Immunology 1979 37 547
A new approach to the thermodynamic study of ABO antibodies
PH. ROUGER & CH. SALMON National Blood Group Reference Laboratory, C.N.T.S., INSERM. U76, 53, boulevard Diderot, 75571 Paris Cedex 12, France
Received 6 September 1978; acceptedfor publication 18 December 1978
Summary. Enthalpy change was determined for natural anti-A (B, 0 subjects) and anti-B (Al, A2, 0 subjects). Entropy change, free energy change and association constant were calculated according to the law of mass action and the Wurmser method. The concentrations of allohaemagglutinins were measured by the Wilkie and Becker method using an autoanalyser. 2-Mercaptoethanol was used to estimate the proportions of IgG and IgM and their respective contribution to the thermodynamic properties. The following results and conclusions were obtained. Individual enthalpy and entropy changes are different for each subject so that only the average values of these thermodynamic parameters represent a characteristic of the phenotype. There is a correlation between enthalpy and entropy changes and the relative proportion of anti-A or anti-B IgG. There is heterogeneity of the values of association constant. Free energy change is about 10 kcal mol I for all anti-A and anti-B; this result confirms the low energy binding between antigen and antibody. All these results confirm the role of environment and red-cell phenotype in the synthesis of allohaemagglutinins.
very individual population of antibodies; the presence of anti-A and anti-B is a function of erythrocyte phenotype, so the antibody is present when the specific erythrocyte antigen is missing. The explanation of the production of so-called natural antibodies is still a subject of discussion. According to Springer et al. (Springer, Horton & Forbes, 1958; 1959; Springer & Horton, 1969) the role of the environment (bacterial intestinal flora) is a dominant factor. According to Filitti-Wurmser, Jacquot-Armand, Aubel Lesure & Wurmser (1954), Wurmser & Filitti-Wurmser (1957), Salmon, Salmon & Saint Paul (1965) anti-B allohaemagglutinins are determined by the ABO phenotype. In fact, these theories are not incompatible, but seem complementary. The purpose of this thermodynamic study is to analyse the behaviour of different molecular classes of specific immunoglobulins to elucidate the mechanism of the production of antibodies.
MATERIAL AND METHODS The concentration of anti-A and anti-B was determined by the probit method of Wilkie & Becker (1955a) using an autoanalyser (Rosenfield & Haber, 1965; Marsh, 1968). This method was based on artificial agglutination and used bromelin-treated human red cells and methyl cellulose. A timing programme controlled the 90 s cycle which consisted of 30 s sampling followed by 60 s washing. This mixture was flowed for 20 min in a manifold. The temperature was
INTRODUCTION Anti-A and anti-B natural allohaemagglutinins are a Correspondence: Dr Ph. Rouger, Centre National de Transfusion Sanguine, Etablissement Saint-Antoine, 53 bd Diderot 75571 Paris Cedex 12, France. 0019-2805/79/0700-0547 $02.00
© 1979 Blackwell Scientific Publications 547
Ph. Rouger & Ch. Salmon
548
kept at 15°. After the addition of hypertonic salt solution (NaCl 0-2 M at pH 7) aggregates were decanted in double settling coils. Free red cell lysis was obtained by triton solution. A colorimeter determined the haemoglobin content. A correction was necessary for the determination of the real agglutination percentage because residual optical density was not equal to zero (Doinel, Ropars & Salmon, 1975). The determination of the agglutination percentage of five different dilutions allowed the regression line probit (agglutination percentage), logarithm (serum dilution) to be drawn. The relative concentration of allohaemagglutinins was calculated by the serum dilution giving 50% agglutination (haemagglutination dose 50% = HD 50). The concentration of anti-B was obtained by using B red blood cells and the concentration of anti-A by using A2 red blood cells.
