RANKL, the receptor activator of nuclear factor kappa B ligand, is an essential factor for the formation of mature osteoclasts. Together with its receptor RANK and its antagonist Osteoprotegerin (OPG) RANKL is a key regulator in bone metabolism1. RANKL is a membrane-bound protein that can be segregated to a soluble form (sRANKL), whereas only the latter has been reported to be bioactive2. Due to its low circulating levels and the nature of the analyte binding to OPG, free sRANKL has been proven difficult to measure: the accuracy of sRANKL measurement is compromised by the very low or undetectable levels, as observed in some patient cohorts3.
Hence, our aim was to develop a highly sensitive and specific assay that enables the direct measurement of free, bioactive, soluble RANKL in serum and plasma samples. We have taken advantage of the high affinity and specificity protein-protein interaction between sRANKL and OPG and used immobilized, recombinant OPG to capture free sRANKL, which subsequently is detected with a biotin labeled anti-sRANKL antibody.
The data presented here, demonstrate that 98% of all samples from an unselected healthy population (n=210) had detectable free sRANKL values within the calibration range of the assay (02 pmol/l). The median of serum samples, prepared immediately after blood collection and stored at −25°C until measurement, was 0.14 pmol/l, with a lower limit of quantification (LLOQ) of 0.01 pmol/l. Assay characteristics, such as intra/inter-assay precision, dilution linearity and spike/recovery as well as sample stability have been analysed.
Our novel ELISA provides a reliable and accurate tool for the quantitative determination of free, soluble, bioactive RANKL in human samples.
References: 1. Walsh M.C. and Choi Y. Cytokine growth factor Rec. 2003; 14(34):25163.
2. Proell V. et al., Bone 2009; 45(4):67781.
3. Anastasilakis A. et al., J. Clin. Endocrinol. Metab. 2013; 98(8):320612.
17 May 2014 - 20 May 2014