The neo-epitope specific PRO-C3 ELISA measures true formation of type III collagen associated with liver and muscle parameters

Abstract

Aim: The present study describes the assessment of true formation of type III collagen in different pathologies using a neo-epitope specific competitive Enzyme-linked immunosorbent assay (ELISA) towards the N-terminal propeptide of type III collagen (PRO-C3).

Methods: The monoclonal antibody was raised against the N-protease mediated cleavage site of the N-terminal propeptide of type III collagen and a competitive ELISA was developed using the selected antibody. The assay was evaluated in relation to neo-epitope specificity, technical performance, and as a marker for liver fibrosis and muscle mass using the rat carbon tetrachloride (CCl4) model and a study of immobilization induced muscle loss in humans, respectively.

Results: The ELISA was neo-epitope specific, technically stable and can be assessed in serum and plasma samples. In the CCl4 liver fibrosis model it was observed that serum PRO-C3 were significantly elevated in rats with liver fibrosis as seen by histology (56% elevated in the highest quartile of total hepatic collagen compared to control rats, p<0.001) and correlated significantly to total hepatic collagen in the diseased rats (r=0.46, p<0.01) and not in control rats, suggesting the pathological origin of the epitope. Human plasma PRO-C3 correlated significantly to muscle mass at baseline (R(2)=0.44, p=0.036).

Conclusion: The developed neo-epitope specific serum ELISA for type III procollagen (PRO-C3) reflects true formation as it is specific for the propeptide cleaved off the intact collagen molecule. In a clinical and in a rodent study we showed that this marker was highly related to liver fibrosis and muscle mass.

Keywords: Biochemical markers; formation; liver fibrosis; muscle mass; neo-epitope; type III collagen.

Figure 1

Alignment of the targeted PRO-C3 α1 chain sequence in human, and rat species (red box). Position of the corresponding human PRO-C3 (▪) and rat PRO-C3 (~) sequences within the alpha 1 chain of the N-terminal pro-peptide of type III collagen. The alignment was performed using the NLP CLUSTALW software.

Figure 2

Western Blot showing the specific bands of N-terminal propeptide of type III collagen in Amniotic fluid from a) rat and b) human recognized by the PRO-C3 monoclonal antibody NB61N62 (lane 1 and 3) and NB61N62 + specific peptide (lane 2+4). Two bands around 52-60 kDA was observed for the rat, whereas one band was observed for human. Addition of specific peptide resulted in weakness of band intensity for both rat and human.

Figure 3

PRO-C3 ELISA runs showing typical calibration curves and native reactivity against human, rodent, and mouse material. A. Calibration curve and inhibition of the competitive ELISA using healthy human serum, plasma, and amniotic fluid (AF). The calibrator curve was diluted in 2-fold from 76.31 ng/mL, whereas native material was run diluted 1:2 to 1:16 as indicated (--), B. Calibration curve and inhibition of the competitive ELISA using healthy rat serum, plasma, and AF. The calibrator curve was diluted in 2-fold from 200 ng/mL, whereas native material was run undiluted to 1:8 as indicated (--), C. Calibration curve and inhibition of the competitive ELISA using healthy mouse serum and plasma. The calibrator curve was diluted in 2-fold from 200 ng/mL, whereas native material was run undiluted to 1:4 as indicated (--), D. Neo-epitope specificity of the PRO-C3 antibody using elongated peptide, i.e. peptide sequence of calibration peptide with one additional amino acid in the C-terminal end. The calibration curve, elongated peptide, and non-sense peptide were diluted in 2-fold from 76.31 ng/mL. The signal is seen as the optical density at 450 nm, subtracting the background at 650 nm, as a function of peptide concentration.

Figure 4

Comparison between PRO-C3 and UniQ PIIINP serum levels in 20 randomly selected serum samples from healthy human individuals; ns=non-significant difference.

Figure 5

PRO-C3 levels measured in the CCl₄ inhalation rat model: A. Serum levels of PRO-C3 was assessed in vehicle rats at termination (controls) as well as in CCl₄ treated rats at termination (CCl₄) at week 8, 12, 16, and 20. Results shown are mean ± standard error of the mean (SEM); B. Serum levels of PRO-C3 in vehicle rats and CCl₄ rats stratified in quartiles according to total collagen in the liver; C, D) Correlation between PRO-C3 and Sirius red in CCl₄ rats and in vehicle rats. Asterisks indicate statistical significance as indicated by bars. (*=p<0.05; **=p<0.01; ***=p<0.001,ns= non-significant difference).

Figure 6

Serum PRO-C3 titers at baseline correlate significantly with quad muscle mass measured by thigh MRI using Pearson correlation. Data have not been transformed.