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Independence of anti-allotype antibody titers and “allotype interference” properties of anti-allotype antisera of rabbits
Immunology Letters, 1 (1979) 85-91
© Elsevier/North-HollandBiomedicalPress
I N D E P E N D E N C E O F A N T I - A L L O T Y P E A N T I B O D Y T I T E R S AND " A L L O T Y P E INTERFERENCE" PROPERTIES OF ANTI-ALLOTYPE ANTISERA OF RABBITS Orsola PUGLIESE, O. ACUTO and R. TOSI Laboratory of Cell Biology, C.N.R., Via Romagnosi 18A, 00196 Rome and Institute of General Physiology, University of Rome, Italy
(Accepted 30 May 1979)
1. Summary Allotypic interference, i.e. the capacity of antiallotype antisera to inhibit antigen binding, is not correlated with the anti-allotype antibody titer of antisera. This rules out the possibility that all antiaUotype antibodies are endowed with interfering activity. It is suggested that this activity may be possessed by a minor subpopulation of anti-allotype-associated antibodies, possibly by those directed against variable region determinant(s).
ties can be envisaged: either all anti-allotype antibodies can interfere, or there is a subset of interfering antibodies which might be directed against a special type of allotypic determinant. In order to distinguish between these two alternatives, we compared the anti-allotype and interfering titer of 11 anti-b4 and 11 anti-b9 alloantisera. We reasoned that the interfering activity of antisera would be strictly proportional to their content of antiaUotype antibodies only if the former possibility was true.
3. Materials and methods 2. Introduction 3.1. A n ti-allotype antisera
The phenomenon of allotypic interference was previously described as follows: alloantisera directed against rabbit b-locus allotype are able to inhibit the binding of antibody molecules. The phenomenon is specific, i.e. only the antigen binding to molecules carrying the appropriate allotype is affected. Interference was observed in different systems such as BSA-anti-BSA; DNP-anti-DNP and activation by anti~-galactosidase antibodies of defective gene product and thus seems to have general relevance. The degree of anti-allotype interference was inversely correlated with the affinity of the antibodies towards their antigen, which suggested competition between binding of antigen to the combining site and anti-aUotype antibodies to the antibody molecule [ 1]. As for the mechanism of interference, two possibili-
Anti b-locus allotype antisera were raised by immunization of rabbits of appropriate phenotype either by subcutaneous,intradermal and intramuscular injection of pooled IgG in Freund's complete adjuvant [2], or by intravenous injection of bacterial agglutinates [3]. 3.2. Test o f interfering activity
The ability of anti-aUotype antisera to inhibit the BSA-anti-BSA reaction was tested as previously described [1]. Briefly, 0.4/~g aliquots of purified anti-BSA antibody from either a b4[b4 or a b9/b9 rabbit were incubated with increasing volumes of corresponding anti-allotype antiserum (either anti-b4 or anti-b9). After 30 min incubation at room temperature, [12SI]labelled BSA was added and the mixture incubated for a further 60 min at room temperature, 85
ammonium sulphate at half saturation was added, and the proportion of [12SI]BSA bound was quantitated by a previously described procedure [4].
tional to the amount of antiserum. Different dilutions of the strongest antiserum were included in each test, to provide a standard curve.
3.3. Tests of anti-allotype activity
3.3.3. Passive haemagglutination with antibody.coated SRBC Anti-SRBC was obtained from a b4/b4 and a b9/b9 rabbit after 5 i.v. injections, at weekly intervals of a 5% SRBC suspension. IgG were prepared from the two antisera by ammonium sulphate precipitation, followed by DEAE chromatography. Papain digestion was performed by the method of Porter [9] and Fab fragments were isolated by G-200 gel filtration. Coated-SRBC were prepared by incubating a 2.5% SRBC suspension with 100/ag of Fab-anti-SRBC for 30 min at room temperature with constant shaking. The red cell suspension was then washed 3 times with cold PBS and brought to a final concentration of 1.25%. The anti-allotype antibodies were tested for their ability to agglutinate SRBC coated with Fab of the corresponding allotype in V type microtiter plates, using 50 #1 of antiserum dilution and 50/al of coatedSRBC suspension. Each test was performed in duplicate and reading was done after overnight incubation at 4°C.
