Binding of Tamm-Horsfall protein to complement 1q measured by ELISA and resonant mirror biosensor techniques under various ionic-strength conditions.
The purpose of the present study was to quantify the binding affinity between Tamm-Horsfall protein (THP) and complement 1q (C1q) using ELISA and a resonant mirror biosensor. In ELISA, immobilized THP was incubated with soluble C1q under both low and physiological ionic-strength conditions. Tamm-Horsfall protein bound C1q with an equilibrium dissociation constant (KD) of 1.9 +/- 0.6 nmol/L in low ionic-strength Tris buffers (20 mmol/L NaCl, pH 7.5) and with a lower affinity (KD of 13.4 +/- 4.7 nmol/L) in physiological-strength Tris buffers (154 mmol/L NaCl, pH 7.5). A resonant mirror biosensor, which monitors binding events in real-time, was used to quantify the KD of this reaction, as well as to estimate the kinetic parameters. In these studies, THP and C1q bound with an association rate constant, kass, of 1.25 x 105 L/mol per s and a dissociation rate constant, kdiss, of 0.002-0.005/s. The calculated KD for the THP/C1q binding in low ionic-strength buffers was higher (averages of 10-15 nmol/L) than that obtained by the ELISA, while physiological ionic-strength buffers still reduced the affinity of this binding by an order of magnitude. In conclusion, THP consistently bound C1q with high affinity using several techniques. At least a portion of this interaction involved electrostatic events, as demonstrated by the influence of ionic strength on the binding affinity.
Rhodes
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Department of Anatomy, Kirksville College of Osteopathic Medicine, Kirksville, Missouri, USA. drhodes@kcom.edu
D000276:Adjuvants, Immunologic / Q000378:metabolism* / Q000652:urine; D000328:Adult; D015374:Biosensing Techniques / Q000379:methods; D015922:Complement C1q / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D009088:Mucoproteins / Q000378:metabolism* / Q000652:urine; D009994:Osmolar Concentration; D011485:Protein Binding; D015203:Reproducibility of Results; D055672:Static Electricity; D058949:Uromodulin
D000276:Adjuvants, Immunologic; D009088:Mucoproteins; C548612:UMOD protein, human; D058949:Uromodulin; D015922:Complement C1q