Immunology 1976 30 289

Quantitative and thermodynamic measurements on I and i antigens of human red blood cells C. DOINEL, C. ROPARS & C. SALMON Groupe de Recherche U 76 de l'INSERM et Service d'Immunologie du Centre National de Transfusion Sanguine, Boulevard Diderot, Paris Cedex 12, France

Received 27 June 1975; acceptedfor publication 23 July 1975

Summary. Different homogeneous IgM cold agglutinins (two anti-I, two anti-i and one anti-Ii crossreacting antibodies) have been used to determine the antigen site densities of adult I and i erythrocytes and of cord red cells. The equilibrium constants and the thermodynamic constants of these reactions have been determined. The two anti-I antibodies, which did not combine with i or cord red blood cells, recognized two different determinants on I red cells. The antigen density of the I Fla. receptor was 120,000 sites per erythrocyte and the standard enthalpy change (-AH0) of the reaction was 18 to 25 kcal/mole. The antigen density of the I Loi. determinant varied according to the red cells tested and the enthalpy change ( HH0) of these reactions was 50-65 kcal/mole. The i and cord erythrocytes reactive structures were more heterogeneous than those present on I erythrocytes. The equilibrium constants rapidly decreased as the temperature rose and the standard enthalpy changes (-AH0) ranged from 50 to 90 kcal/mole. Two types of i determinants were observed; one of the anti-i antibodies reacted mainly with an i component present on cord erythrocytes, the other antibody reacted with a different i component present on i adult red-cells. The determinants, recognized by the cross-reacting

antibody on I red cells, differed from those on i or cord red cells in equilibrium constant, thermodynamic constants, index of heterogeneity of the reaction and in their sensitivity to formalin treatment. INTRODUCTION

Many attempts of classification of the I and i antigens into subgroups have been proposed (Wiener, Unger, Cohen and Feldman, 1956; Jenkins and Marsh, 1961; Marsh, Nichols and Reid, 1971; Marsh and Jenkins, 1960; Marsh, 1961). The interpretation of the serological reactions is restricted by the semiquantitative nature of the measurements and by the apparently heterogeneous nature of the I and i antigens (Crookston, Dacie and Rossi, 1956; Maas and Schubothe, 1968). Moreover, the antibodies seldom have simple specificities in haemagglutination. They are often mixtures of antibodies which are difficult to separate or cross-reacting antibodies (Jackson, Issitt, Francis, Garris and Sanders, 1968; Boissezon, Marty, Bierme and Ducos, 1970; Dzierzkowa-Borodej, Seyfried and Lisowska, 1975; Doinel, Ropars and Salmon, 1974). It cannot be specified whether the cross-reacting antibodies recognize other different or common antigens on I Correspondence: Dr C. Doinel, Groupe de Recherche and i red blood cells. U 76 de 1'INSERM, 53, Boulevard Diderot, 75571 Paris Cedex 12, France. Feizi and Kabat (1972) studied various anti-I and 289

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C. Doinel, C. Ropars & C. Salmon

anti-i, using a quantitative precipitation method, and attempted to group them according to their reactivity with blood group substances. They characterized six different anti-I specificities and four groups for anti-i. Roelcke, Ebert, Metz and Weickert (1971) found three determinants in the fraction of glycoproteins with I activity, obtained from the erythrocyte membrane. Feizi, Kabat, Vicari, Anderson and Marsh (1971) described the biochemical structure of an oligosaccharide detected by the anti-I (Ma) but the specificities of the other antibodies were different and are not yet known (Feizi and Kabat, 1974). Most antibodies cross-react with the numerous determinants of the I antigen complex, each one combining with a varying number of these antigenic determinants. In this work, we have studied several antigenantibody combinations in the I, i blood group system. To avoid the problem of mixtures of antibodies we used homogeneous IgM from cold agglutinin disease. The purification and the biochemical characterization of these compounds have also enabled us to attempt a quantitative study with radiochemical methods. The five antibodies we have studied belong to three distinct groups. Two react with I red blood cells only, two others with the red blood cells of adult i subjects, and one with both types. In each case, we have measured the antigen site densities on the red cells, the equilibrium and thermodynamic constants of the reactions. MATERIALS AND METHODS Red cells I erythrocytes were freshly drawn from healthy adult blood donors. The erythrocytes from Caucasian i adults and from cord blood were frozen in liquid nitrogen until used. Sera These were obtained from subjects with chronic cold agglutinin disease. Two monoclonal macro-globulins without any known antibody activity were used as a negative control. All the homogeneous IgM had been purified by delipidation with dextran sulphate, 2 M ammonium sulphate precipitation and preparative ultracentrifugation in a Beckman L 3-50 ultracentrifuge at 110,000 g maximum, for 18 h at 40 in a 5-20 per cent linear gradient of sucrose in

