Applied Biochemistry and Biotechnology

, Volume 48, Issue 2, pp 117–123 | Cite as

Immunoassay for native enzyme quantification in biological samples

  • Christelle de Bellefontaine
  • Flore Josse
  • Martine Domurado
  • Dominique Domurado


In order to detect low levels of enzyme activity, specifically glucose oxidase, in biological samples, an immunoenzymatic assay was developed since currently available methods could not be used because of either their lack of sensitivity or the conditions prevailing in our samples: turbidity of the medium, presence of redox systems other than glucose oxidase, and high concentration of proteins.

The principle of the method is to coat a polystyrene surface with a fragment Fc-specific anti-IgG, then with an antibody directed against the looked-for enzyme, which is simultaneously the antigen and the enzyme activity required for immunoenzymatic detection. We applied this concept to biological samples after glucose oxidase administration to mice. This method achieves specificity and sensitivity (20 ng/mL or 1 ng) with samples of biological origin. No marker is needed since the antigen itself possesses an enzyme activity. This method, which requires a small sample volume (50 ΜL, 20 ΜL, if necessary), can be extended easily to the many enzymes currently used as markers. It could also be applied to the native enzymes of medical interest for which antibodies and a colorimetric reaction are available.

Index Entries

Immunoglobulin rabbit anti-mouse fragment Fc-specific phosphate buffered saline PBS-Tween o-phenylenediamine ABTS 




anti-Fc IgG

rabbit antimouse fragment Fc-specific IgG


phosphate buffered saline


Tween 20 0.1% dissolved in PBS




2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt


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Copyright information

© Humana Press Inc 1994

Authors and Affiliations

  • Christelle de Bellefontaine
    • 1
  • Flore Josse
    • 1
  • Martine Domurado
    • 2
  • Dominique Domurado
    • 2
  1. 1.Groupe de Pharmacocinétique des Conjugés Macromoléculaires, Laboratoire de Technologie EnzymatiqueUniversite de Technologie de CompiègneCompiègne cedexFrance
  2. 2.Groupe de Pharmacocinétique des Conjuges Macromoléculaires (INSERM)Centre de Recherche sur les Biopolymères Artificiels, U.R.A. no. 1465 du CNRS, Faculté de PharmacieMontpellier cedex 1France

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