True total growth hormone (hGH) measurement: Elimination of hGH binding protein (hGHBP) interference by a hGH receptor blocking monoclonal antibody

Abstract

Discrepancies in results of growth hormone measurements (hGH) in one serum sample by different laboratories using different methods has been recognized as a major problem in diagnosis of hGH related diseases. Among the factors leading to this heterogeneity are the choice of calibrator, differences in the epitopes and spectrum of hGH molecular isoforms recognized by various antibodies used. In addition, the interference caused by human growth hormone binding protein (hGHBP) has been discussed. We developed a simple method to eliminate interference from hGHBP independently of the immunoassay used: Screening a large panel of monoclonal antibodies (mAb) raised against hGHBP revealed that one antibody (code ZMC1) binds with high affinity to an epitope inside the hormone-binding domain of hGHBP, and thereby competitively displaces hGH from it's binding to hGHBP. We evaluated whether this displacement could be used to achieve a better recognition of hGH by immunoassays known to be prone to hGHBP interference: Sheep serum was spiked with both, recombinant hGH (IRP 88/624) and increasing concentrations of hGHBP (0–6000 pM). Aliquots were kept at 4°C over night to allow complex formation and subsequently assayed. A concentration dependent negative bias of hGHBP on the hGH results was observed in a variety of GH immunoassays, ranging from -30 to –80% at the highest hGHBP concentration. This negative bias, however, was diminished in a concentration-dependent manner by an additional incubation step with ZMC1 for 12 hours before the assay. At a concentration of 0.1mg/ml, ZMC1 completely abolished the negative bias caused by hGHBP in all hGH assays tested. The displacement of hGH from hGHBP in serum samples by the addition of ZMC1 therefore provides a simple method to eliminate the negative bias of hGHBP in hGH immunoassays and may serve to reduce heterogeneity of hGH immunoassay results. Addition of ZMC1 effectively facilitates measurement of total hGH in serum samples regardless of hGHBP content.