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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

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Abstract

15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) is a biologically active molecule serving as a pro-adipogenic factor or an anti-inflammatory regulator. This compound is one of naturally occurring derivatives formed by the non-enzymatic dehydration of PGD2. To determine the endogenous synthesis of 15d-PGJ2, a convenient immunological approach is useful. At first, we established a cloned hybridoma cell line to secrete a monoclonal antibody specific for 15d-PGJ2. For the development of a solid-phase enzyme-linked immunosorbent assay (ELISA), the immobilized antigen using a protein conjugate of 15d-PGJ2 was allowed to react competitively with a monoclonal antibody in the presence of free 15d-PGJ2. Under the optimized conditions, a sensitive calibration curve was generated able to determine the amount of 15d-PGJ2 from 0.5 pg to 9.7 ng with 71 pg of 50% displacement in one assay. Our monoclonal antibody did not recognize other related prostanoids except PGJ2 with cross-reaction of 4%. Our ELISA was demonstrated to be reliable for the quantification of 15d-PGJ2 in the maturation medium of cultured adipocytes by confirming the accuracy and specificity of its determination. The application of our assay revealed that the non-enzymatic formation of 15d-PGJ2 became more evident after several hours of incubation with authentic PGD2 at 37 °C. The results indicate the usefulness of our developed solid-phase ELISA with the monoclonal antibody for further studies on the endogenous synthesis of 15d-PGJ2 and its roles in various cells and tissues.

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Abbreviations

15d-PGJ2 :

15-deoxy-Δ12,14-Prostaglandin J2

ELISA:

Enzyme-linked immunosorbent assay

PPAR:

Peroxisome proliferator-activated receptor

COX:

Cyclooxygenase

PGDS:

PGD synthase

BSA:

Bovine serum albumin

DME-HEPES:

Dulbecco’s modified Eagle medium with 25 mM HEPES

FBS:

Fetal bovine serum

IBMX:

3-Isobutyl-1-methylxanthine

PBS(−):

Phosphate-buffered saline without Ca2+ and Mg2+ ions

RP-HPLC:

Reverse-phase high-performance liquid chromatography

GC–MS:

Gas chromatography–mass spectrometry

LC–MS/MS:

Liquid chromatography–mass spectrometry/mass spectrometry

References

  1. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P., Spiegelman, B. M., & Evans, R. M. (1995). 15-Deoxy-Δ12,14-prostaglandin J2 is a ligand for adipocyte determination factor PPARγ. Cell, 83, 803–812.

    Google Scholar 

  2. Kliewer, S. A., Lenhard, J. M., Wilson, T. M., Patel, I., Morris, D. C., & Lehman, J. M. (1995). A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor γ and promote adipocyte differentiation. Cell, 83, 813–819.

    Google Scholar 

  3. Mazid, M. A., Chowdhury, A. A., Nagao, K., Nishimura, K., Jisaka, M., Nagaya, T., et al. (2006). Endogenous 15-deoxy-Δ12,14-prostaglandin J2 synthesized by adipocytes during maturation phase contributes to upregulation of fat storage. FEBS Letters, 580, 6885–6890.

    Article  CAS  Google Scholar 

  4. Hossain, M. S., Chowdhury, A. A., Rahman, M. S., Nishimura, K., Jisaka, M., Nagaya, et al. (2011). 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 Mediators, 94, 73–80.

  5. Straus, D. S., Pascual, G., Li, M., Welch, J. S., Ricote, M., & Hsiang, C. H. (2000). 15-Deoxy-Δ12,14-prostaglandin J2 inhibits multiple steps in the NF-κB signaling pathway. Proceedings of the National Academy of Sciences, USA, 97, 4844–4849.

    Article  CAS  Google Scholar 

  6. Rossi, A., Elia, G., & Santoro, M. G. (1996). 2-Cyclopenten-1-one, a new inducer of heat shock protein 70 with antiviral activity. Journal of Biological Chemistry, 271, 32192–32196.

    Article  CAS  Google Scholar 

  7. Bui, T., & Straus, D. S. (1998). Effects of cyclopentenone prostaglandins and related compounds on insulin-like growth factor-I and Waf1 gene expression. Biochimica et Biophysica Acta, 1397, 31–42.

    Article  CAS  Google Scholar 

  8. Chowdhury, A. A., Rahman, M. S., Nishimura, K., Jisaka, M., Nagaya, T., Ishikawa, T., et al. (2011). 15-Deoxy-Δ12,14-prostaglandin J2 interferes inducible synthesis of prostaglandins E2 and F2α that suppress subsequent adipogenesis program in cultured preadipocytes. Prostaglandins & Other Lipid Mediators, 95, 53–62.

    Article  CAS  Google Scholar 

  9. Hayaishi, O. (1988). Sleep-wake regulation by prostaglandins D2 and E2. Journal of Biological Chemistry, 263, 14593–14596.

    CAS  Google Scholar 

  10. Fukushima, M. (1992). Biological activities and mechanisms of action of PGJ2 and related compounds: An update. Prostaglandins, Leukotrienes and Essential Fatty Acids, 47, 1–12.

    Article  CAS  Google Scholar 

  11. Fitzpatrick, F. A., & Wynalda, M. A. (1983). Albumin-catalyzed metabolism of prostaglandin D2. Identification of products formed in vitro. Journal of Biological Chemistry, 258, 11713–11718.

    CAS  Google Scholar 

  12. Shibata, T., Kondo, M., Osawa, T., Shibata, N., Kobayashi, M., & Uchida, K. (2002). 15-Deoxy-Δ12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes. Journal of Biological Chemistry, 277, 10459–10466.

