title: Noncompetitive immunoassay detection system for haptens on the basis of antimetatype antibodies.
abstract: BACKGROUND
  Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies.
  
  
  METHODS
  We generated antimetatype monoclonal antibodies against a hapten-antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH)D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated.
  
  
  RESULTS
  The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH)D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%-12.6% (intraassay) and 6.2%-21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH)D were 1.0%-2.3% (intraassay) and 1.9%-3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH)D showed correlations of r = 0.99 with both LC-MS/MS and a commercially available (125)I RIA.
  
  
  CONCLUSIONS
  Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins.
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Omi
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ando
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Sakyu
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shirakawa
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Oka
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Ise
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan. km-aoyagi@fujirebio.co.jp.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Aoyagi
authors.affiliation: Biotechnology Research Group, Fundamental Research Department, Fujirebio Inc., Tokyo, Japan.
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Goishi
mesh_terms: D000911:Antibodies, Monoclonal; D004958:Estradiol / Q000097:blood*; D006241:Haptens / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D057230:Limit of Detection; D012680:Sensitivity and Specificity; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D000911:Antibodies, Monoclonal; D006241:Haptens; D014807:Vitamin D; D004958:Estradiol; C104450:25-hydroxyvitamin D