Binding of Tamm‐Horsfall protein to complement 1q measured by ELISA and resonant mirror biosensor techniques under various ionic‐strength conditions

Abstract

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.

Intact protein core structure is essential for protein-binding, mononuclear cell proliferating, and neutrophil phagocytosis-enhancing activities of normal human urinary Tamm–Horsfall glycoprotein

“…However, Moonen et al [37] reported that THP could not bind with native cytokines including TNF-α. In addition, the binding of THP to complement 1 and complement 1q depends on the electrostatic evens as demonstrated by the influence of hydrogen concentration and ionic-strength on THP-C1q binding affinity [16,38]. In this study, we found that enzymatic digestion of THP with neuraminidase or β-galactosidase did not significantly affect the binding activity with different protein molecules, although a tendency of decrease was shown in Figs.…”

Qualification and application of an ELISA for the determination of Tamm Horsfall Protein (THP) in human urine and its use for screening of Kidney Stone Disease

“…At 1:10 urine dilution, the optimum WGA concentration for plate coating was found to be 10 μg/mL, where binding of urinary THP to WGA was a linear function from 1 – 10 μg/mL WGA, increasing in WGA concentration after 10 μg/mL does not improve the OD of the assay. Bovine serum albumin (BSA) was used to block the non-lectin binding sites on the plate since THP does not bind to BSA 19. The WGA microtiter plates were sealed and stored at 4 o C where they were stable for at least 6 weeks.…”

Interaction of C1q with IgG1, C-reactive Protein and Pentraxin 3: Mutational Studies Using Recombinant Globular Head Modules of Human C1q A, B, and C Chains

“…C1q recognizes several charged molecules, including IgG (22), IgM (23), spectrin (24), OmpK36 (25), Tamm-Horsfall protein (26), decorin (27), and fibromodulin (28). Many of these interactions have been shown to be sensitive to the ionic strength of the binding buffer.…”