The kinetics of antibody binding to membrane antigens in solution and at the cell surface

Abstract

The reaction kinetics of 251I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/13 and W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+ ), dissociation rate constants (k-) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-i antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-, for the univalent interaction, in which case all binding was bivalent.(ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 105-1.1 x 106M-1s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently.However, the k-, values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.

Interferon-γ Augments Acute Macrophage-Mediated Renal Injury Via a Glucocorticoid-Sensitive Mechanism

“…The following monoclonal antibodies (mAb) were used in this study: ED1, anti-rat CD68 labels monocytes and macrophages (16); PC-10, anti-proliferating cell nuclear antigen (PCNA) labels cells in the G1, S, and G2 phase of the cell cycle (Dako Ltd, Glostrup, Denmark); M744 mAb, anti-BrdU (Dako Ltd); OX-7, anti-Thy-1 antigen (CD90), which labels rat thymocytes and glomerular mesangial cells (17,18); OX-42, anti-rat CD11b/c (iC3b) (19); 1A29, anti-rat ICAM-1 (CD54) (Serotec, Oxford, UK) (20); WT.3, anti-rat CD18 (LFA-1 ␤-chain) (Serotec); and 73.5, mouse monoclonal antibody against human leukocytes that does not react with rat tissues, was used as negative control. Peroxidase-and alkaline phosphatase-conjugated goat anti-mouse IgG, peroxidase-conjugated mouse anti-peroxidase complexes (PAP), and alkaline phosphatase-conjugated mouse antialkaline phosphatase complexes (APAAP) were purchased from Dako Ltd. FITC-conjugated sheep anti-mouse IgG F(ab') 2 fragment (AM-RAD Biotec., Boronia, Victoria, Australia) was used in flow cytometry.…”

Affinity and Kinetics of the Interaction between Soluble Trimeric OX40 Ligand, a Member of the Tumor Necrosis Factor Superfamily, and Its Receptor OX40 on Activated T Cells

“…Cell Binding Assays-sCD4-OX40L was radiolabeled with [ 125 I]iodide (Amersham International plc, Little Chalfont UK) using the chloramine-T method as described (27). The specific activity of 125 I-sCD4-OX40L was determined by competition binding experiments using unlabeled sCD4-OX40L to be 1100 Ci/mmol.…”

“…A value for k on of 1.9 ϫ 10 5 M Ϫ1 s Ϫ1 was determined from the initial reaction rates of 125 I-sCD4-OX40L binding to activated cells ( Fig. 5A; Table I) as described (27,33). The dissociation rate was measured by incubating activated cells with near-saturating concentrations of 125 I-sCD4-OX40L until binding reached equilibrium (2 h).…”

Local opsonization by secreted macrophage complement components. Role of receptors for complement in uptake of zymosan.

“…Binding of MOI to human monocytes was detected by using a radioiodinated F(ab')~ rabbit anti-mouse ab, a gift from Dr. A. F. Williams, University of Oxford. Both first and second stage ab were used at saturation and the number of sites per cell determined (28).…”