3.3.1. Acid elution from insoluble antigen Normal sera from either b4/b4 or b9/b9 homozygous rabbits were insolubilized with ethylchloroformate [5] by a previously described procedure. The insoluble material was washed 4 times with cold PBS. 150/al of anti-allotype antisera (or 150/11 of normal rabbit serum in controls) were mixed with 350/al of the appropriate insolubilized serum suspension. This amount provided a great antigen excess. After 24 h at room temperature, on a revolving wheel, the absorbant was washed 4 times with 0.5 ml of H C I KC1 buffer 0.9 M, pH 1.5, constantly shaking for 5 min at room temperature, and centrifuging for 5 min at 2000 g. This procedure was repeated 3 times and the O.D.28o of each eluted fraction was determined. Preliminary experiments showed that this procedure resulted in maximal recovery. The content of anti-allotype antibodies in each antiserum was estimated by adding the absorption values of the 3 elutions, and then subtracting the O.D.28o obtained in the control (normal rabbit serum instead of anti-allotype antiserum). An extinction coefficient of 15.0 [7] was used for conversion to weight units. 3.3.2. PEG radial immunodiffusion A modification of Mancini Technique was used [8]. Four ml of 3% Special Agar Noble were melted in a boiling waterbath, then cooled to 60°C. Two ml of a 1:500 dilution of normal rabbit serum (either b4/b4 or b9/b9) in Veronal buffer, pH 8.6, were warmed at 55°C and mixed with the Agar. Polyethylene glycol (PEG) 6000 to a f'mal concentration of 2% was then added to the mixture. After shaking, the A g a r antigen-PEG mixture was poured into an appropriate mold, providing 1 mm thickness of the Agar plate. After solidification, wells of 2 mm diameters were punched out of the gel and then each well was filled to the brim with antiserum. Plates were incubated for 19 h at room temperature in a humidified box, then washed and stained with Amido black. Diameters of precipitation circles were shown to be linearly propor86
4. Results
4.1. A llotype interference by an tisera Eleven different anti-b4 antisera and I 1 anti-b9 antisera were tested for their interfering activity. All antisera significantly inhibited the BSA-anti-BSA reaction, when the anti-BSA antibody carried the corresponding allotype. However, the different antisera varied markedly from the point of view of maximum per cent inhibition reached: from 100% to 29% for anti-b4, and from 88.4 to 57.5% for anti-b9. The inhibition curves of the anti-b4 antisera are shown in fig.1. The antisera were classified in a rank order according to the maximum per cent of inhibition reached, regardless of the corresponding concentration. A second classification of antisera was made according to the antiserum concentration required for half the maximal inhibition (tables 1 and 2).
• T8 O L-542
(lag/b9 ) (b61b 6 )
•
A J ' l l T / 3 ( b g / b 9)
A
753
(bglb 9 )
O L-550 s F 3.-5
Ib61b 6 )
S2t A-21 • L'$43 • S8 O L'53B v
tna,,na~ (bS/b 91 (nst ha) (bS/b s) (bS/b5)
(bS/b s)
100"
i 1/
ii
s d
I
/
I
/
2;'-,
/ I----
/ / / / / / /
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;
,~
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Fig.1. Inhibition of the BSA-anti-BSA reaction by a set of anti-b4 alloantisera. The anti-BSA antibody was derived from a b4/b4 rabbit. The b-locus phenotypes of the antiserum donors are indicated next to the antiserum designation.
