Introduction Liver biopsy is the reference standard for assessing liver fibrosis and serum biomarkers can be used as a less invasive approach. Various antibody-based assays for serum biomarkers are currently in use to help diagnose fibrosis stage. However, these immunoassays have potential disadvantages such as the inability to detect degraded proteins and often these assays are time consuming. We have developed an antibody-free method to detect and quantify novel liver fibrosis biomarkers in human plasma/serum which overcomes these disadvantages.

Method Novel liver fibrosis biomarkers were identified by analysing proteins in plasma/serum samples from controls and patients with varying stages of liver fibrosis using a proteomics technique: two dimensional gel electrophoresis (2DE). For the most promising liver fibrosis biomarkers, an antibody-free assay (parallel reaction monitoring using mass spectrometry) was used which detects tryptic peptides of the biomarkers and their fragments. A calibration curve, established from known amounts of synthetic isotopically labelled peptides, was used to determine the concentrations of our biomarkers in serum/plasma samples from patients with varying stages of liver fibrosis.

Results Several candidate biomarkers for hepatic fibrosis were identified using 2DE. Our best biomarkers were promising when compared by Western blotting to the proteins used in existing serum biomarker tests for liver fibrosis. Using parallel reaction monitoring, antibody-free assays were developed for our most promising biomarkers which were able to successfully discriminate between neighbouring stages of liver fibrosis.

Conclusion We have developed a fast, sensitive and robust antibody-free method to detect and quantify novel liver fibrosis biomarkers in human plasma/serum. Unlike immunoassays which are restricted on the number of biomarkers due to antibody cost, our method can successfully detect and quantify more than 50 biomarkers in a single 30 min run. This novel assay may help clinicians to assess hepatic fibrosis and reduce the need for invasive liver biopsies.

Disclosure of interest None Declared.

References

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2. Bevin Gangadharan et al. Two dimensional gel electrophoresis using pH3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers. Nature Protocol Exchange, 2011:doi:10.1038/protex.2011.261

3. Bevin Gangadharan et al. New approaches for biomarker discovery: The search for liver fibrosis markers in hepatitis C patients. J Proteome Res 2011;10:2643

4. Bevin Gangadharan et al. Novel serum biomarker candidates for liver fibrosis in hepatitis C patients. Clin Chem 2007;53:1792