Subtraction method for determination of N-terminal connective tissue growth factor

Abstract

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.

The diagnostic value of plasma N-terminal connective tissue growth factor levels in children with heart failure

“…16 Anti-connective tissue growth factor antibodies were prepared, and each sandwich enzyme-linked immunosorbent assay was performed as described previously. 16,20 The full-length connective tissue growth factor levels were detected by a sandwich enzyme-linked immunosorbent assay using two monoclonal antibodies against modules 1 and 4. Total connective tissue growth factor levels were determined by a sandwich enzyme-linked immunosorbent assay using two monoclonal antibodies against modules 1 and 2.…”

Rapid hepatic clearance of full length CCN-2/CTGF: a putative role for LRP1-mediated endocytosis

“…Increased platelet activation in advanced liver disease (Panasiuk et al 2001) might also contribute to elevated CCN-2, since platelets contain large amounts of full length CCN-2 that is released upon activation (Cicha et al 2004; Miyazaki et al 2010). We here show that hepatic metabolism is the main elimination route of full length CCN-2.…”

Connective Tissue Growth Factor Neutralization Aggravates the Psoriasis Skin Lesion: The Analysis of Psoriasis Model Mice and Patients

“…Human CTGF enzyme-linked immunosorbent assay (ELISA) analysis was performed as described before 10 11 . Briefly, plasma samples were added to 96 well-plates (ThemoFisher Scientific, Waltham, MA, USA) that were coated with a monoclonal antibody against human CTGF modules.…”