The physiological and pathophysiological significance of hGH isoforms remains to be fully elucidated. In order to study the two most abundant hGH isoforms 20 and 22 kDa hGH, we have generated monoclonal antibodies (mAbs) against 20 and 22 kDa hGH. The mAbs against 20 and 22 kDa were characterized for their specificity and epitopes with different binding assays as well as by Western blot. The mAb 1G12 against 20 kDa with lower than 0.05% cross reactivity to 22 kDa hGH combined with the detection mAb 5C4 were chosen to construct the time resolved fluorescence sandwich assay for 20 kDa hGH. The assay has a working range of 0.02 to 20 ng/ml and the cross-reactivity to 22 kDa hGH is <0.2%. The intra- and inter-assay CVs are 3.54.6 and 10.716.6% respectively. The recovery is 102.9% and the linearity is 96.3%. The mAb 5E1 against 22 kDa hGH with lower than 0.01% cross reactivity to 20 kDa hGH combined with the detection mAb 8B11 were chosen for the 22 kDa sandwich assay construction. The assay has a working range of 0.02 to 50 ng/ml and the cross-reactivity to 20 kDa hGH is <0.1%. The intra- and inter-assay CVs are 4.66.7 and 4.29.4%, respectively. The recovery is 99.1% and the linearity is 91%. Spiking recombinant growth hormone binding protein (hGHBP) to the hGH samples reduced the concentration measured by both 20 and 22 kDa hGH assays as hGHBP concentration increased. However, the ratios between 20 and 22 kDa hGH remained stable (<15% for hGHBP from 0 to 2 nM). There is a good correlation between 20 and 22 kDa hGH concentrations in the serum samples from 105 healthy donors (20 kDa=0.041+0.189×22 kDa, R2=0.935), indicating the ratios between 20 and 22 kDa hGH are quite constant.
25 - 29 Apr 2009
European Society of Endocrinology