A novel bioassay for human somatogenic activity in serum samples supports the clinical reliability of immunoassays.
OBJECTIVE
Because there is discordance between different immunoassay values for serum hGH, and because clinical state may not correlate with immunoreactive hGH, we have developed an assay to accurately measure serum hGH somatogenic bioactivity. The results of this assay were compared with the Elegance two-site ELISA assay across 135 patient samples in a variety of clinical states.


METHODS
The somatogenic assay was based on stable expression of hGH receptor in the murine BaF line, allowing these cells to proliferate in response to hGH. To eliminate interference by other growth factors in serum, we created a specific antagonist of the hGH receptor (similar to Trovert or Pegvisomant) which allowed us to obtain a true measure of hGH somatogenic activity by subtraction of the activity in the presence of the antagonist. The assay was carried out in microtiter plates over 24 h, with oxidation of a chromogenic tetrazolium salt (MTT) as the endpoint.


METHODS
These encompassed a number of different clinical conditions related to short stature, including idiopathic short stature, neurosecretory dysfunction and renal failure, as well as obese patients on dietary restriction and normal volunteers.


METHODS
In addition to the colourimetric (MTT) response to hGH, we measured free hGH by stripping out GHBP-bound hGH using beads coupled to a monoclonal antibody to the GHBP (GH binding protein). All samples were measured in both bioassay and ELISA assay.


RESULTS
This bioassay was sensitive (5 mU/l or 2 microg/l) and precise, and not subject to interference by the GHBP. There was a good correlation (r = 0.95) between bioactivity and immunoactivity across clinical states. There was, however, an increased bioactivity during secretory peaks (over 25 mU/l), which has been reported previously for the Nb2 bioassay. Free hGH did not correlate with clinical state.


CONCLUSIONS
Because the results of the Elegance ELISA and the bioassay correlate well, even though there is greater bioactivity at higher hormone concentrations, it is evident that an appropriate immunoassay is able to act as a reliable indicator for clinical assessment. In those rare cases where bio-inactive GH exists, our bioassay should provide an appropriate means to demonstrate this.
Rowland
Jennifer E
JE
Department of Physiology & Pharmacology and Centre for Molecular & Cellular Biology, University of Queensland, St Lucia, Australia 4072.
Marshall
Nicholas J
NJ
Leung
Kin-chuen
KC
Ho
Ken K Y
KK
Cotterill
Andrew M
AM
Rowlinson
Scott W
SW
Waters
Michael J
MJ
D000328:Adult; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006130:Growth Disorders / Q000097:blood*; D019382:Human Growth Hormone / Q000037:antagonists & inhibitors / Q000097:blood*; D006801:Humans; D051379:Mice; D011986:Receptors, Somatotropin / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results
D011986:Receptors, Somatotropin; D019382:Human Growth Hormone