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(5): 1107-18, 2012 Jul.
Article
em En
| MEDLINE
| ID: mdl-22391692
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Ensaio de Imunoadsorção Enzimática
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Prostaglandina D2
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Adipócitos
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Meios de Cultura
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Anticorpos Monoclonais
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article