4.2. Anti-allotype antibodies concentration of antisera The concentration of anti-allotype antibodies is difficult to determine by the classical method of quantitative precipitation analysis, since the antisera are known to produce variable amount of soluble complexes. As an alternative, the following methods were used, on each of the 22 anti-allotype sera. 4.2.1. Acid elution of antibodies from insoluble antigen-antibody complexes This was obtained by reacting the antiserum against normal rabbit serum of appropriate aUotype, rendered insoluble by cross-linking with ethylchloroformate. The antibody eluted from 1 ml of antiserum varied from 8.88 mg to 31 mg.
4.2.2. PEG radial immunodiffusion As compared with the classical Mancini method, two modifications were introduced: first the antigen (i.e. normal rabbit serum of defined allotype) was included in the Agar instead of the antibody, and secondly 2% PEG was added to the Agar in order to maximize immune precipitation. The results were expressed as the mean of two orthogonal diameters of the precipitation circles. 4.2.3. Agglutination titer against SRBC coated with Fab, isolated from anti-SRBC antisera of appropriate phenotype (either b4/b4 or b9/b9) The antisera gave appreciable agglutination titers varying from 1 : 128 to 1:2048. 87
Table 1 Comparison between anti-allotype activity and interfering activity of anti-b4 alloantisera Antiserum (phenotype of recipient)
AH-177-3(b9/b9) T-8(b9/b9) A-21(b5/b9) S-8(b5/b5) F-311-5(b5/b5) L-538(b5/b5) L-542(b6/b6) 753(b9/b9) L-543(bS/b6) L-550(b6/b6) S-21(b9/b9)
Anti-allotype activity
Interfering activity
Acid
elution
PEG radial immunodiffusion
Passive haemagglutination
mg/ml antiserum
Rank order
diameters (mm)
Rank titer order
Rank order
per cent
Rank order
tzl of antiserum
Rank order
8.88 7.46 3.93 3.46 2.53 2.13 1.96 1.77 1.77 1.33 1.33
1 2 3 4 5 6 7 8.5 8.5 10.5 10.5
10.2 10.0 6.7 8.0 7.2 4.0 4.5 5.2 6.5 5.2 5.7
1 2 5 3 4 11 10 8.5 6 8.5 7
1.5 1.5 4 4 4 8.5 11 6 8.5 8.5 8.5
91.8 96.0 29.0 68..2 99.9 97.3 99.4 93.0 35.4 57.3 94.0
7 4 11 8 1 3 2 6 10 9 5
0.08 0.13 0.45 0.55 0.04 1.23 0.03 0.13 0.82 1.50 0.38
3 4.5 7 8 2 10 1 4.5 9 11 6
2048 2048 1024 1024 1024 256 128 512 256 256 "256
Maximum inhibition 50% inhibition
Spearman Rank test statistic analysis Acid elution PEG rad. imm. Passive haem.
0.716 0.823
0.889
-
Acid elution
PEG rad. imm.
Passive haem.
Results from these 3 tests are summarized in tables 1 and 2, where the antisera are classified in rank orders according to immunoglobulin concentrations of eluates, diameters of precipitation circles and passive haemagglutination titers.
