Generation of Monoclonal Antibody for 15-deoxy-Δ12,14-Prostaglandin J 2 and Development of Enzyme-Linked Immunosorbent Assay for Its Quantification in Culture Medium of Adipocytes
2012
Syeda, Pinky Karim | Hossain, Mohammad Salim | Chowdhury, Abu Asad | Rahman, Mohammad Sharifur | Nishimura, Kohji | Jisaka, Mitsuo | Nagaya, Tsutomu | Shono, Fumiaki | Yokota, Kazushige
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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