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We have found that amino acid residues necessary for Clq and FcγR binding of human IgGl are located in the N-terminal region of the CH2 domain, residues 231--238, using a matched set of engineered antibodies based on the anti-HLA-DR antibody L243. Changing the leucine 235 in the CH2 region of IgG3 and IgG4 to glutamic acid was already known to abolish FcγRl binding. We have confirmed this for IgG1 and also found a concomitant abolition of human complement lysis with retention of FcγRIII-mediated function. Changing the glycine at 237 to alanine of IgG1 also abolished FcγRl binding and reduced human complement lysis and FcγRIII-mediated function. Exchanging the whole region 233-236 with the sequence found in human IgG2, abolished FcγRI binding and human complement lysis and reduced FcγRIII-mediated function of IgG1. In contrast, a change in the previously described C1q-binding motif, from lysine at 320 to alanine, had no effect on IgG1-mediated complement lysis. [ABSTRACT FROM AUTHOR]

The strength of the bond between 125I-labelled Clq and immune complexes, Fc piece, dextran sulphate, polyglutamic acid and polylysine has been investigated. The binding of Clq to Fc piece, small molecular weight (< 10,000) dextran sulphate, polyglutamic acid and polylysine have values for the functional affinity constant (Ko) in the range 0.2-1.5 × 104 M-1. In contrast, the binding of Clq to immune complexes and large molecular weight polyions (> 100,000 is much greater and lies in the range 3×107 - 4×108 M-1. The differences in the binding constants between the two groups can be explained if the Fc piece and small molecular weight compounds bind to only 1 head of the Clq molecule but the immune complexes and large molecules bind to 2 heads. There are probably 6 binding sites on the Clq molecule for dextran sulphate. The enhancement of the binding affinity of Clq by reduction in ionic strength and the reaction with polyions, indicate that ionic groups are present near or within the binding sites. [ABSTRACT FROM AUTHOR]

The strength of the bond between 125I-labelled Clq and immune complexes, Fc piece, dextran sulphate, polyglutamic acid and polylysine has been investigated. The binding of Clq to Fc piece, small molecular weight (< 10,000) dextran sulphate, polyglutamic acid and polylysine have values for the functional affinity constant (Ko) in the range 0.2-1.5 × 104 M-1. In contrast, the binding of Clq to immune complexes and large molecular weight polyions (> 100,000 is much greater and lies in the range 3×107 - 4×108 M-1. The differences in the binding constants between the two groups can be explained if the Fc piece and small molecular weight compounds bind to only 1 head of the Clq molecule but the immune complexes and large molecules bind to 2 heads. There are probably 6 binding sites on the Clq molecule for dextran sulphate. The enhancement of the binding affinity of Clq by reduction in ionic strength and the reaction with polyions, indicate that ionic groups are present near or within the binding sites. [ABSTRACT FROM AUTHOR]

Estimates have been made of the value of the equilibrium constant and of the heterogeneity index of the reaction between anti-D and Rh positive red cells, using anti-D immunoglobulin preparations made from pools of donor plasma. The values of the equilibrium constant all fell within the range 1 × 108 to 10 × 108 l/mole, and in eighteen of the twenty-five samples the values fell within the narrow range of 2 × 108 to 4 × 108 l/mole. Estimates of the value of the heterogeneity index varied from 0.5 to 0.8. Calculations made using these values of the equilibrium constant indicate that if 200 µg of anti-D from each preparation were injected into an Rh-negative recipient with approximately 3 ml of R1r cells in the circulation, between 8 and 28 per cent (average 17 per cent) of the D antigen sites on the red cells would be bound by anti-D if equilibrium conditions were reached. [ABSTRACT FROM AUTHOR]

The reactivity of native IgM anti-B and of the monomer subunits with B cells was investigated using 125I-labelled antibody. A proportion of the monomers produced by mercaptoethanol treatment retained antibody activity, but the value of the equilibrium constant of the subunits was reduced by a factor of 36–170 compared to that of the native IgM molecule. It is concluded that native IgM anti-B is attached to red cells by at least two combining sites and evidence is given which indicates that IgM anti-A behaves similarly. [ABSTRACT FROM AUTHOR]

