Related Experiment Video
Updated: Jun 13, 2026
A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors
Published on: March 15, 2016
Subtraction method for determination of N-terminal connective tissue growth factor
Osamu Miyazaki1, Syunsuke Kurashita, Isamu Fukamachi
1Tsukuba Research Institute, Sekisui Medical Co Ltd, 3-3-1, Koyodai, Ryugasaki, Ibaraki 301-0852, Japan. miyazaki084@sekisui.jp
Insights
A new method accurately measures N-terminal connective tissue growth factor (CTGF) levels in plasma and serum. This technique distinguishes true plasma CTGF from platelet-derived CTGF, improving fibrosis marker accuracy.
Area of Science:
- Biochemistry
- Molecular Biology
- Biomarker Discovery
Background:
- Connective tissue growth factor (CTGF) is a potential fibrosis marker.
- Platelet activation can release full-length CTGF into plasma, confounding measurements.
- Plasma CTGF exists as an N-terminal fragment, distinct from platelet-derived full-length CTGF.
Purpose of the Study:
- To develop a method distinguishing N-terminal CTGF from full-length CTGF.
- To enable accurate determination of true plasma and serum CTGF levels.
Main Methods:
- Developed two sandwich ELISAs: M1/4 ELISA for full-length CTGF and M1/2 ELISA for total CTGF.
- Calculated N-terminal CTGF levels by subtracting full-length CTGF from total CTGF.
Main Results:
- Both M1/2 and M1/4 ELISAs demonstrated good analytical performance.
- N-terminal CTGF levels were consistent between plasma and serum using the subtraction method, regardless of full-length CTGF presence.
Conclusions:
- The subtraction method accurately quantifies N-terminal CTGF in plasma and serum.
- This method overcomes interference from platelet-derived full-length CTGF released during sample collection.
Background:
Connective tissue growth factor (CTGF) may be a potential marker of fibrosis. However, platelet-derived CTGF may be released into the plasma by platelet activation during or after blood collection, thereby interfering with accurate determination of the true plasma CTGF level. Plasma CTGF exists as the N-terminal CTGF fragment (N-fragment), composed of modules 1 and 2, whereas platelet CTGF exists as full-length CTGF (full-length), composed of modules 1-4. We perceived the need to develop a method for distinguishing between the N-fragment and full-length CTGF levels, so that the true plasma and serum CTGF (N-fragment) levels could be accurately determined.
Methods:
Full-length levels were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies recognizing modules 1 and 4, respectively (M1/4 ELISA). Total CTGF (full-length CTGF plus N-terminal CTGF) levels were determined by a sandwich ELISA using two monoclonal antibodies recognizing modules 1 and 2, respectively (M1/2 ELISA). N-terminal CTGF levels were determined by subtracting the full-length levels from the total CTGF levels.
Results:
Both the M1/2 and M1/4 ELISAs showed good analytical performance. When the CTGF levels of plasma and serum collected simultaneously from the same subject were compared, the N-fragment levels determined by the subtraction method were the same, in spite of the fact that full-length CTGF was present in the sample.
Conclusion:
N-fragment levels in plasma and serum can be accurately determined by this subtraction method, even if full-length CTGF in platelets is released during or after blood collection.
