Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay

Norihiro Kobayashi, Kazuki Shibusawa, Kazuyuki Kubota, Noriko Hasegawa, Pi Sun, Toshifumi Niwa, Junichi Goto

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Anti-idiotype antibodies recognizing the variable regions of a particular anti-hapten antibody are valuable tools, which can be used in sensitive hapten immunoassays based on a noncompetitive format. Here, we describe the production and characterization of monoclonal anti-idiotype antibodies against idiotopes on the variable regions of an antibody showing high affinity and specificity to 11-deoxycortisol (11-DC). 11-DC is the biosynthetic precursor of cortisol and a diagnostic index for the assessment of pituitary-adrenal function. BALB/c or A/J mice were repeatedly immunized with the anti-11-DC antibody conjugated with keyhole limpet hemocyanin and their spleen cells were then fused with P3/NS1/1-Ag4-1 myeloma cells. Seven kinds of anti-idiotype antibodies were generated, one of which was a β-type antibody recognizing the paratope and others which were α-type antibodies recognizing the framework region. A noncompetitive ELISA based on idiotype-anti-idiotype reactions was established using one of these α-type antibodies in combination with the β-type antibody and with the anti-11-DC antibody. This noncompetitive assay system provided improved sensitivity (detection limit: 1.0 pg=2.9 fmol), which is approximately 10 times higher than the corresponding competitive enzyme immunoassay, and offered a practical specificity for clinical use. Appropriate serum 11-DC levels were obtained for normal subjects [0.16±0.09 (S.D.) μg/l (n=6), ranging from 0.086 to 0.316 μg/l] using the present assay system.

Original languageEnglish
Pages (from-to)63-75
Number of pages13
JournalJournal of Immunological Methods
Volume274
Issue number1-2
DOIs
Publication statusPublished - 2003 Mar 1

Keywords

  • 11-Deoxycortisol
  • Anti-idiotype antibody
  • ELISA
  • Hapten
  • Monoclonal antibody
  • Noncompetitive immunoassay

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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