The rate constants for association and dissociation, and the equilibrium constants, were determined for 125I-labelled anti-A and anti-B of both IgG and IgM molecular types. The following results and conclusions were obtained: 1. The equilibrium constants were within the range 0·6 × 108–13·0 × 108 l/mole, and were of the same order for both IgG and IgM antibodies. 2. The initial rate constants for association were in the range 2·1 × 105–4·8 × 105 1/mole/sec, and the energy of activation (Ea) 6700–9000 cal/mole. These results indicate that the rate of association is approaching the limit set by the rate of diffusion of the reactants. 3. The initial rate constants for dissociation were 1 × 10-4–5 × 10-4/sec and Ea = 20,000–36,000 cal/mole. These latter values suggest that more than one bond must be broken simultaneously during dissociation. 4. Ionic strcngth and pH changcs have only a minor effect on the constants; this indicates absence of ionic groups on A and B antigen sites. 5. The changes in enthalpy were -5400 to -21,800 cal/mole; the reactions are mainly enthalpy driven and this accounts for the fact that anti-A and anti-B agglutination titres increase as the temperature is decreased. 6. There was heterogeneity of the values of the standard change in free energy, enthalpy and entropy within each example of antibody. 7. The approximate concentrations of antibody at the end-points of the agglu- tination titres were: for IgG antibody, 0·2 μg/ml; for IgM antibody, 0·01 μg/ml. [ABSTRACT FROM AUTHOR]

A method is described for estimating the concentration and equilibrium constant of the blood group antibody, anti-D. The principle of the method is based on the fact that the reaction between anti-D and red cells is reversible and obeys the law of mass action. D-positive red cells at five different concentrations were added to aliquots of antisera containing anti-D and the reaction allowed to come into equilibrium. The amount of anti-D bound to the red ceils in each case was estimated using 125I-labelled anti-γ-globulin. The results of these estimates were then analysed according to two derivations of the law of mass action. Twenty-one examples of antisera containing anti-D were examined and the estimated range of values was as follows: concentration, 0.3–7.2 μg/ml; equilibrium constant, 1.4 × 107 to 1.2 × 109 l/mole; index of heterogeneity, a = 0.6–0.9.

Ten previously untransfused Rh(D)-negative subjects were given an intravenous injection of approximately 0·3 ml of Rh(D)-positive red cells and at about the same time, or 24-48 hours previously, were given an intramuscular injection of 1-1000 μg of anti-D. Following the injection of the red cells there was a variable period before the onset of red cell destruction; when the antibody was injected at the same time as the red cells the delay was due partly to the time taken for the antibody to reach the circulation; when the antibody was injected at least 24 hours before the red cells there was still some delay due to the time required for the antibody to be taken up by the red cells; the delay before the onset of a maximum rate of red cell destruction varied from about 0·2 hours following the injection of 1000 μg of antibody to approximately 100 hours following the injection of 1 μg. The maximum rate of red cell destruction was calculated to be approximately proportional to the square root of the amount of antibody on the cells. After the injection of the smallest amount of antibody (1 μg) the amount on the red cells was calculated to be about 0·03 μg/ml corresponding to about ten molecules of antibody per cell. This dose produced clearance with a T½ of the order of 100 hours. The significance of these observations in relation to protection against Rh-immunization is briefly discussed. [ABSTRACT FROM AUTHOR]

The effect of pH and ionic strength on the reaction between anti-D and erythrocytes was investigated using 1311-labelled antibody. The following observations were made. 1. The association constant of the reaction was influenced by the pH of the suspending medium. The highest values were obtained in the pH range 6·0–8·0. The association constant was considerably reduced above and below these values. 2. There was considerable heterogeneity of the rate of dissociation of the antigen-antibody complex at pH values below 6·0 3. The antibody was separated into fractions which, at neutral pH, had different rate constants for dissociation. 4. The rate of association between anti-D and erythrocytes was greatly increased by a reduction in the ionic strength of the suspending medium. [ABSTRACT FROM AUTHOR]

