title: 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.
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.
authors.affiliation: Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan.
authors.forename: Pinky Karim
authors.initials: PK
authors.lastname: Syeda
authors.forename: Mohammad Salim
authors.initials: MS
authors.lastname: Hossain
authors.forename: Abu Asad
authors.initials: AA
authors.lastname: Chowdhury
authors.forename: Mohammad Sharifur
authors.initials: MS
authors.lastname: Rahman
authors.forename: Kohji
authors.initials: K
authors.lastname: Nishimura
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Jisaka
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nagaya
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Shono
authors.forename: Kazushige
authors.initials: K
authors.lastname: Yokota
mesh_terms: D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D003470:Culture Media / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism
chemical_list: C097240:15-deoxy-delta(12,14)-prostaglandin J2; D000911:Antibodies, Monoclonal; D003470:Culture Media; D015230:Prostaglandin D2