Description

Nori Human Pro-C3 ELISA Kit Summary

Alternative names for Pro-C3: N-terminal pro-collagen III peptide

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Pro-C3 (N-terminal pro-collagen III peptide) is a marker of fibrogenesis and formation of type III collagen neo-epitopes. During liver fibrosis, extracellular matrix proteins are degraded by matrix metalloproteinases into soluble fragments. PRO-C3 correlates to fibrosis stage and degree of portal hypertension in HIV/HCV co-infected patients [1]. A study showed that PRO-C3 correlates to histologic fibrosis progression in patients with chronic hepatitis C.[2]. PRO-C3 levels are associated with grade of fibrosis in Fibroscan in HIV mono-infected individuals.[3] Liver fibrosis and liver injury are reflected by PRO-C3 levels in HIV infected patients and can rule-out hepatic fibrosis in these patients. Moreover, PRO-C3 may be used to monitor patients at risk of fibrosis in centers where Fibroscan is not available. Pro-C3 is also associated with inflammation.[2] Pro-C3 differentiated mild/moderate from severe disease and, importantly, can identify CHC patients with fibrosis progression more accurately than the broadly used FibroTest.[4] Pro-C3 represents a rational blood parameter to be included in future studies in combination with other non-invasive modalities for monitoring disease progression for example in NASH, and eventually for assessing efficacy of potential antifibrotic therapies. PRO-C3 may be more reflective of the synthesis of type III collagen and fibrogenesis.[5] Patients with chronic hepatitis C who had higher baseline PRO-C3 levels (≥20.2 ng/ml) showed progression (worsening) of fibrosis while those with lower PRO-C3 did not progress.[5, 2] Further, PRO-C3, PRO-C5 and C4M have also been correlated with hepatic venous pressure gradient which is an invasive diagnostic and prognostic marker in cirrhosis for portal hypertension.[6,7] Higher PRO-C3 levels were associated with higher grade of lobular inflammation and ballooning, suggesting that PRO-C3 levels may be reflective of active disease. Furthermore, decrease of PRO-C3 over time was associated with improvement (or regression) in fibrosis.[8] unlike elastography which measures liver stiffness, PRO-C3 measures active collagen formation reflective of fibrogenesis, but not exactly the fibrosis area in the liver. PRO-C3 may be more valuable for identifying patients with active fibrogenesis than diagnosing static disease stages. Some patients with advanced fibrosis may have less active disease with old scars, and thus may have low levels of PRO-C3. This may explain the normal levels of PRO-C3 in some F3-4 patients.

References

1. Jansen C, et al. (2014) PLoS One 2014, 9:e108544. https://doi.org/10.1371/journal.pone.0108544.
2. Nielsen MJ, et al. (2015) Liver Int, 35:429–437.
3. Dold L et al. (2019) PLOS ONE https://doi.org/10.1371/journal.pone.0219526
4. Boyle M et al. (2019) JHEP Reports 1:188-198.
5. Karsdal, M. A. et al. (2016) Am J Physiol Gastrointest Liver Physiol 311, G1009–G1017.
6. Leeming, D. J. et al. (2013). Aliment Pharmacol Ther 38, 1086–1096.
7. Leeming, D. J. et al. (2015) Scand J Gastroenterol 50, 584–592.
8. Luo Y et al. (2018) Nature 8:12414. DOI:10.1038/s41598-018-30457-y.

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