title: Quantitative analysis of serum procollagen type I C-terminal propeptide by immunoassay on microchip.
abstract: BACKGROUND
  Sandwich enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip.
  
  
  RESULTS
  The microchip was made of cyclic olefin copolymer with straight microchannels that were 300 µm wide and 100 µm deep. For the construction of a sandwich ELISA for procollagen type I C-peptide (PICP), a biomarker for bone formation, we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-PICP antibody on the surface of the microchannel. After the infusion of the mixture of 2.0 µl of peroxidase-labeled 2nd anti-PICP antibody and 0.4 µl of sample to the microchannel and a 30-min incubation, the substrate for peroxidase was infused into the microchannel; and the luminescence intensity of each spot of 1st antibody was measured by CCD camera. A linear relationship was observed between PICP concentration and luminescence intensity over the range of 0 to 600 ng/ml (r(2) = 0.991), and the detection limit was 4.7 ng/ml. Blood PICP concentrations of 6 subjects estimated from microchip were compared with results obtained by the conventional method. Good correlation was observed between methods according to simple linear regression analysis (R(2) = 0.9914). The within-day and between-days reproducibilities were 3.2-7.4 and 4.4-6.8%, respectively. This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method.
  
  
  CONCLUSIONS
  This assay enabled us to determine serum PICP with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis.
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology, Japan.
authors.forename: Shouki
authors.initials: S
authors.lastname: Yatsushiro
authors.forename: Rie
authors.initials: R
authors.lastname: Akamine
authors.forename: Shohei
authors.initials: S
authors.lastname: Yamamura
authors.forename: Mami
authors.initials: M
authors.lastname: Hino
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Kajimoto
authors.forename: Kaori
authors.initials: K
authors.lastname: Abe
authors.forename: Hiroko
authors.initials: H
authors.lastname: Abe
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Kido
authors.forename: Masato
authors.initials: M
authors.lastname: Tanaka
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Baba
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ooie
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kataoka
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D056656:Lab-On-A-Chip Devices; D010446:Peptide Fragments / Q000097:blood*; D011347:Procollagen / Q000097:blood*
chemical_list: D010446:Peptide Fragments; D011347:Procollagen; C042813:procollagen type I carboxy terminal peptide