , nuclear antigen , recombinant antigen , tumor cells
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