.5. Discussion The aim of this work was a classification of the antisera according to their strength in different types of reactions, either classical anti-aUotype reactions or interfering reactions. Such classification was expressed in rank orders, which can then be compared by non-parametric statistical analysis, such as the Spearman rank test, as 88
0.041 0.202 0.075
0.275 0.319 0.284
Maximum 150 Interfering activity
shown in tables 1 and 2. This test yields an r s parameter, that, like the ordinary correlation coefficient, r, can range from - 1 (complete discordance) to +1 (complete concordance), 0 corresponding to a random distribution of the series of observations studied. An r s > 0.602 corresponds (for series of 11 samples, as in our case) to a P < 0.05, an r s > 0.735 to a P < 0.01 [10]. First we can observe that the results obtained with series of anti-b4 antisera are similar to those obtained with anti-b9 antisera. Therefore we will examine them together. As expected the data obtained by the 3 different determinations of anti-allotype activity-acid elution, PEG radial immunodiffusion and passive haemagglutination, are closely related, with r~ ranging from
Table 2 Comparison between anti-aUotype activity and interfering activity of anti-b9 alloantisera Antisera (phenotype of recipient)
F-15-5(b5/b5) F-15-6(b5/b5) 743(b4/b4) E-98-5(b4/b4) K-136-6(b5/b5) D-124-1(b4/b4) F-196-4(b4/b4) S-61(b4/b4) 745(bS/bS) S-40(b4/b4) L-574(b6/b6)
Interfering activity
Anti-aUotype activity Passive haemagglutination
Maximum inhibition 50% inhibition
Acid
elution
PEG radial immunodiffusion
mg/ml antiserum
Rank order
diameters (mm)
Rank titer order
Rank order
per cent
Rank order
tzl of antisera
Rank order
5.68 4.86 3.75 2.86 2.06 1.75 1.07 1.00 0.80 0.73 0.31
1 2 3 4 5 6 7 8 9 10 11
9.7 8.5 6.5 8.0 6.5 5.5 6.7 5.0 4.5 4.7 4.0
1 2 5.5 3 5.5 7 4 8 10 9 11
1 3 3 3 9.5 6 6 6 9.5 9.5 9.5
75.0 61.0 80.0 83.0 70.0 64.5 57.5 75.7 88.4 67.3 66.7
5 10 3 2 6 9 11 4 1 7 8
0.08 0.07 0.21 0.20 0.15 0.34 0.13 0.36 0.44 0.14 0.15
2 1 8 7 5.5 9 3 10 11 4 5.5
2048 1024 1024 1(124 256 512 512 512 256 256 256
Spearman Rank test statistic analysis Acid elution PEG rad. imm. Passive haem.
0.911 0.868
0.841
-
Acid elution
PEG rad. imm.
Passive haem.
0.716 to 0.911, corresponding i n a l l cases except one, to P < 0.01. A complete concordance of the 3 tests was not expected since they are probably measuring somewhat different antibody populations. For instance, IgM antibodies should be comparatively more effective in the haemagglutination reactions than in the Agar-precipitation method and antibodies of either very low or very high avidities would not be detected by the acid elution method. Taken together, these tests give a plausible indication of the antiallotype activity of the antisera. Considering the interfering activity of the different antisera from each inhibition curve, two quantitative parameters were derived, i.e. the maximum inhibition reached, and the amount of antiserum required for inhibition. The latter is more conveniently referred to
0.089 0.105 0.105
0.391 0.600 0.330
Maximum 150 Interfering activity
as the point on the curve corresponding to 50% of the maximum inhibition. When the antisera were classified according to either of these criteria and the corresponding rank orders compared with those expressing general antiallotype activity, no significant correlation was found. As seen in table 1 and 2, only in one case was a value o f P close to 5% significance reached. The conclusion that can be drawn is that the ability of an anti-allotype antiserum to interfere in a n t i g e n - a n t i b o d y reaction is independent of the total amount of anti-allotype antibodies it contains. Still, since the interference is specific (Acuto et al. [ 1]), it is reasonable to think that it is due to anti-allotype antibodies. This contradiction can be solved only if we assume that n o t all anti-allotype antibodies possess 89
