Md. Mazharul Islam Chowdhury , Nafisa Kabir , Rezwana Ahmed , Kazushige Yokota , Randy Mullins , Hasan Mahmud Reza
{"title":"Generation of monoclonal antibody against 6-Keto PGF1α and development of ELISA for its quantification in culture medium","authors":"Md. Mazharul Islam Chowdhury , Nafisa Kabir , Rezwana Ahmed , Kazushige Yokota , Randy Mullins , Hasan Mahmud Reza","doi":"10.1016/j.bbrep.2024.101748","DOIUrl":null,"url":null,"abstract":"<div><p>Prostacyclin or prostaglandin I<sub>2</sub> (PGI<sub>2</sub>), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI<sub>2</sub> in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGF<sub>1</sub>α, a stable PGI<sub>2</sub> metabolite, and its quantification to determine the role of PGI<sub>2</sub> in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGF<sub>1</sub>α and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGF<sub>1</sub>α was found. Then, fusion of antibody-producing spleen lymphocytes with SP-2 myeloma cells and thymocytes was performed and cultured in HAT-medium supplemented with hypoxanthine, aminopterin, and thymine. Specific antibody-producing cells (M2-A4-B8-D10) against 6-Keto PGF<sub>1</sub>α were identified and separated. A standard ELISA calibration curve was developed with 100% reactivity for 6-Keto-PGF 1 α ranging from 0.26 pg to 6.44 ng corresponding to 90% and 10% of the maximum binding capacity for the immobilized antigen respectively. This method can easily be applied to monitor PGI<sub>2</sub> regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI<sub>2</sub> in maintaining homeostasis and adipocyte differentiation.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.3000,"publicationDate":"2024-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001122/pdfft?md5=13a159d6eb5def427e7325a56862fb03&pid=1-s2.0-S2405580824001122-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry and Biophysics Reports","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2405580824001122","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Prostacyclin or prostaglandin I2 (PGI2), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI2 in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGF1α, a stable PGI2 metabolite, and its quantification to determine the role of PGI2 in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGF1α and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGF1α was found. Then, fusion of antibody-producing spleen lymphocytes with SP-2 myeloma cells and thymocytes was performed and cultured in HAT-medium supplemented with hypoxanthine, aminopterin, and thymine. Specific antibody-producing cells (M2-A4-B8-D10) against 6-Keto PGF1α were identified and separated. A standard ELISA calibration curve was developed with 100% reactivity for 6-Keto-PGF 1 α ranging from 0.26 pg to 6.44 ng corresponding to 90% and 10% of the maximum binding capacity for the immobilized antigen respectively. This method can easily be applied to monitor PGI2 regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI2 in maintaining homeostasis and adipocyte differentiation.
期刊介绍:
Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.