Obtaining Specific Hybridomas for Ki-67 Protein Immunodetection

Aigerim Turgimbayeva, Sailau Abeldenov, Nurgul Sarina, Bekbolat Khassenov, Saule Eskendirova


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.

KEYWORDS: breast cancer, Ki-67, monoclonal antibodies, nuclear antigen, recombinant antigen, tumor cells

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Harbeck NC, Penault-Llorca FN, Cortes JA, Gnant MS, Houssami NF, Poortmans P, et al. Breast cancer. Nat Rev Dis Primers. 2019; 5: 66, CrossRef.

Savitri M, Bintoro UY, Sedana MP, Diansyah MN, Romadhon PZ Amrita PNA, et al. Circulating plasma miRNA-21 as a superior biomarker compared to CA 15-3: assessment in healthy age matched subjects and different stage of breast cancer patients. Indones Biomed J. 2020; 12: 157-64, CrossRef.

Meiliana A, Dewi NM, Wijaya A. Cancer immunotherapy: a review. Indones Biomed J. 2016; 8: 1-20, CrossRef.

International Agency for Research on Cancer [Internet]. Kazakhstan, Source: Globocan 2020 [updated Mar 2021; cited Mar 2021]. Available from: https://gco.iarc.fr/.

Mansani FP, Bertucci MF, Montemór NP, Collaço LM, Pereira dos Santos JP. Evaluation of immunocytochemistry on the diagnosis of papillary mammary tumors by fine-needle aspiration biopsy.Mastology; 2019: 29: 90-6, CrossRef.

Garbar CB,Curé HP. Fine-needle aspiration cytology can play a role in neoadjuvantchemotherapy in operable breast cancer. Oncology. 2015; 92: 34-9, CrossRef.

Geethamala KN, Murthy VS, Vani BR, Rao MS, Thejaswini MU, Padmaja KP. Comparison of immunocytochemistry and immunohistochemistry on breast carcinoma: A boon or a bane? J Lab Physicians. 2017; 9: 5-10, CrossRef.

Pampa CT, Siddaraju NS, Kadambari DG, Kumar SD. Evaluation of estrogen and progesterone receptors and Her-2 expression with grading in the fine-needle aspirates of patients with breast carcinoma. J Cytol. 2018; 35: 223-8, CrossRef.

Osale CG, Mungania MS, Mutinda CK. Validation of fine needle aspiration cytology in the evaluation of human epidermal growth factor receptor-2 and hormonal receptor expressionpatterns in breast cancer patients. J Med Science Clin Research. 2019; 7: 1079-85, CrossRef.

Booth DC, Takagi MB, Sanchez-Pulido LA, Petfalski ED, Vargiu GR, Samejima K, et al. Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery. Elife. 2014; 2014: e01641, CrossRef.

Scholzen TS, Gerdes JG. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000; 182: 311-22, CrossRef.

Sun XV, Kaufman PD. Ki-67: more than a proliferation marker. Chromosoma. 2018; 127: 175-86, CrossRef.

Cuylen SV, Blaukopf CS, Politi AA, Müller-Reichert TN, Neumann B, Poser I, et al. Ki-67acts as a biological surfactant to disperse mitotic chromosomes. Nature. 2016; 535: 308-12, CrossRef.

Robertson SV, Stеlhammar GS, Darai-Ramqvist EA, Rantalainen MF, Tobin NP, Bergh J, et al. Prognostic value of Ki67 analysed by cytology or histology in primary breast cancer. J Clin Pathol. 2018; 22: 1-8, CrossRef.

Ács BF, Kulka JB, Kovács KC, Teleki IG, Tőkés AF, Meczker Á, et al. Comparison of 5 Ki-67 antibodies regarding reproducibility and capacity to predict prognosis in breast cancer: does the antibody matter? Hum Pathol. 2017; 65: 31-40, CrossRef.

Maloy SS, Valley JB, Taylor RR. Genetic Analysis of Pathogenic Bacteria : A Laboratory Manual. Plainville: Cold Spring Harbor Laboratory Press; 1995, article.

Oi VL, Herzenberg LS. Immunoglobulin-producing hybrid cell lines. In: Mishell BA, Shiigi SS, editors. Selected Methods in Cellular Immunology. San Francisco: Freeman; 1980. p.363-5, NLMID.

