Julia Dittrich (Presenter)
University Hospital Leipzig
Authorship: Julia Dittrich, Jürgen Kratzsch, Uta Ceglarek
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany
Human growth hormone (hGH), which is expressed in various isoforms, is essential for growth stimulation. However, commercial immunoassays are insufficiently selective and sensitive to interferences. We developed a Mass Spectrometry ImmunoAssay (MSIA) for the simultaneous quantitation of total hGH and its isoforms from 200 µL serum applying a polyclonal antibody coupled to magnetic beads. An elaborated sample preparation including protein internal standards was combined with a 5 min LC MS/MS analysis. The assay’s quantitation limits facilitated a reliable analysis in the lower diagnostic decision range of <1 ng/mL.
Human growth hormone (hGH) is a key player in the age-adjusted development of infants, children and adolescents. By virtue of alternative splicing, hGH is expressed in various isoforms whose detailed functions are still unknown since commercially available immunoassays cannot perform an exact quantitation of these hGH isoforms. Additionally, such immunoassays are known to suffer from interferences with growth hormone-binding protein and may have a lack of specificity due to antibody cross-reactivity.
Consequently, we developed a Mass Spectrometry ImmunoAssay (MSIA) for a reliable concurrent quantitation of total hGH and its isoforms.
In a targeted proteomics assay U-15N-labeled analogues of 20 kDa hGH and 22 kDa hGH were used for internal standardization. hGH isoforms and internal standards were enriched from 200 µL human serum applying polyclonal rabbit anti-hGH antibodies coupled to Protein A-modified magnetic beads. Omitting an elution of captured hGH isoforms, basic proteomics sample preparation steps comprising denaturation, reduction, alkylation and in solution tryptic digestion were performed. Subsequently, samples were purified in an on-line SPE prior to reversed-phase chromatographic separation performed at 1.5 mL/min resulting in a total run time of 5 min. A hybrid triple quadrupole mass spectrometer was used for MS detection.
The single steps of the complex sample preparation procedure including bead incubation, alkylation and tryptic digestion were optimized for the application in a 96-well format facilitating a highly sensitive high-throughput application. The methodology proved to be linear as low as a total hGH concentration of 0.5 ng/mL. In a first comparison with a commercial immunoassay (hGH from IDS-iSYS) it could be shown that in average by 30% higher concentrations were determined by our LC-MS/MS approach. For this comparison values of hGH ranged from 0.9 ng/mL to 70 ng/mL and a Pearson correlation coefficient of 0.995 was obtained. In a ring trial for MS-based hGH quantitation the target value was missed by only 22%.
Conclusions & Discussion
The presented MSIA hGH assay enables the sensitive quantitation of hGH by the combination of polyclonal anti-hGH antibodies and highly specific MS/MS detection. Furthermore, it allows the detailed investigation of hGH isoform functions in growth disorders.
References & Acknowledgements:
This research project was supported by the Foundation for Pathobiochemistry and Molecular Diagnostics of the German Society of Clinical Chemistry and Laboratory Medicine (DGKL).
|Grants||yes||Foundation for Pathobiochemistry and Molecular Diagnostics of the DGKL|
IP Royalty: no
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||