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Research Article

Academia Journal of Biotechnology 6(8): 225-235, August 2018
DOI: 10.15413/ajb.2018.0128
ISSN: 2315-7747
2018 Academia Publishing

Abstract


Application of flow cytometry to study receptor binding potency of FITC and/or biotin conjugated human growth hormone in comparison with radio receptor assay
 

Accepted 9th August, 2018
 
Abdolkhaleg Deezagi

Department of Molecular Medicine and Biochemistry, National Institute of Genetic Engineering and Biotechnology, Tehran-Iran.

Radio receptor assay (RRA) is the most sensitive and useful method for studying bioactivity of therapeutic proteins. Some of the disadvantages and limitations of the RRA are the biosafety and biohazardous problems. The aim of this paper was to investigate and compare receptor binding potential of non-radioactive conjugated human growth hormone (hGH) by flow cytometry versus to 125I hGH in binding to hGH receptors. hGH was separately labeled with 125I and/or non-radioactive ligands (FITC and/or biotin). The efficiency of labeling was calculated. Thereafter, the receptor binding potential and fraction of the radio labeled and non-radioactive ligand conjugated hGH were assayed by IM-9 human lymphocyte cell lines and pregnant rabbit liver hepatocytes microsomes. Receptor assay was done in a competitive manner and in immunoneutralizing conditions. The fluorescence was observed by fluorescent microscope and quantified by flow cytometry. Labeling of hGH by biotin in theoric 5 molar ratio (5:1; Biotin: hGH) showed good efficiency of labeling and quantitative data of receptor assays comparable with radio receptor assay with hGH-125I. In a competitive manner, the profile of binding to these receptors indicated change in the affinity of labeled hGH and the affinity of FITC conjugated hGH decreased in comparison with biotin conjugated and 125I labeled hGH. In a competitive manner, the profile of binding to these receptors indicated the affinity of labeled hGH was slightly changed. In general, this flow cytometry based receptor assay is simple to perform and comparable with RRA and provides a safer and reliable alternative to radioactive methods.

Key words: Biological activity, biotinylation, human growth hormone, FITC conjugation, flow cytometry, radio- receptor Assay.
 

This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cite this article as:
Deezagi A (2018). Application of flow cytometry to study receptor binding potency of FITC and/or biotin conjugated human growth hormone in comparison with radio receptor assay. Acad. J. Biotechnol. 6(8): 225-235.

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