title: A monoclonal antibody specific for Δ12-prostaglandin J2 and its utilization in the immunological assay in cell culture system of adipocytes.
abstract: Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to PGs of J(2) series, including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ(12)-PGJ(2) in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ(12)-PGJ(2). Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ(12)-PGJ(2) and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA). The immobilized antigen using a conjugate of Δ(12)-PGJ(2) and γ-globulin was competitively allowed to react with the monoclonal antibody in the presence of free Δ(12)-PGJ(2). The assay provided a sensitive calibration curve for Δ(12)-PGJ(2), allowing us to determine a range from 0.16 pg to 0.99 ng with a value of 13 pg at 50% displacement in one assay. The monoclonal antibody showed almost no cross-reactivity with other related prostanoids since PGJ(2) and 15d-PGJ(2) were only recognized with much lower values of 0.5% and 0.2%, respectively. The accuracy for determining Δ(12)-PGJ(2) in the culture medium of adipocytes was confirmed by measurement after the culture medium was fortified with known amounts of authentic Δ(12)-PGJ(2) in a range from 10 to 200 pg/mL. The application of our ELISA revealed that the formation of Δ(12)-PGJ(2) became more pronounced after several hours of incubation of PGD(2) at 37°C in fresh maturation medium of cultured adipocytes. Furthermore, we provide evidence for the increased ability of cultured adipocytes to synthesize endogenous Δ(12)-PGJ(2) during the progression of adipogenesis. These results indicate the reliability and usefulness of our solid-phase ELISA for stable Δ(12)-PGJ(2), reflecting the biosynthesis of unstable PGD(2) in the culture system of adipocytes.
authors.affiliation: Department of Life Science and Biotechnology, Shimane University, Matsue, Shimane, 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: Ferdous
authors.initials: F
authors.lastname: Khan
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: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D050156:Adipogenesis / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002999:Clone Cells / Q000276:immunology; D017077:Culture Media, Conditioned / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006241:Haptens / Q000737:chemistry*; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D018796:Immunoconjugates / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D015230:Prostaglandin D2 / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D013388:Succinimides / Q000737:chemistry; D005719:gamma-Globulins / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D006241:Haptens; D018796:Immunoconjugates; D013388:Succinimides; C499810:delta(12)-prostaglandin J(2); D005719:gamma-Globulins; C001426:N-hydroxysuccinimide; D015230:Prostaglandin D2