the ability to interfere, but only a small minority of them do, so that their concentration in the antisera may not be strictly proportional to the overall antiallotypic activity of the antisera. In fact there is solid evidence that aUotypic determinants are multiple. Experiments by Mage et al. [11 ] have demonstrated that at least 3 Fab fragments of anti-b4 bind to each L-chain of b4 IgG. In addition amino acid sequences show differences at multiple sites [12-14]. If only some of the multiple allotypic determinants are involved in the interference phenomenon, the problem arises of whether they are located in the C- or the V-portion of the chain. Since antigen binding is a property of the V-region, its inhibition by anti-b allotypes antisera would be better explained by assuming the existence of b-locusassociated determinants in the V-region. Although the V L-portion of the molecule has not yet been separated in intact form to detect possible reactivity with antiallotype antisera, there is some evidence of allotyperelated substitutions in V k sequence [ 15-17]. An extensive analysis of 66 k-chain N-terminal sequences [18] has shown that no "allotype-specific" residues exist in this portion of the V-region, i.e. no amino acid residue was found that is unique to all L-chains having a given allotype. However, among the first 21 residues, 6 positions were found to possess 'allotype-associated" residues, i.e. residues occurring only in the chain of a given allotype, though not in all of them. It is conceivable that these allotype-associated structural differences may be immunogeneic, thus contributing to the set of multiple b-determinants. These structural differences involve only a subset of L-chains, and they would probably vary according to the individual rabbit and to the kind ofimmunoglobulin used for allo-immunization. Antibodies against such V-determinants would then be produced with widely different ratios with respect to the antibodies against C-determinants, since the latter are represented on all molecules. This would explain the observed lack of correlation between anti-aUotype activity and interfering activity of antisera. The possibility that there are b-locus determinant(s) in the variable part poses genetic problems for interpretation of b-locus aUotypes. If the 'two genes-one polypeptide chain hypothesis" is correct, the " b " determinants would be controlled by different genes, 90
i.e. the genes for the V L-pOrtiOn and the genes for the CL-portion. If V L and C L genes are separated by a distance similar to that found between V H and V C genes (0.3 map units) [19], the two sets of determinants should be reshuffled relatively rapidly, their association decreasing by a factor 1-0.003 = 0.997 at each generation [20] unless some special mechanism is assumed by which their linkage disequilibrium is maintained. On the other hand, even the "b" determinants which are certainly present on the constant portion require some ad hoc genetic hypothesis. In fact, at least two isotypic forms of k-chains are found in rabbits: K A possessing 5 cystein residues, and K B having two additional cystein residues [ 14,21 ]. Both forms carry as yet serologically indistinguishable b-locus determinants. It is hard to explain why they are never separated by crossing over yielding "mixed" b-phenotypes. An analogous problem is posed by the "a" locus allotypes, which are present on the V-region of most rabbit H-chains and hence should be shared by at least several different loci while behaving as simple Mendelian factors (although a special gene insertion mechanism has been proposed which may explain this fact [22]). The fixation in a given phase (cis or trans) of genetic information controlling different structures can be viewed as an extreme degree of linkage disequilibrium and it may represent, like allelic exclusion, a peculiarity of the immunoglobulin system.
Acknowledgement Thanks are due to Dr. Rose Mage for revising the manuscript and to Dr. Franco Celada for stimulating discussion. O-P. is the recipient of a fellowship from the Instituto Superiore di Sanit~ Viale Regina Elena 299, Rome.
References [1] Acuto, O., Manzo, C., Pane, A., Fontana, S., Zappacosta, S. and Tosi, R. (1975) J. lmmunol. 144, 1430. [2] Dray, S., Young, G. O. and Gerald, L. (1963) J. Immunol. 91,403.
[3] Dubiski, S., Dudziak, Z., Skalba, D. and Dubiska, A. (1959) Immunology 2, 84. [4] Celada, F. (1966) J. Exp. Med. 124, 1. [5] Avrameas, S. and Ternynck, T. (1967) J. Biol. Chem. 242, 1651. [6] Tosi, R. and Landucci Tosi, S. (1973) in: Contemporary Topics in Molecular Immunology (Reisfeld, R. A. and Mandy, W. J. eds.) vol. 2 p. 79, Plenum Press, New York. [7] Little, J. R. and Donahue, H. (1968) in: Methods in Immunology and Immunochemistry, (Williams, C. A. and Chase, M. A. eds.) vol. II p. 171, Academic Press, New York and London. [8] Mancini, G., Carbonara, A. O. and Heremans, J. F. (1965) Immunochemistry 2,235. [9] Porter, R. R. (1959) Biochem. J. 73, 119. [ 10] Snedecor, G. W. and Co chram, W. G. (1969) in: Statistical Methods p. 194. Iowa State University Press, Ames, Iowa. [ 11 ] Mage, R. G., Reisfeld, R. A. and Dray, S. (1966) Immunochemistry 3,299.