    Article  CAS  Google Scholar 

  13. Lu, S., Nishimura, K., Hossain, M. A., Jisaka, M., Nagaya, T., & Yokota, K. (2004). Regulation and role of arachidonate cascade during changes in life cycle of adipocytes. Applied Biochemistry and Biotechnology, 118, 133–153.

    Article  CAS  Google Scholar 

  14. Xu, L., Nishimura, K., Jisaka, M., Nagaya, T., & Yokota, K. (2006). Gene expression of arachidonate cyclooxygenase pathway leading to the delayed synthesis of prostaglandins E2 and F2α in response to phorbol 12-myristate 13-acetate and action of these prostanoids during life cycle of adipocytes. Biochimica et Biophysica Acta, 1761, 434–444.

    Article  CAS  Google Scholar 

  15. Jowsey, I. R., Murdock, P. R., Moore, G. B., Murphy, G. J., Smith, S. A., & Hayes, J. D. (2003). Prostaglandin D2 synthase enzymes and PPARγ are co-expressed in mouse 3T3-L1 adipocytes and human tissues. Prostaglandins & Other Lipid Mediators, 70, 267–284.

    Article  CAS  Google Scholar 

  16. Xie, Y., Kang, X., Ackerman, W. E., IV, Belury, M. A., Koster, C., Rovin, B. H., et al. (2006). Differentiation-dependent regulation of the cyclooxygenase cascade during adipogenesis suggests a complex role for prostaglandins. Diabetes, Obesity & Metabolism, 8, 83–93.

    Article  CAS  Google Scholar 

  17. Yokota, K., Morishima, T., Nagaya, T., Jisaka, M., & Takinami, K. (1996). Modification of cultured Madin-Darby canine kidney cells with dietary unsaturated fatty acids and regulation of arachidonate cascade reaction. Bioscience Biotechnology and Biochemistry, 60, 1096–1103.

    Article  CAS  Google Scholar 

  18. Chu, X., Ageishi, Y., Nishimura, K., Jisaka, M., Nagaya, T., Shono, F., et al. (2009). Development of enzyme-linked immunosorbent assay for 8-iso-prostaglandin F2α, a biomarker of oxidative stress in vivo, and its application to the quantification in aged rats. Journal of Pharmaceutical and Biomedical Analysis, 50, 911–916.

    Article  CAS  Google Scholar 

  19. Shono, F., Yokota, K., Horie, K., Yamamoto, S., Yamashita, K., Watanabe, K., et al. (1988). A heterologous enzyme immunoassay of prostaglandin E2 using a stable enzyme-labeled hapten mimic. Analytical Biochemistry, 168, 284–291.

    Article  CAS  Google Scholar 

  20. Goding, J. W. (1980). Antibody production by hybridomas. Journal of Immunological Methods, 39, 285–308.

    Article  CAS  Google Scholar 

  21. Oi, V.T., & Herzenberg, L.A. (1980). Immunoglobulin-producing hybrid cell lines. In B. B. Mishell & S. M. Shiigi (Eds.), Selected methods in cellular immunology (pp. 351–372). San Francisco, CA: Freeman.

  22. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. L. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  23. Markwell, M. A., Hass, S. M., Toleret, N. E., & Bieber, L. I. (1981). Protein determination in membrane and lipoprotein samples: Manual and automated procedures. Methods in Enzymology, 72, 296–303.

    Article  CAS  Google Scholar 

  24. Green, H., & Kehinde, O. (1974). Sublines of mouse 3T3 cells that accumulate lipid. Cell, 1, 113–116.

    Article  CAS  Google Scholar 

  25. Green, H., & Kehinde, O. (1975). An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell, 5, 19–27.

    Article  CAS  Google Scholar 

  26. Yokota, K., Kusaka, M., Ohshima, T., Yamamoto, S., Kurihara, N., Yoshino, T., et al. (1986). Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor. Journal of Biological Chemistry, 261, 15410–15415.

    CAS  Google Scholar 

  27. Mazid, M. A., Nishimura, K., Nagao, K., Jisaka, M., Nagaya, T., & Yokota, K. (2007). Development of enzyme-linked immunosorbent assay for prostaglandin D2 using the stable isosteric analogue as a hapten mimic and its application. Prostaglandins & Other Lipid Mediators, 83, 219–224.

    Article  CAS  Google Scholar 

  28. Bell-Parikh, L. C., Ide, T., Lawson, J. A., McNamara, P., Reilly, M., & FitzGerald, G. A. (2003). Biosynthesis of 15-deoxy-Δ12,14-prostaglandin J2 and the ligation of PPARγ. The Journal of Clinical Investigation, 112, 945–955.

    CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan

    Pinky Karim Syeda, Mohammad Salim Hossain, Abu Asad Chowdhury, Mohammad Sharifur Rahman, Mitsuo Jisaka, Tsutomu Nagaya & Kazushige Yokota

  2. Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan

    Kohji Nishimura

  3. Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima, Tokushima, 770-8514, Japan

    Fumiaki Shono

Authors
  1. Pinky Karim Syeda
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  2. Mohammad Salim Hossain
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  3. Abu Asad Chowdhury
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  4. Mohammad Sharifur Rahman
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  5. Kohji Nishimura
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  6. Mitsuo Jisaka
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  8. Fumiaki Shono
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  9. Kazushige Yokota
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Correspondence to Kazushige Yokota.

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Syeda, P.K., Hossain, M.S., Chowdhury, A.A. et al. 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 167, 1107–1118 (2012). https://doi.org/10.1007/s12010-012-9616-9

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  • DOI: https://doi.org/10.1007/s12010-012-9616-9

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