The strength of the bond between C1 and C1 binders (as measured by C1q binding) has been correlated with the ability of the binders to activate C1. The rate of activation of C1 has been studied by following the extent of hydrolysis of the C1r and C1s subcomponents, using a purified preparation of C1 labelled with 125I. The rate of activation of C1 was not correlated with the binding strength between C1q and the C1 binders. Immune complexes were found to activate C1 rapidly, whereas glutaraldehyde-aggregated IgG .failed to activate faster than the spontaneous activation seen on incubation of C1 alone; the strength of the bond between C1q and the binders was similar in the two cases, it is suggested that an interaction other than the binding between C1q and C1 binders is necessary for activation of C1. C1 bound to immune complexes was not activated in the presence of C1 inhibitor, indicating that the inhibitor can prevent the hydrolysis of C1r under the test conditions. [ABSTRACT FROM AUTHOR]

Seven IgG1 and seven IgG3 human monoclonal antibodies derived from heterohybridoma or Epstein-Barr virus-transformed lymphocytes and specific for the D antigen of the human Rh blood group system were tested for their ability to bring about red cell attachment to and phagocytosis by monocytes. The antibodies produced by the heterohybridomas were also investigated for their potency to mediate antibody-dependent cellular cytotoxicity (ADCC) by man ocytes. When red cells were sensitized with any of the IgG I anti-D antibodies, most of them were ingested by the phagocytes. By contrast, many of the red cells coated with any of the IgG3 antibodies remained attached to the monocyte surface while only few underwent phagocytosis. Some of the attached red cells remained on the phagocyte exterior for a considerable length of time. The ADCC activities of the IgG3 anti-D antibodies was greater than that of the IgG1 and D antibodies. The results mean that in vitro IgG 1 anti-D mediates red cell destruction mainly by phagocytosis, while IgG3 anti-D causes their destruction predominantly by prolonged cytolysis. These differences between the effector functions of human monoclonal IgG1 and IgG3 anti-D antibodies might have important implications for their use in the prophylaxis of haemolytic disease of the new-born. [ABSTRACT FROM AUTHOR]

Four IgG1 and three IgG3 human monoclonal antibodies specific for the blood group D(Rh) antigen were tested for their ability to mediate red cell-binding to macrophages in vitro. The IgG3 monoclonals were found to opsonize at a density of approximately 100 molecules per rut cell, whereas the IgG1 antibodies were only active at a level of 10,000 molecules per cell. There was no substantial difference between the two IgG subclasses in their ability to bind to Fc receptors on macrophages and it is suggested that the more potent opsonic activity of IgG3 is the result of the relatively long hinge, leading to greater accessibility to the Fc receptor binding site on the Fc piece. [ABSTRACT FROM AUTHOR]

Heterohybridomas secreting human IgM and IgG anti-D antibodies of the rhesus blood group system have been established by fusion of EBV-transformed anti-D secreting cells with the mouse myeloma cells X63-Ag8.653. Both classes of antibody reacted with all Rh-positive cells, some Du cells but not with Rh-negative or DB cells. Concentrations of both antibodies reached between 25 μg/ml and 50 μg/ml in the culture supernatants. The cell lines have been maintained in culture for 14 months and have been shown to be suitable for large-scale production of antibody. [ABSTRACT FROM AUTHOR]

Antibodies directed against immunoglobulin (anti-Ig) were found to inhibit the spontaneous incorporation of [3H]thymidine by Epstein-Burr virus (EBV) transformed lymphoblastoid cell lines (LCL). The degree of inhibition was variable but could exceed 80% following a 24 hr exposure of cells to anti-Ig. While a short pulse of antibody was sufficient to cause inhibition, maintainence of the effect required the continual presence of anti-Ig in culture. Maximal suppression by anti-Ig of [3H]thymidine uptake was reached between days 3 and 5, but cells eventually escaped from inhibition. For a line coexpressing IgM and IgD at the cell surfaces it was found that antibodies specific for the individual isotypes were equally effective at inducing suppression. Successful inhibition of DNA synthesis by F(ab′)2 fragments of anti-Ig demonstrated that the Fc portion was not required for this effect. Anti-Ig immobilized on plastic surfaces was as effective as soluble antibody in inducing suppression. The implications of these findings for the generation of EBV-transformed LCL producing specific antibody are discussed. [ABSTRACT FROM AUTHOR]