IMMUNOASSAY FOR MEASURING THE HEPARIN-BINDING GROWTH FACTORS HARP AND MK IN BIOLOGICAL FLUIDS

ABSTRACT

Heparin-affin regulatory peptide (HARP) and Midkine (MK) belong to a family of growth/differentiation factors that have a high affinity for heparin. The involvement of these molecules in various proliferative diseases prompted us to develop an assay for measuring the concentrations of these factors in biological fluids and culture media. This report describes an immunoassay that uses only commercially available materials, based on the high affinity of certain molecules for heparin. It consists of adsorbing heparin-BSA covalent complexes to microtiter plate wells and to quantify the heparin bound HARP or MK by using appropriate antibody. The method is specific and measures concentrations ranging from 40–1200 pg/mL HARP and from 25–1200 pg/mL MK and various parameters are investigated. The within-assay coefficient of variation was less than 5% for both assays. The method was checked by measuring the concentrations of these growth factors in the sera of healthy humans and in patients with cancer. As previously reported, we confirmed that the serum concentrations of MK are higher in patients with tumours (n = 139) than in controls (n = 19). The synthesis of HARP and MK by various cells in culture was also analysed.

ACKNOWLEDGMENTS

This work was supported by grants from Association pour la Recherche sur le Cancer (J. Courty, N°5257) and Ministère de l’Education Nationale et de la Recherche (DRED/CNRS). M.H. is a recipient of a grant from Association pour la Polyarthrite Rhumatoide.