Efficiency of activation of complement by anti-hapten antibodies at the red cell surface: effect of patchy vs random distribution of hapten

Binding and activation of complement (C) by anti-hapten IgG and IgM antibodies (Abs) bound to a cell surface are dependent on the density and presumably on the distribution of cell-bound hapten. The purpose of this study was to find out if altering the distribution of the hapten on a red cell surface could modify the ability of anti-hapten IgG or IgM Ab to activate C. To test this we devised methods for comparing the C binding and activating efficiency of Abs bound to a hapten distributed randomly or in patches on cells. Random distribution was achieved by the covalent binding of methotrexate (MTX) directly to sheep red blood cells (E), while patchy distribution was achieved by the convalent binding to E of bovine serum albumin-MTX complexes. We considered bound albumin molecules as patches of MTX molecules. The results showed that, for IgG Ab, the number of hapten/E and the number of anti-hapten Ab molecules/cell required to generate one C-activating IgG complex were about an order of magnitude lower for hapten bound in patches than for randomly bound hapten. In contrast, IgM Ab bound to a hapten distributed in patches on an E surface lacked the ability to activate the lytic sequence of C, although maintaining a full ability to binding C1.     

Activation of hemolytic complement by mouse monoclonal IgG1 antibody: comparison with rabbit IgG

We investigated the ability of a mouse anti-hapten monoclonal IgG1 antibody (Ab) to bind to cell-bound specific hapten and to fix and activate C1 and thus the lytic sequence of complement (C). In a comparative study with polyclonal rabbit anti-hapten IgG Ab, we found that about 6 times more monoclonal Ab molecules than polyclonal were necessary for the generation of 1 hemolytic site/cell: the data were interpreted to mean that a cluster of four cell-bound monoclonal Ab molecules was necessary to bind C1 and activate C-mediated hemolysis. Experiments performed under conditions of low density of cell-bound hapten and excess of antibody showed that both monoclonal and polyclonal IgG Abs were able to react only with 20-30% of the cell-bound hapten and that both Abs recognized the same hapten specificity. We also found that even though monoclonal IgG1 Ab was able to bind strongly to a protein A-Sepharose column and could be eluted only by a low-pH buffer, the purified Ab, when bound to cell surface hapten, showed a weak ability to react with free protein A.     

Binding and activation of C1 by cell bound IgG: Activation depends on cell surface hapten density

We have investigated the binding and activation of C1 by IgG-anti methotrexate antibody at cell surfaces. Under conditions where variation in cell surface hapten density had no effect on binding of IgG. the number of C1 (or its active form, C1) bound by the IgG was independent of hapten density. The ability of the C1 binding IgG complex to activate C1, however, was decreased with decreasing density of the hapten. The decreased ability to activate bound C1 was paralelled by decreased ability to activate the hemolytic sequence in whole complement. The results were interpreted to mean that binding of C1 was the result of aggregation (doublet formation) by IgG while activation of the bound C1 depended on changes induced in the IgG molecule by straddling hapten molecules at varying distances.     

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand alpha-galactosylceramide bound to mouse CD1d

The alpha-galactosylceramide (alpha-GalCer) known as KRN7000 remains the best studied ligand of the lipid-binding MHC class I-like protein CD1d. The KRN7000:CD1d complex is highly recognized by invariant natural killer T (iNKT) cells, an evolutionarily conserved subset of T lymphocytes that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. To facilitate the study of glycolipid antigen presentation to iNKT cells by CD1d, we undertook the production of mouse monoclonal antibodies (mAbs) specific for complexes of KRN7000 bound to mouse CD1d (mCD1d) proteins. Three such monoclonal antibodies were isolated that bound only to mCD1d proteins that were loaded with KRN7000 or closely-related forms of alpha-GalCer. These mAbs showed no reactivity with mCD1d proteins that were not loaded with alpha-GalCer, nor did they bind to complexes formed by loading mCD1d with the self-glycolipid and putative iNKT cell ligand isoglobotrihexosylceramide. These complex-specific monoclonal antibodies allow the direct detection and monitoring of complexes formed by the binding of KRN7000 and other alpha-GalCer analogues to mCD1d. The availability of these mAbs should facilitate a wide range of studies on the biology and potential clinical applications of CD1d-restricted iNKT cells.     

A rapid quantitative staphylococcal co-agglutination assay. Utilization for the assay of bovine factor VIII-related antigen

A simple and rapid technique to measure bovine factor VIII-related antigen has been developed which utilizes protein A-bearing staphylococci and monospecific rabbit antiserum to bovine factor VIII. Staphylococci coated with a specific antibody agglutinate when they are mixed with the specific antigen. We have used an aggregometer to detect an quantitate the agglutination of the antibody-coated staphylococci. The assay has been optimized with respect to amount of antiserum needed for coating staphylococci, concentration of antibody-coated staphylococci, pH and ionic strength of the assay system, and stirring speed of the aggregometer. The staphylococcal co-agglutination assay as monitored by an aggregometer is at least 10 times more sensitive than the conventional slide agglutination method, and can detect as little as 0.1 microgram/ml of factor VIII antigen. It however, cannot be used to quantitate factor VIII-related antigen in plasma, since plasma contains some components which can non-specifically agglutinate staphylococci.     

Effect of reduction and alkylation on structure and function of rabbit IgG antibody--II. Effects on classical pathway C3 convertase formation

Anaerobic reduction of purified rabbit IgG antibody (Ab) with 1.5 moles of dithiothreitol per mole of Ab at pH 8.0, followed by alkylation, cleaves 39% of the inter-heavy-chain (H-H) disulfide (SS) bonds. This treatment has the following effects on the ability of the Ab to activate the classical pathway of complement. Compared to control Ab, reduced and alkylated (RA) Ab retained 4-5.6% of overall hemolytic activity and 55% of complement-fixing activity at 0 degrees C. Complexes of RA Ab and equivalent amounts of soluble Ag consumed C4, C2 and C3 at 37, 51 and 44%, respectively, of the rate at which these components were consumed by equal concns of complexes containing control Ab. Complexes made with RA Ab bound 18% as much C-1 as those made with native Ab. These data indicate that the principal, if not the only, effect of RA is on C-1 binding. Measurements of the ability of complexes of Ab with cell-bound Ag to bind C-1 showed at most a 20% loss of C-1 binding sites and a ca two-fold decrease in affinity for C-1. Similar results were obtained with purified (activated) C-1 and with native C1 in serum. No significant difference could be detected in the rate of activation of bound C1. Normal rabbit IgG which was reduced and alkylated under the same conditions retained 52% of its H-H SS bonds and 30% of its ability to bind C-1. This finding suggests that the impairment in C-1 binding results from an effect on the C1 binding site itself, rather than from an effect on the ability of the RA Ab to transmit a putative conformational "signal" from the Ag-binding site to the C1 binding site. Finally, our data show that the observed functional effect of reduction and alkylation depends strongly on the assay used to evaluate that effect.