phosphate-buffered saline. On average, 5 per cent of a2-macroglobulin contaminated these IgM preparations. Quantitative agglutination The agglutinating activity of the various IgM antibodies was determined by a quantitative method using a Technicon Autoanalyzer (Doinel et al., 1975). The titres were expressed as the reciprocal of the dilution able to agglutinate 50 per cent of a suspension of 800,000 red cells per mm3. Treatment of the red cells with formalin One volume of packed cells was treated with twenty volumes of 20 per cent formalin in PBS, pH 7.3, for 20 h at 200, then washed, filtered through glass-wool and stored at 40 until used.

Labelling of the purified antibodies (Bocci, 1974) To 10 mg of purified IgM in PBS (pH 7 2) were successively added 10 p1 of 3 x 10-3M KI, 110 pCi of 125I-labelled Na, 300 pg of chloramine T, and 30 min later, 300 pg of Na metabisulphite. The free iodine was eliminated by dialysis. On an average, two atoms of iodine were fixed per IgM molecule.

Non-specific fixation Analytical ultracentrifugation of the purified cold agglutinins has shown the presence of components with sedimentation coefficients over 20S; the proportion of aggregates was increased after the labelling procedure. Consequently, just before use, the labelled IgM preparations were ultracentrifuged under the conditions described above. The peak of the aggregates, representing about 20 per cent of the proteins, was eliminated. In all experiments, the use of a monoclonal IgM without any known antibody activity, labelled with 1251I, made it possible to determine the amount of non-specific fixation included in each measurement. The amount of non-specific fixation was the same for both IgM preparations. The specificity of the cold agglutinins was determined by agglutination at 30 of red cells with various phenotypes in the I, i system. It was confirmed by comparing the percentage of purified antibody that could be absorbed onto these cells. Quantitative and thermodynamic measurements (a) Determination of the number of bound IgM molecules per erythrocyte: the experiments were

I and i antigens

carried out in PBS (pH 7 2) containing 1 per cent bovine albumin (Miles-Pentex) and 0-15 M glycine. The concentrations of specific antibodies ranged from 0 5 to 50 ,ug in 0-5 ml. The red cell concentration determined with a Coultronics Counter, model B, varied between 1 x 107 and 2 5 x 107 cells/ml. All experiments were carried out in duplicate, in plastic tubes stored at 30 under continuous agitation for 4 h. The packed cells, centrifuged and washed three times at 30, were transferred into new tubes; counting was performed in a CGR-SAIP gamma counter. The maximum number of IgM molecules, n, which could be bound per erythrocyte was evaluated from the expressions of the mass action law derived by Scatchard (1949) or by Ekins, Newman and O'Riordan (1967). (b) Determination of the equilibrium constants: to 5 pg of specific antibodies in 1 ml of PBS containing 1 per cent albumin, various cell concentrations ranging from 5 x 106 to 250 x 106 cells/ml were added. The samples were incubated in duplicate, at 30 and at 100, under continuous mild shaking, for 18 h. The equilibrium constants were calculated from the Karush derivation (1962). The calculation was performed assuming that antibodies behave like univalent coumpounds. (c) The standard free energy change AF0, the standard enthalpy change AHO and the standard entropy change AS0 were calculated from the equilibrium constants at 30 and 100.

RESULTS (1) Cold agglutinins specificities The specificities of the cold agglutinin auto-antibodies which have been studied, are shown in Table 1. Two antibodies (Fla., Loi.) have anti-I specificities. The Fou. and Min. antibodies are anti-i. The Abg. antibody agglutinated the I erythrocytes from adults and, but more weakly, the erythrocytes from cord and i adults. The agglutination titres were the same with the cord and i adult red cells. The two reactivities disappeared simultaneously and in the same proportion on partial absorption with I or i erythrocytes. After absorption and elution from I erythrocytes, the same ratio of agglutination titres on I and i red cells was obtained as for the initial antibody. The same result was obtained when the antibody was absorbed on and

291 Table 1. Specificities of the cold agglutinins

Sera

Percentage purified antibody

agglutination titres at 30

absorbed at 3 'C by:

Specificity Fla. Loi. Fou. Min. Abg.

I RBC

i RBC*

475,000 180,000

10,000 10,000

250 250

8000 32,000 58,500

232,500

I RBC i RBC* 73 37

Quantitative and thermodynamic measurements on I and i antigens of human red blood cells.

Immunology 1976 30 289 Quantitative and thermodynamic measurements on I and i antigens of human red blood cells C. DOINEL, C. ROPARS & C. SALMON Grou...
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