Skip to Main content Skip to Navigation
Journal articles

Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

Abstract : Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision < or =20% CV and accuracy within +/-20% of the nominal values. Intra- and inter-assay coefficients of variation were within 9% and accuracies within +/-10% of the nominal values. The validated method was applied to the study of the cellular uptake of native and cationized anti-tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.
Document type :
Journal articles
Complete list of metadatas

Cited literature [27 references]  Display  Hide  Download

https://www.hal.inserm.fr/inserm-00259343
Contributor : Jean-Michel Scherrmann <>
Submitted on : Wednesday, February 27, 2008 - 4:14:58 PM
Last modification on : Saturday, September 19, 2020 - 4:26:59 AM
Long-term archiving on: : Friday, November 25, 2016 - 10:28:15 PM

File

 Restricted access
To satisfy the distribution rights of the publisher, the document is embargoed until : jamais

Please log in to resquest access to the document

Identifiers

Collections

Citation

Frédéric Welfringer, Philippe d'Athis, Jean-Michel Scherrmann, Françoise Hervé. Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.. Journal of Immunological Methods, Elsevier, 2005, 307 (1-2), pp.82-95. ⟨10.1016/j.jim.2005.09.015⟩. ⟨inserm-00259343⟩

Share

Metrics

Record views

901