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.

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.