HD 50, relative concentration of total allohaemagglutinins This relation could be used only if the percentage of bound sites was weak (Wurmser & Filitti-Wurmser, 1957; Kabat, 1956). The equilibrium was obtained after 105 min at 25 and 37°. The number of red cells was constant and equal to 300,000/mm3 the serum concentration varied as shown in Table 1. The separation of free antibodies from the cells carrying bound antibodies was obtained by centrifugation. In this manner, thirty-eight normal anti-B human sera and twenty normal anti-A human sera were studied. The straight lines obtained at two temperatures (25 and 370) by plotting Nt/HDf against l/HD'50 allowed us to determine the ratio of the two slopes and the values of Ah by the Van't Hoff relation:
Thermodynamic method The law of mass action can be applied to the thermodynamic studies of immunological reactions (FilittiWurmser & Jacquot Armand, 1947; Filitti-Wurmser, Jacquot-Armand & Wurmser, 1950; Filitti-Wurmser et al.,1954). We used the following relation (Wurmser & FilittiWurmser, 1957; Wurmser, 1972): Nt
HDf
=
6x 1017
-AH=4-575 log slope 25 x slope
cT25Cal mol T37
1
HD'50
Nt, number of red blood cells per mm3 m, total number ofsites by red blood cell K, association constant HD' 50, relative concentration of free allohaemagglutinins HDf, relative concentration of bound allohaemagglutinins
Determination of relative proportion of anti-A or anti-B IgG To estimate the relative proportion of IgG and IgM allohaemagglutinins we used the 2-mercaptoethanol *
T37xT25 310x298 = 7-698 T37-T25 310-298 =
Table 1. Serum and red blood cell concentrations used in this study
RBC Solution: 900,000 RBC/mm3 (A2 or B)
Serum 90 u. HD 50/ml
Dilution Volume (ml) HD50 Dilution Volume (ml) Final Nt
250 and
370
ND 0-8 0-6 0-5 0-3
1 1 1 1 1
T25
The number of specific sites was calculated from the work of Economidou, Hughes-Johns & Gardner (1967), Cartron (1975) and Greenbury, Moore & Nunn (1963). B red cells were found to possess 720,000 sites and A red cells to possess 270,000 sites. We could thus calculate other thermodynamic parameters: association constant, entropy change and free energy change.
x
mK
250
60 48 36 30 18
ND ND ND ND
ND
ND, No dilution.
0-5 0-5 0-5 0-5 0-5
3x105 3x 105 3x 105 3x 105 3 x 105
Thermodynamic study of ABO antibodies
549
Table 2. Results of the thermodynamic investigation of serum No. 22 (A20); the slope ratio is 1 676 and the enthalpy change is equal to -8 kcal mol - l. Temperature Dilution HD 50 HD'50 HDf 252
37c
HDf l0'4
Nt 10-410 HDf HD'50
HDf Nt
1 89
0393
1514
0398 0422 0-377 0-433 0 404
P 08 06 05 0-3
64 512 384 32 19-2
48 3-8 27 25 1-3
592 474 35-7 29-5 17 9
05287 06603 0876 1-061 1 748
0208 0263 0370 0-400 0-760
P 08 0-6 05 03
64 512 38 4 32 192
7-6 6 4-4 4 2-1
56 4 452 34 28 17-1
0-555 0692 0-920 1 117 1 83
0 131 0166 0 227 025 0-476
0
Nt HD'50
1-140 0-942 0 57 Average 1 802 1444 1 086 0894 0546
0-236% 0239 0-246 0223 0260 0-241
Average
treatment method described by Beale & Feinstein (1969) and Deutsch & Morton (1957). The same volumes of serum and 2-mercaptoethanol solution (0-2 M) were mixed for 2 h at room temperature; then, this mixture was dialysed and the residual HD50 was measured. The ratio of HD50 after treatment to HD50 before treatment by 2-mercaptoethanol was referred to as the G coefficient.
