title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Tsukuba Research Institute, Sekisui Medical Co Ltd, 3-3-1, Koyodai, Ryugasaki, Ibaraki 301-0852, Japan. miyazaki084@sekisui.jp
authors.forename: Osamu
authors.initials: O
authors.lastname: Miyazaki
authors.forename: Syunsuke
authors.initials: S
authors.lastname: Kurashita
authors.forename: Isamu
authors.initials: I
authors.lastname: Fukamachi
authors.forename: Koki
authors.initials: K
authors.lastname: Endo
authors.forename: Poh-Sing
authors.initials: PS
authors.lastname: Ng
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Takehara
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal; D055513:Connective Tissue Growth Factor / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D055815:Young Adult
chemical_list: D000911:Antibodies, Monoclonal; D055513:Connective Tissue Growth Factor