[12] Appella, E., Rejnek, J. and Reisfeld, A. (1969) J. Mol. Biol. 41,473. [13] Frangione, B. (1969) FEBS Lett. 3,341. [14] Chen, K. C. S., Kindt, T. J. and Krause, R. M. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1995. [ 15] Waterfield, M. D., Morris, J. E., Hood, L. E. and Todd, C. W. (1973) J. Immunol. 110,227. [ 16] Thunberg, A. L., Lackland, H. and Kindt, T. J. (1973) J. Immunol. 111,1755. [17] Thunberg, A. L. and Kindt, T. J. (1975) Scand. J. Immunol. 4,197. [18] Cannon, E. L., Margolies, M. N., Strosberg, A. D., Chen, F. W., NeweU, J. and Haber, E. (1976) J. Immunol.
117,164. [ 19] Mage, R., Lieberman, R., Potter, M. and Terry, W. D. (1973) in: The Antigens (Sela, M.ed.) vol. 1 p. 299. Academic Press, New York. [20] Bodmer,W. F. (1973) Israel J. Med. Sci. 9, 1503. [21] Rejnek, J., Appella, E., Mage, R. G. and Reisfeld, R. A. (1969) Biochemistry 8, 2712. [22] Capra, D. and Kindt, T. J. (1975) Immunogenetics 1,5.
91
© Elsevier/North-HollandBiomedicalPress
I N D E P E N D E N C E O F A N T I - A L L O T Y P E A N T I B O D Y T I T E R S AND " A L L O T Y P E INTERFERENCE" PROPERTIES OF ANTI-ALLOTYPE ANTISERA OF RABBITS Orsola PUGLIESE, O. ACUTO and R. TOSI Laboratory of Cell Biology, C.N.R., Via Romagnosi 18A, 00196 Rome and Institute of General Physiology, University of Rome, Italy
(Accepted 30 May 1979)
1. Summary Allotypic interference, i.e. the capacity of antiallotype antisera to inhibit antigen binding, is not correlated with the anti-allotype antibody titer of antisera. This rules out the possibility that all antiaUotype antibodies are endowed with interfering activity. It is suggested that this activity may be possessed by a minor subpopulation of anti-allotype-associated antibodies, possibly by those directed against variable region determinant(s).
ties can be envisaged: either all anti-allotype antibodies can interfere, or there is a subset of interfering antibodies which might be directed against a special type of allotypic determinant. In order to distinguish between these two alternatives, we compared the anti-allotype and interfering titer of 11 anti-b4 and 11 anti-b9 alloantisera. We reasoned that the interfering activity of antisera would be strictly proportional to their content of antiaUotype antibodies only if the former possibility was true.
3. Materials and methods 2. Introduction 3.1. A n ti-allotype antisera
The phenomenon of allotypic interference was previously described as follows: alloantisera directed against rabbit b-locus allotype are able to inhibit the binding of antibody molecules. The phenomenon is specific, i.e. only the antigen binding to molecules carrying the appropriate allotype is affected. Interference was observed in different systems such as BSA-anti-BSA; DNP-anti-DNP and activation by anti~-galactosidase antibodies of defective gene product and thus seems to have general relevance. The degree of anti-allotype interference was inversely correlated with the affinity of the antibodies towards their antigen, which suggested competition between binding of antigen to the combining site and anti-aUotype antibodies to the antibody molecule [ 1]. As for the mechanism of interference, two possibili-
Anti b-locus allotype antisera were raised by immunization of rabbits of appropriate phenotype either by subcutaneous,intradermal and intramuscular injection of pooled IgG in Freund's complete adjuvant [2], or by intravenous injection of bacterial agglutinates [3]. 3.2. Test o f interfering activity
The ability of anti-aUotype antisera to inhibit the BSA-anti-BSA reaction was tested as previously described [1]. Briefly, 0.4/~g aliquots of purified anti-BSA antibody from either a b4[b4 or a b9/b9 rabbit were incubated with increasing volumes of corresponding anti-allotype antiserum (either anti-b4 or anti-b9). After 30 min incubation at room temperature, [12SI]labelled BSA was added and the mixture incubated for a further 60 min at room temperature, 85
ammonium sulphate at half saturation was added, and the proportion of [12SI]BSA bound was quantitated by a previously described procedure [4].