Coding JG. Antibody production by hybridoma. J Immunol Meth. 1980; 39: 285-308.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-54, CrossRef.

Mahmood TS, Yang PC. Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 2012; 4: 429-34, CrossRef.

Lestadi ID, Siregar NC, Wuyung PE. The correlation between TP53 expression and Ki-67 proliferation with Bartl malignancy degree of plasma cell neoplasm. Indones Biomed J. 2017; 9: 35-42, CrossRef.

Gerdes JS, Li LN, Schlueter CJ, Duchrow MA, Wohlenberg CB, Gerlach C, et al. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 1991; 138: 867-73, PMID.

Schmidt MF, Broll RN, Bruch HS, Finniss SX, Bögler OM, Duchrow M. Proliferation marker pKi-67 occurs in different isoforms with various cellular effects. J Cell Biochem. 2004; 91: 1280-92, CrossRef.

Key GS, Becker MK, Duchrow MJ, Schlüter CV,Gerdes JR. New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope. Lab Invest. 1993; 68: 629-36, PMID.

Ross WS, Hall PA. Ki67: from antibody to molecule to understanding? Clin Mol Pathol. 1995; 48: M113-7, CrossRef.

Kubbutat MH, Key GG, Duchrow MA, Schlüter CS, Gerdes JN. Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein), J Clin Pathol. 1994; 47: 524-28, CrossRef.

Roche [Internet]. CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody, Cat No. Cat. N. 790-4286 [cited Mar 2021]. Available from: https://pim-eservices.roche.com .

Abcam [Internet]. Recombinant Anti-Ki67 antibody [SP6] (ab16667) [cited Mar 2021]. Available from: https://www.abcam.com/.

Chung BK, Lee DY. Computational codon optimization of synthetic gene for protein expression, BMC Syst Biol. 2012; 6: 134-9, CrossRef.

Hames BD. Gel Electrophoresis of Proteins: A Practical Approach. 3rd ed. Oxford: Oxford University Press; 1998, NLMID.

Dowsett MA, Nielsen TK, A'Hern RN, Bartlett JS, Coombes RC. Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. J Natl Cancer Inst. 2011; 103: 1656-64, CrossRef.

Viale GS, Hanlon AE, Walker EG, Harlow GK, Bai IO, Russo L, et al. Ki-67 (30-9) scoring and differentiation of luminal A- and luminal B-like breast cancer subtypes. Breast Cancer Res Treat. 2019; 178: 451-8, CrossRef.

Zabaglo LF, Salter JA, Anderson HN, Quinn ED, Hills MK, Detre S, et al. Comparative validation of the SP6 antibody to Ki67 in breast cancer. J Clin Pathol. 2010; 63: 800-4, CrossRef.

NordiQC [Internet]. Immunohistochemical Quality Control. [cited Mar 2021]. Available from: https://www.nordiqc.org/.

United Kingdom National External Quality Assessment Service (UK NEQAS). Breast HER2 ICC Module and Breast Steroid gormone receptor Module. London: UK NEQAS ICC ISH; 2021.

de Azambuja ED, Cardoso FA, de Castro GJr, Colozza MH , Durbecq V, Sotiriou C, et al. Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients. Br J Cancer. 2007; 96: 1504-13, CrossRef.

Stuart-Harris RK, Caldas CB, Pinder SE, Pharoah PS. Proliferation markers and survival in early breast cancer: a systematic review and meta-analysis of 85 studies in 32,825 patients. Breast. 2008; 17: 323-34, CrossRef.

Ragab HM, Samy N, Afify ME. Maksoud NA, Shaaban HM. Assessment of Ki-67 as a potential biomarker in patients with breast cancer. J Genet Eng Biotechnol. 2018; 16: 479-84, CrossRef.

Prayogo AA, Wijaya AY, Hendrata WM, Looi SS, I’tishom R, HakimL, et al. Dedifferentiation of MCF-7 breast cancer continuous cell line, development of breast cancer stem cells (BCSCs) enriched culture and biomarker analysis. Indones Biomed J. 2020; 12: 115-23, CrossRef.

DOI: https://doi.org/10.18585/inabj.v13i3.1531

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The Prodia Education and Research Institute