Novel reference method developed for the quantification of proteins in a complex matrix
LGC scientists have developed a novel method for the quantification of human growth hormone in serum, furthering LGC’s status internationally as a leading National Measurement Institute (NMI) in protein quantification.
In order for clinicians to make informed diagnostic or therapeutic decisions, it is vital for measurement results to be accurate, specific and comparable between laboratories.
The major challenges associated with this for proteins are the definition of the measurand (the quantity being measured), the value-assignment of the reference standard used, and assessing the commutability of the reference standard. Once the measurand is defined, the next step in improving standardisation is the development of quantitative methods which give results that are traceable to recognised references. These methods can then be used to characterise reference standards which, ideally, match patient samples as closely as possible to reduce measurement bias and improve commutability of the materials.
Scientists from the Organic Analysis team at LGC have published a paper describing the development of a novel SI-traceable reference method for the quantification of human growth hormone (rhGH) in serum. The reference method uses multi-step sample clean-up at the protein level, combined with tryptic digestion, and isotope dilution mass spectrometry using a labelled protein as the internal standard. The critical considerations for using isotopically labelled growth hormone as the internal standard are described in the paper.
To assess the performance of the method, a bulk serum sample containing rhGH at the clinically relevant level of 10 ng/g was analysed. The results obtained were traceable to the SI with a total measurement uncertainty of <20 %. To demonstrate the validity of the method, an inter-laboratory study was performed with the German NMI (Physikalisch Technische Bundesanstalt, PTB), which used an alternative SI-traceable method for the analysis of the bulk serum sample. The results of the study demonstrated that the two methods were comparable with results in good agreement.
Dr Caroline Pritchard, researcher in the Organic Analysis team at LGC and lead author of the paper, said, “Approximately 10-15 % of in vitro diagnostics currently used in clinical laboratories are for protein biomarkers. Standardisation of these measurements requires the use of reference materials that mimic real samples as closely as possible to ensure consistency of clinical diagnostic and therapeutic results across both time and space. The development of traceable methods is critical to enabling the production of such materials.
“Improved standardisation of measurements will ultimately help to prevent potential misclassification of disease risk or administration of inappropriate intervention strategies, potentially saving the healthcare system millions of pounds in ineffective treatments.”
The work described demonstrates LGC's first traceable method for the quantification of proteins in a complex matrix, furthering LGC’s status as a leading NMI internationally in protein quantification. Ongoing work in protein quantification includes the development of a traceable method for quantification of brain-natriuretic peptide (BNP) in plasma, and supporting CCQM studies in this area in collaboration with the German NMI. Having had this paper accepted ensures that we continue to raise awareness of the considerable technical challenges that are faced in achieving accurate low level SI-traceable quantification of clinically relevant proteins.
The paper, "The quantification of human growth hormone in serum with a labelled protein as an internal standard: essential considerations", written in collaboration with PTB, is published in Analytical Chemistry. To read an abstract and download the paper, visit the ACS Publications website.