Generation and characterisation of Rhd and Rhag null mice.

Mouse Rhd* and Rhag* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. Rhag glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of Rhag expression. Double knockout phenotype was similar to that of Rhag targeted mice. Rhd and Rhag deficient mice exhibited neither the equivalent of human Rh(null) haemolytic anaemia nor any clinical or cellular abnormalities. Rhd-/- and Rhag-/- erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in Rhag-/- but only slightly in Rhd-/- animals that significantly expressed Rhag, supporting the view that RhAG and Rhag, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and Rhag proteins in the red cell membrane.

Measurement of the affinity of anti-D in the serum of immunized mothers and in immunoglobulin preparations with unlabeled antibodies.

BACKGROUND: Few data are available on the affinity of maternal anti-D responsible for hemolytic disease of the fetus and the newborn (HDN) and of anti-D used for the prophylaxis of that disease. A method was recently described to measure the affinity (K(a)) of untagged anti-D monoclonal antibodies (MoAbs). In this work, the same method was applied to determine the K(a) of polyclonal anti-D. STUDY DESIGN AND METHODS: O R(1)r red blood cells (RBCs) were sensitized with increasing concentrations of native anti-D in serum samples from immunized mothers and donors and in RhIG preparations. At equilibrium, the amount of anti-D bound to RBCs was measured by enzyme-linked immunosorbent assay. Scatchard and Langmuir equations were used to determine Ka. RESULTS: The experimental data fitted well with the Scatchard equation (mean r2=0.95) but a better correlation was observed with the Langmuir equation (mean r2=0.99). The mean Ka of anti-D in 11 maternal serum samples, in 6 immunized donors, and in 5 lots of RhIG were 5.6x10(8) per M (from 2.8x10(8) to 12x10(8)/M), 3.9x10(8) per M (from 1.5x10(8) to 6.8x10(8)/M), and 3.4x10(8) per M (from 3.1x10(8) to 4.2x10(8)/M), respectively. The comparison of anti-D affinity in 5 cases of HDN with fetal anemia and in 6 cases of HDN with postnatal anemia showed no significant difference. CONCLUSION: The method previously described for anti-D MoAbs was applied to polyclonal anti-D present in the serum of immunized subjects and in immunoglobulin preparations. The experimental data fitted well with the Langmuir equation, and the affinity of polyclonal of anti-D was measured with accuracy.

Measurement of anti-D intrinsic affinity with unlabeled antibodies.

BACKGROUND: The method most commonly used for measuring the affinity of MoAbs specific for the D antigen is a binding assay using 125I-labeled MoAb. Although the method is relatively simple, there are several drawbacks that can lead to inaccurate results. The objective of the present work was to develop a method for the determination of the affinity of anti-D MoAb using unlabelled antibodies. STUDY DESIGN AND METHODS: Rh-positive RBCs were sensitized with varying amounts of unlabeled anti-D MoAb, and, at equilibrium, the amount of anti-D bound to the RBCs was measured by ELISA. The affinity and the number of antigenic sites were determined with the Scatchard and Langmuir equations. RESULTS: The method was applied to determine the affinity of several MoAbs specific for the D antigen on RBCs of different Rh-positive phenotypes. Similar results were observed with both equations. The affinity constant (Ka) for 3 MoAbs specific for the D antigen on group O R1r RBCs ranged from 1.3 to 7.4 108 M-1, depending on the antibody. When measured by a binding assay with 125I-labeled anti-D MoAbs, the Ka were significantly lower, indicating that 125I-labeling diminishes anti-D affinity, even when the labeling is at a low level. CONCLUSION: A simple ELISA method was developed for the measurement of the affinity of anti-D MoAbs for the D antigen and for the number of antigenic sites per RBC. As a result, the affinity of anti-D can be estimated accurately, thus avoiding the drawbacks inherent in modification of the antibody by 125I-labeling.

IgG1 and IgG3 anti-D in maternal serum and on the RBCs of infants suffering from HDN: relationship with the severity of the disease.

BACKGROUND: Anti-D IgG antibodies that are responsible for severe cases of HDN belong chiefly to IgG1 and IgG3 subclasses. The relationship between the concentrations of IgG1 anti-D and IgG3 anti-D in maternal serum and the amount bound to the surface of infants' RBCs is not known. In addition, the contribution of the two subclasses to the severity of HDN is not well established. STUDY DESIGN AND METHODS: Blood samples from 40 infants suffering from severe forms of HDN due to anti-D were collected before transfusion together with sera from their respective mother. The amount of total anti-D IgG as well as IgG1 anti-D and IgG3 anti-D on infants' RBCs and the concentration in maternal sera were determined by ELISA. RESULTS: The median percentages of IgG1 anti-D and of IgG3 anti-D in maternal sera were 90 and 10 percent, respectively, whereas on infants' RBCs they were 97 and 3 percent, respectively. The differences between maternal and infantile percentages were significant (p < 0.001). IgG1 and IgG3 anti-D bound to infants' RBCs increased concomitantly with the concentration of IgG1 and IgG3 anti-D in maternal sera. The severity of HDN correlated positively with the concentration of IgG1 anti-D in maternal sera, but negatively with the amount of IgG3 anti-D bound to infants' RBCs. In addition, the existence of a high proportion of IgG3 anti-D in maternal serum was associated with a delayed risk of fetal anemia. CONCLUSION: The proportion of IgG3 anti-D relative to the total anti-D IgG on infants' RBCs is only one- third of the proportion present in maternal serum. The study of the correlations between the amount of IgG1 anti-D and IgG3 anti-D and the severity of HDN suggests that IgG1 anti-D are more important than IgG3 anti-D in the pathogenesis of fetal anemia.