Kinetic analysis of TNF-α oligomer-monomer transition by surface plasmon resonance and immunochemical methods

Claudio Poiesi, Alberto Albertini, Salvatore Ghielmi, Giovanni Cassani, Angelo Corti

Research output: Contribution to journalArticle

Abstract

In this work we have studied the kinetic parameters of oligomeric tumour necrosis factor α (TNF-α) dissociation using biospecific interaction analysis (BIA), based on surface plasmon resonance (SPR) of TNF-α immobilized on a sensor chip, and by an ELISA technique able to detect TNF-α oligomers in solution. Validation studies, carried out with sensor chips bearing TNF-α oligomers or bovine albumin monomers, verified that: (a) TNF-α can be immobilized in the oligomeric form onto sensor chips; (b) the covalent linkage between TNF-α and sensor chips is stable under the experimental conditions: (c) TNF-α monomers are present on the sensor chips after dissociation; (d) immobilization and dissociation rate constant (kdiss) measurements are reproducible. The kdiss of recombinant TNF-α, measured under non denaturing conditions at pH 7.4 by BIA and ELISA were in good agreement, being 0.92×10-5/s and 1.1×10-5/s respectively (corresponding to a half life of about 20.9 h and 17.5 h, respectively). The dissociation rate was found to be significantly affected by the presence of detergents and by the pH of the solution, suggesting that TNF-α, at low concentrations, exists in solution with different molecular forms depending on the time of storage and buffer composition. Real-time BIA is rapid and does not require particular antibodies or reagents. Thus, the stability of the quaternary structure of natural or recombinant TNF-α from human or animal species as well as that of other oligomeric cytokines can probably be studied using this method.

Original languageEnglish
Pages (from-to)539-545
Number of pages7
JournalCytokine
Volume5
Issue number6
DOIs
Publication statusPublished - 1993

Keywords

  • biospecific interaction assay
  • cytokine
  • quaternary structure
  • surface plasmon resonance
  • TNF-α

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Immunology
  • Hematology
  • Immunology and Allergy
  • Endocrinology

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