RESULTS
0~~~~~
Results of one thermodynamic study Results of a thermodynamic study on one serum (A20) are given in Table 2. The enthalpy change was 8 kcal mol -'. The relationship between Nt/HDf x l0-4 and l/HD'50 at 250 and 37° is shown in Fig. 1. Thermodynamic analysis of anti-A allohaemagglutinins The slope of Nt/HDf x 10-4=f (lI/HD'50) is a characteristic of each serum; similar curves referring to antibodies in B phenotype and 0 phenotype sera are shown in Figs 2 and 3 respectively. The association constant was greater at 250 than at 37°. At 370, the average association constant of anti-A(O) was greater than that of anti-A(B). Enthalpy change was more characteristic, the difference between the average enthalpy change of anti-A(B) (- 20-2 + 6 kcal mol -') and anti-A (0) (-8 + 2-4 kcal mol -') was significant (P
A new approach to the thermodynamic study of ABO antibodies
PH. ROUGER & CH. SALMON National Blood Group Reference Laboratory, C.N.T.S., INSERM. U76, 53, boulevard Diderot, 75571 Paris Cedex 12, France
Received 6 September 1978; acceptedfor publication 18 December 1978
Summary. Enthalpy change was determined for natural anti-A (B, 0 subjects) and anti-B (Al, A2, 0 subjects). Entropy change, free energy change and association constant were calculated according to the law of mass action and the Wurmser method. The concentrations of allohaemagglutinins were measured by the Wilkie and Becker method using an autoanalyser. 2-Mercaptoethanol was used to estimate the proportions of IgG and IgM and their respective contribution to the thermodynamic properties. The following results and conclusions were obtained. Individual enthalpy and entropy changes are different for each subject so that only the average values of these thermodynamic parameters represent a characteristic of the phenotype. There is a correlation between enthalpy and entropy changes and the relative proportion of anti-A or anti-B IgG. There is heterogeneity of the values of association constant. Free energy change is about 10 kcal mol I for all anti-A and anti-B; this result confirms the low energy binding between antigen and antibody. All these results confirm the role of environment and red-cell phenotype in the synthesis of allohaemagglutinins.
very individual population of antibodies; the presence of anti-A and anti-B is a function of erythrocyte phenotype, so the antibody is present when the specific erythrocyte antigen is missing. The explanation of the production of so-called natural antibodies is still a subject of discussion. According to Springer et al. (Springer, Horton & Forbes, 1958; 1959; Springer & Horton, 1969) the role of the environment (bacterial intestinal flora) is a dominant factor. According to Filitti-Wurmser, Jacquot-Armand, Aubel Lesure & Wurmser (1954), Wurmser & Filitti-Wurmser (1957), Salmon, Salmon & Saint Paul (1965) anti-B allohaemagglutinins are determined by the ABO phenotype. In fact, these theories are not incompatible, but seem complementary. The purpose of this thermodynamic study is to analyse the behaviour of different molecular classes of specific immunoglobulins to elucidate the mechanism of the production of antibodies.
MATERIAL AND METHODS The concentration of anti-A and anti-B was determined by the probit method of Wilkie & Becker (1955a) using an autoanalyser (Rosenfield & Haber, 1965; Marsh, 1968). This method was based on artificial agglutination and used bromelin-treated human red cells and methyl cellulose. A timing programme controlled the 90 s cycle which consisted of 30 s sampling followed by 60 s washing. This mixture was flowed for 20 min in a manifold. The temperature was
INTRODUCTION Anti-A and anti-B natural allohaemagglutinins are a Correspondence: Dr Ph. Rouger, Centre National de Transfusion Sanguine, Etablissement Saint-Antoine, 53 bd Diderot 75571 Paris Cedex 12, France. 0019-2805/79/0700-0547 $02.00
© 1979 Blackwell Scientific Publications 547
Ph. Rouger & Ch. Salmon
548
kept at 15°. After the addition of hypertonic salt solution (NaCl 0-2 M at pH 7) aggregates were decanted in double settling coils. Free red cell lysis was obtained by triton solution. A colorimeter determined the haemoglobin content. A correction was necessary for the determination of the real agglutination percentage because residual optical density was not equal to zero (Doinel, Ropars & Salmon, 1975). The determination of the agglutination percentage of five different dilutions allowed the regression line probit (agglutination percentage), logarithm (serum dilution) to be drawn. The relative concentration of allohaemagglutinins was calculated by the serum dilution giving 50% agglutination (haemagglutination dose 50% = HD 50). The concentration of anti-B was obtained by using B red blood cells and the concentration of anti-A by using A2 red blood cells.
