Generation of Monoclonal Antibody Against 6-Keto PGF and Development of ELISA for Its Quantification in Culture Medium

Overview

Journal Biochem Biophys Rep

Specialty Biochemistry

Date 2024 Jun 28

PMID 38939124

Authors

Md Mazharul Islam Chowdhury

Nafisa Kabir

Rezwana Ahmed

Kazushige Yokota

Randy Mullins

Hasan Mahmud Reza

Affiliations

Soon will be listed here.

Abstract

Prostacyclin or prostaglandin I (PGI), 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 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 PGFα, a stable PGI metabolite, and its quantification to determine the role of PGI in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGFα and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGFα 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 PGFα 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 regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI in maintaining homeostasis and adipocyte differentiation.

References

Xu L, Nishimura K, Jisaka M, Nagaya T, Yokota K . Gene expression of arachidonate cyclooxygenase pathway leading to the delayed synthesis of prostaglandins E2 and F2alpha in response to phorbol 12-myristate 13-acetate and action of these prostanoids during life cycle of adipocytes. Biochim Biophys Acta. 2006; 1761(4):434-44. DOI: 10.1016/j.bbalip.2006.03.017. View

Syeda P, Hossain M, Chowdhury A, Rahman M, Khan F, Nishimura K . A monoclonal antibody specific for Δ12-prostaglandin J2 and its utilization in the immunological assay in cell culture system of adipocytes. Hybridoma (Larchmt). 2012; 31(5):364-71. DOI: 10.1089/hyb.2012.0027. View

Mazid M, Chowdhury A, Nagao K, Nishimura K, Jisaka M, Nagaya T . Endogenous 15-deoxy-Delta(12,14)-prostaglandin J(2) synthesized by adipocytes during maturation phase contributes to upregulation of fat storage. FEBS Lett. 2006; 580(30):6885-90. DOI: 10.1016/j.febslet.2006.11.049. View

Chu X, Ageishi Y, Nishimura K, Jisaka M, Nagaya T, Shono F . Development of enzyme-linked immunosorbent assay for 8-iso-prostaglandin F2alpha, a biomarker of oxidative stress in vivo, and its application to the quantification in aged rats. J Pharm Biomed Anal. 2009; 50(5):911-6. DOI: 10.1016/j.jpba.2009.06.015. View

Hopkins N, Gorman R . Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2). Biochim Biophys Acta. 1981; 663(2):457-66. DOI: 10.1016/0005-2760(81)90174-0. View

Goding J . Antibody production by hybridomas. J Immunol Methods. 1980; 39(4):285-308. DOI: 10.1016/0022-1759(80)90230-6. View

Syeda P, Hossain M, Chowdhury A, Rahman M, Nishimura K, Jisaka M . Generation of monoclonal antibody for 15-deoxy-Δ¹²,¹⁴-prostaglandin J₂ and development of enzyme-linked immunosorbent assay for its quantification in culture medium of adipocytes. Appl Biochem Biotechnol. 2012; 167(5):1107-18. DOI: 10.1007/s12010-012-9616-9. View

Shono F, Yokota K, Horie K, Yamamoto S, Yamashita K, Watanabe K . A heterologous enzyme immunoassay of prostaglandin E2 using a stable enzyme-labeled hapten mimic. Anal Biochem. 1988; 168(2):284-91. DOI: 10.1016/0003-2697(88)90320-x. View

Chu X, Xu L, Nishimura K, Jisaka M, Nagaya T, Shono F . Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms. Biochim Biophys Acta. 2009; 1791(4):273-80. DOI: 10.1016/j.bbalip.2009.01.022. View

10.

LOWRY O, ROSEBROUGH N, FARR A, RANDALL R . Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265-75. View

11.

Yokota K, Morishima T, Nagaya T, Jisaka M, Takinami K . Modification of cultured Madin-Darby canine kidney cells with dietary unsaturated fatty acids and regulation of arachidonate cascade reaction. Biosci Biotechnol Biochem. 1996; 60(7):1096-103. DOI: 10.1271/bbb.60.1096. View

12.

Kelton J, Blajchman M . Prostaglandin I2 (prostacyclin). Can Med Assoc J. 1980; 122(2):175-9. PMC: 1801769. View

13.

Negrel R, Gaillard D, Ailhaud G . Prostacyclin as a potent effector of adipose-cell differentiation. Biochem J. 1989; 257(2):399-405. PMC: 1135593. DOI: 10.1042/bj2570399. View

14.

Hossain M, Chowdhury A, Rahman M, Nishimura K, Jisaka M, Nagaya T . Development of enzyme-linked immunosorbent assay for Δ12-prostaglandin J2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase. Prostaglandins Other Lipid Mediat. 2011; 94(3-4):73-80. DOI: 10.1016/j.prostaglandins.2010.12.005. View

15.

Hyman B, Stoll L, Spector A . Prostaglandin production by 3T3-L1 cells in culture. Biochim Biophys Acta. 1982; 713(2):375-85. DOI: 10.1016/0005-2760(82)90256-9. View

16.

Tsuboi H, Sugimoto Y, Kainoh T, Ichikawa A . Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation. Biochem Biophys Res Commun. 2004; 322(3):1066-72. DOI: 10.1016/j.bbrc.2004.08.018. View

17.

Rahman M, Khan F, Syeda P, Nishimura K, Jisaka M, Nagaya T . Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes. Cytotechnology. 2013; 66(4):635-46. PMC: 4082781. DOI: 10.1007/s10616-013-9616-9. View

18.

Catalioto R, Gaillard D, Maclouf J, Ailhaud G, Negrel R . Autocrine control of adipose cell differentiation by prostacyclin and PGF2 alpha. Biochim Biophys Acta. 1991; 1091(3):364-9. DOI: 10.1016/0167-4889(91)90201-8. View

19.

Lu S, Nishimura K, Hossain M, Jisaka M, Nagaya T, Yokota K . Regulation and role of arachidonate cascade during changes in life cycle of adipocytes. Appl Biochem Biotechnol. 2004; 118(1-3):133-53. DOI: 10.1385/abab:118:1-3:133. View

20.

Reginato M, Krakow S, Bailey S, Lazar M . Prostaglandins promote and block adipogenesis through opposing effects on peroxisome proliferator-activated receptor gamma. J Biol Chem. 1998; 273(4):1855-8. DOI: 10.1074/jbc.273.4.1855. View