Kinetic analysis of TNF-alpha oligomer-monomer transition by surface plasmon resonance and immunochemical methods.
In this work we have studied the kinetic parameters of oligomeric tumour necrosis factor alpha (TNF-alpha) dissociation using biospecific interaction analysis (BIA), based on surface plasmon resonance (SPR) of TNF-alpha immobilized on a sensor chip, and by an ELISA technique able to detect TNF-alpha oligomers in solution. Validation studies, carried out with sensor chips bearing TNF-alpha oligomers or bovine albumin monomers, verified that: (a) TNF-alpha can be immobilized in the oligomeric form onto sensor chips; (b) the covalent linkage between TNF-alpha and sensor chips is stable under the experimental conditions; (c) TNF-alpha monomers are present on the sensor chips after dissociation; (d) immobilization and dissociation rate constant (kdiss) measurements are reproducible. The kdiss of recombinant TNF-alpha, measured under non denaturing conditions at pH 7.4 by BIA and ELISA were in good agreement, being 0.92 x 10(-5)/s and 1.1 x 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-alpha, 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-alpha from human or animal species as well as that of other oligomeric cytokines can probably be studied using this method.
Poiesi
C
C
Institute of Chemistry, School of Medicine, University of Brescia, Italy.
Albertini
A
A
Ghielmi
S
S
Cassani
G
G
Corti
A
A
D000818:Animals; D015374:Biosensing Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012710:Serum Albumin, Bovine; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry*
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D012710:Serum Albumin, Bovine