tional to the amount of antiserum. Different dilutions of the strongest antiserum were included in each test, to provide a standard curve.
3.3. Tests of anti-allotype activity
3.3.3. Passive haemagglutination with antibody.coated SRBC Anti-SRBC was obtained from a b4/b4 and a b9/b9 rabbit after 5 i.v. injections, at weekly intervals of a 5% SRBC suspension. IgG were prepared from the two antisera by ammonium sulphate precipitation, followed by DEAE chromatography. Papain digestion was performed by the method of Porter [9] and Fab fragments were isolated by G-200 gel filtration. Coated-SRBC were prepared by incubating a 2.5% SRBC suspension with 100/ag of Fab-anti-SRBC for 30 min at room temperature with constant shaking. The red cell suspension was then washed 3 times with cold PBS and brought to a final concentration of 1.25%. The anti-allotype antibodies were tested for their ability to agglutinate SRBC coated with Fab of the corresponding allotype in V type microtiter plates, using 50 #1 of antiserum dilution and 50/al of coatedSRBC suspension. Each test was performed in duplicate and reading was done after overnight incubation at 4°C.
3.3.1. Acid elution from insoluble antigen Normal sera from either b4/b4 or b9/b9 homozygous rabbits were insolubilized with ethylchloroformate [5] by a previously described procedure. The insoluble material was washed 4 times with cold PBS. 150/al of anti-allotype antisera (or 150/11 of normal rabbit serum in controls) were mixed with 350/al of the appropriate insolubilized serum suspension. This amount provided a great antigen excess. After 24 h at room temperature, on a revolving wheel, the absorbant was washed 4 times with 0.5 ml of H C I KC1 buffer 0.9 M, pH 1.5, constantly shaking for 5 min at room temperature, and centrifuging for 5 min at 2000 g. This procedure was repeated 3 times and the O.D.28o of each eluted fraction was determined. Preliminary experiments showed that this procedure resulted in maximal recovery. The content of anti-allotype antibodies in each antiserum was estimated by adding the absorption values of the 3 elutions, and then subtracting the O.D.28o obtained in the control (normal rabbit serum instead of anti-allotype antiserum). An extinction coefficient of 15.0 [7] was used for conversion to weight units. 3.3.2. PEG radial immunodiffusion A modification of Mancini Technique was used [8]. Four ml of 3% Special Agar Noble were melted in a boiling waterbath, then cooled to 60°C. Two ml of a 1:500 dilution of normal rabbit serum (either b4/b4 or b9/b9) in Veronal buffer, pH 8.6, were warmed at 55°C and mixed with the Agar. Polyethylene glycol (PEG) 6000 to a f'mal concentration of 2% was then added to the mixture. After shaking, the A g a r antigen-PEG mixture was poured into an appropriate mold, providing 1 mm thickness of the Agar plate. After solidification, wells of 2 mm diameters were punched out of the gel and then each well was filled to the brim with antiserum. Plates were incubated for 19 h at room temperature in a humidified box, then washed and stained with Amido black. Diameters of precipitation circles were shown to be linearly propor86
4. Results
4.1. A llotype interference by an tisera Eleven different anti-b4 antisera and I 1 anti-b9 antisera were tested for their interfering activity. All antisera significantly inhibited the BSA-anti-BSA reaction, when the anti-BSA antibody carried the corresponding allotype. However, the different antisera varied markedly from the point of view of maximum per cent inhibition reached: from 100% to 29% for anti-b4, and from 88.4 to 57.5% for anti-b9. The inhibition curves of the anti-b4 antisera are shown in fig.1. The antisera were classified in a rank order according to the maximum per cent of inhibition reached, regardless of the corresponding concentration. A second classification of antisera was made according to the antiserum concentration required for half the maximal inhibition (tables 1 and 2).