HD 50, relative concentration of total allohaemagglutinins This relation could be used only if the percentage of bound sites was weak (Wurmser & Filitti-Wurmser, 1957; Kabat, 1956). The equilibrium was obtained after 105 min at 25 and 37°. The number of red cells was constant and equal to 300,000/mm3 the serum concentration varied as shown in Table 1. The separation of free antibodies from the cells carrying bound antibodies was obtained by centrifugation. In this manner, thirty-eight normal anti-B human sera and twenty normal anti-A human sera were studied. The straight lines obtained at two temperatures (25 and 370) by plotting Nt/HDf against l/HD'50 allowed us to determine the ratio of the two slopes and the values of Ah by the Van't Hoff relation:
Thermodynamic method The law of mass action can be applied to the thermodynamic studies of immunological reactions (FilittiWurmser & Jacquot Armand, 1947; Filitti-Wurmser, Jacquot-Armand & Wurmser, 1950; Filitti-Wurmser et al.,1954). We used the following relation (Wurmser & FilittiWurmser, 1957; Wurmser, 1972): Nt
HDf
=
6x 1017
-AH=4-575 log slope 25 x slope
cT25Cal mol T37
1
HD'50
Nt, number of red blood cells per mm3 m, total number ofsites by red blood cell K, association constant HD' 50, relative concentration of free allohaemagglutinins HDf, relative concentration of bound allohaemagglutinins
Determination of relative proportion of anti-A or anti-B IgG To estimate the relative proportion of IgG and IgM allohaemagglutinins we used the 2-mercaptoethanol *
T37xT25 310x298 = 7-698 T37-T25 310-298 =
Table 1. Serum and red blood cell concentrations used in this study
RBC Solution: 900,000 RBC/mm3 (A2 or B)
Serum 90 u. HD 50/ml
Dilution Volume (ml) HD50 Dilution Volume (ml) Final Nt
250 and
370
ND 0-8 0-6 0-5 0-3
1 1 1 1 1
T25
The number of specific sites was calculated from the work of Economidou, Hughes-Johns & Gardner (1967), Cartron (1975) and Greenbury, Moore & Nunn (1963). B red cells were found to possess 720,000 sites and A red cells to possess 270,000 sites. We could thus calculate other thermodynamic parameters: association constant, entropy change and free energy change.
x
mK
250
60 48 36 30 18
ND ND ND ND
ND
ND, No dilution.
0-5 0-5 0-5 0-5 0-5
3x105 3x 105 3x 105 3x 105 3 x 105
Thermodynamic study of ABO antibodies
549
Table 2. Results of the thermodynamic investigation of serum No. 22 (A20); the slope ratio is 1 676 and the enthalpy change is equal to -8 kcal mol - l. Temperature Dilution HD 50 HD'50 HDf 252
37c
HDf l0'4
Nt 10-410 HDf HD'50
HDf Nt
1 89
0393
1514
0398 0422 0-377 0-433 0 404
P 08 06 05 0-3
64 512 384 32 19-2
48 3-8 27 25 1-3
592 474 35-7 29-5 17 9
05287 06603 0876 1-061 1 748
0208 0263 0370 0-400 0-760
P 08 0-6 05 03
64 512 38 4 32 192
7-6 6 4-4 4 2-1
56 4 452 34 28 17-1
0-555 0692 0-920 1 117 1 83
0 131 0166 0 227 025 0-476
0
Nt HD'50
1-140 0-942 0 57 Average 1 802 1444 1 086 0894 0546
0-236% 0239 0-246 0223 0260 0-241
Average
treatment method described by Beale & Feinstein (1969) and Deutsch & Morton (1957). The same volumes of serum and 2-mercaptoethanol solution (0-2 M) were mixed for 2 h at room temperature; then, this mixture was dialysed and the residual HD50 was measured. The ratio of HD50 after treatment to HD50 before treatment by 2-mercaptoethanol was referred to as the G coefficient.
RESULTS
0~~~~~
Results of one thermodynamic study Results of a thermodynamic study on one serum (A20) are given in Table 2. The enthalpy change was 8 kcal mol -'. The relationship between Nt/HDf x l0-4 and l/HD'50 at 250 and 37° is shown in Fig. 1. Thermodynamic analysis of anti-A allohaemagglutinins The slope of Nt/HDf x 10-4=f (lI/HD'50) is a characteristic of each serum; similar curves referring to antibodies in B phenotype and 0 phenotype sera are shown in Figs 2 and 3 respectively. The association constant was greater at 250 than at 37°. At 370, the average association constant of anti-A(O) was greater than that of anti-A(B). Enthalpy change was more characteristic, the difference between the average enthalpy change of anti-A(B) (- 20-2 + 6 kcal mol -') and anti-A (0) (-8 + 2-4 kcal mol -') was significant (P
