Construction of a specific and sensitive sandwich enzyme immunoassay for 20 kDa human growth hormone

Hashimoto, Yoshihide; Ikeda, Ichiro; Ikeda, Miwa; Takahashi, Yuka; Hosaka, Masaharu; Uchida, Hiroshi; Kono, Naoko; Fukui, Hirokazu; Makino, T.; Honjo, Masaru

doi:10.1016/s0022-1759(98)00157-4

Cited by 37 publications

(21 citation statements)

References 30 publications

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“…Recombinant 22-kDa GH (16) was administered subcutaneously at a dose of 0.1 mg/kg. Blood samples were obtained immediately before and at 1,2,3,4,5,6,7,8,9,10,11,12, and 24 h after administration for GH measurement. IGF-I concentrations were measured at 0, 4,8,12, and 24 h.…”

Section: Methodsmentioning

confidence: 99%

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Physiological and pharmacological regulation of 20-kDa growth hormone

Leung

Howe

Gui

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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The 20-kDa growth hormone (GH) is generated from alternative splicing of the primary transcript of full-length 22-kDa GH. We have studied the regulation of 20-kDa GH over a range of pathophysiological conditions and in response to pharmacological stimulation using isoformspecific enzyme-linked immunosorbent assays (ELISAs). Mean 24-h levels of 20-and 22-kDa GH were higher in acromegaly and lower in GH deficiency than in normal subjects, with the 20-to-22-kDa ratio not different between the three groups. In normal subjects, 20-kDa GH was secreted in a pulsatile manner throughout the day, with peaks coinciding with those of 22-kDa GH. However, the half-life of 20-kDa GH (18.7 Ϯ 0.8 min) was significantly longer than that of 22-kDa GH (14.7 Ϯ 0.8 min; P Ͻ 0.02). Insulin-induced hypoglycemia, androgen, and oral estrogen caused a parallel and proportionate increase in both isoforms. Octreotide suppressed 20-kDa less rapidly than 22-kDa GH in blood. Administration of recombinant 22-kDa GH in normal subjects rapidly reduced the 20-kDa GH levels. In conclusion, 20-kDa GH is cosecreted with and circulates at a constant proportion of 22-kDa GH. The 20-kDa GH level is reduced by administration of exogenous 22-kDa GH, suggesting rapid negative feedback regulation on pituitary release.androgen; estrogen; deconvolution analysis; insulin-induced hypoglycemia GROWTH HORMONE (GH) exists as a mixture of multiple molecular forms arising from both posttranscriptional and posttranslational modifications (1, 23). The major isoform of molecular size 22 kDa comprises 191 amino acid residues, whereas the second-most abundant isoform is 20 kDa in size and accounts for ϳ10% of total GH in the pituitary (19). This isoform is generated by alternative splicing within exon 3 of the GH primary transcript, resulting in the deletion of residues 32-46 of 22-kDa GH (1, 6). The 20-kDa GH has growthpromoting and lipolytic activities similar to those of 22-kDa GH (18, 25) but has reduced antinatriuretic activity (21). Although these observations suggest that it has a slightly different metabolic profile, the physiological role of 20-kDa GH is not known.Recently, studies using a highly specific enzymelinked immunosorbent assay (ELISA) for 20-kDa GH have revealed that it circulates at a constant proportion to 22-kDa GH under a variety of physiological and pathophysiological conditions (12). However, it remains to be determined whether 20-kDa GH is regulated differently from 22-kDa GH. To gain more insight in the regulation of 20-kDa GH in blood, we have compared the serum levels of 20-and 22-kDa GH 1) in health and disease, 2) in response to acute stimulation and suppression, 3) in response to gonadal steroids, and 4) in response to exogenous recombinant 22-kDa GH. SUBJECTS AND METHODSSubjects and study design. Five separate studies were performed to investigate the physiological and pharmacological regulation of 20-kDa GH secretion (14-17, 20, 33). The clinical data of some of these studies have been previously published in part. All study protocols w...

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Section: Methodsmentioning

confidence: 99%

“…Recently, studies using a highly specific enzymelinked immunosorbent assay (ELISA) for 20-kDa GH have revealed that it circulates at a constant proportion to 22-kDa GH under a variety of physiological and pathophysiological conditions (12). However, it remains to be determined whether 20-kDa GH is regulated differently from 22-kDa GH.…”

mentioning

confidence: 99%

Physiological and pharmacological regulation of 20-kDa growth hormone

Leung

Howe

Gui

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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“…An alternate isoform-based method to detect exogenous GH measures the specific 20K GH isoform together with measurement of 22K GH, using highly sensitive and selective MAbs for each of the two GH isoforms in an enzyme-linked immunosorbent assay [34,35]. Cosecretion of 20K GH with 22K GH results in peaks of secretion that coincide during the day, with the circulating concentration of 20K in a constant proportion to 22K GH [16,35].…”

Section: The Gh Isoform Approachmentioning

confidence: 99%

Growth Hormone in Sports: Detecting the Doped or Duped

Nelson¹

2011

Horm Res Paediatr

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Background: Doping with growth hormone (GH) is banned; however, there is anecdotal evidence that it is widely abused. GH is reportedly often used in combination with anabolic steroids at high doses for several months. Development of a robust test for detecting GH has been challenging since recombinant human 22-kDa GH used in doping is indistinguishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentrations. Discussion: One approach to GH testing is based on measurement of different circulating GH isoforms using immunoassays that differentiate between 22-kDa and other GH isoforms. Administration of 22-kDa GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method is, however, limited by its short time window of detection. A second approach that extends the time window of detection is based on detection of increased levels of circulating GH-responsive proteins, such as the insulin-like growth factor (IGF) axis and collagen peptides. As age and gender are the major determinants of variability for IGF-I and the collagen markers, a test based on these markers must take these factors into account. Extensive data now validate the GH-responsive marker approach, and implementation is largely dependent on establishing an assured supply of standardized assays. Conclusions: Robust tests are available to detect GH and enforce the ban on its abuse in sports. Novel approaches that include gene expression and proteomic profiling must continue to be pursued to expand the repertoire of testing approaches available and to maintain deterrence of GH doping.

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“…Clearly, this 20K hGH assay is not suitable for the determination of physiological concentrations of 20K hGH circulating in human serum, which is only about 10% of total hGH. Hashimoto et al [16] have generated the mAb D5, a specific mAb to the recombinant 20K hGH isoform. Using mAb D5 combined with HRP-labeled affinity purified polyclonal antibody, they constructed a sensitive sandwich enzyme immunoassay for the 20K isoform, with a working range of 50-5000 pg/mL.…”

Section: Introductionmentioning

confidence: 99%