• T8 O L-542
(lag/b9 ) (b61b 6 )
•
A J ' l l T / 3 ( b g / b 9)
A
753
(bglb 9 )
O L-550 s F 3.-5
Ib61b 6 )
S2t A-21 • L'$43 • S8 O L'53B v
tna,,na~ (bS/b 91 (nst ha) (bS/b s) (bS/b5)
(bS/b s)
100"
i 1/
ii
s d
I
/
I
/
2;'-,
/ I----
/ / / / / / /
/
/
I
/
,/
/
•
/
3
/
/
/ / / fll
/
;
,~
.'1
....
.25'
,J
H
r
.S
~
//
"+
1
~Jl Of
8(~tloe~r'M4'~
Fig.1. Inhibition of the BSA-anti-BSA reaction by a set of anti-b4 alloantisera. The anti-BSA antibody was derived from a b4/b4 rabbit. The b-locus phenotypes of the antiserum donors are indicated next to the antiserum designation.
4.2. Anti-allotype antibodies concentration of antisera The concentration of anti-allotype antibodies is difficult to determine by the classical method of quantitative precipitation analysis, since the antisera are known to produce variable amount of soluble complexes. As an alternative, the following methods were used, on each of the 22 anti-allotype sera. 4.2.1. Acid elution of antibodies from insoluble antigen-antibody complexes This was obtained by reacting the antiserum against normal rabbit serum of appropriate aUotype, rendered insoluble by cross-linking with ethylchloroformate. The antibody eluted from 1 ml of antiserum varied from 8.88 mg to 31 mg.
4.2.2. PEG radial immunodiffusion As compared with the classical Mancini method, two modifications were introduced: first the antigen (i.e. normal rabbit serum of defined allotype) was included in the Agar instead of the antibody, and secondly 2% PEG was added to the Agar in order to maximize immune precipitation. The results were expressed as the mean of two orthogonal diameters of the precipitation circles. 4.2.3. Agglutination titer against SRBC coated with Fab, isolated from anti-SRBC antisera of appropriate phenotype (either b4/b4 or b9/b9) The antisera gave appreciable agglutination titers varying from 1 : 128 to 1:2048. 87
Table 1 Comparison between anti-allotype activity and interfering activity of anti-b4 alloantisera Antiserum (phenotype of recipient)
AH-177-3(b9/b9) T-8(b9/b9) A-21(b5/b9) S-8(b5/b5) F-311-5(b5/b5) L-538(b5/b5) L-542(b6/b6) 753(b9/b9) L-543(bS/b6) L-550(b6/b6) S-21(b9/b9)
Anti-allotype activity
Interfering activity
Acid
elution
PEG radial immunodiffusion
Passive haemagglutination
mg/ml antiserum
Rank order
diameters (mm)
Rank titer order
Rank order
per cent
Rank order
tzl of antiserum
Rank order
8.88 7.46 3.93 3.46 2.53 2.13 1.96 1.77 1.77 1.33 1.33
1 2 3 4 5 6 7 8.5 8.5 10.5 10.5
10.2 10.0 6.7 8.0 7.2 4.0 4.5 5.2 6.5 5.2 5.7
1 2 5 3 4 11 10 8.5 6 8.5 7
1.5 1.5 4 4 4 8.5 11 6 8.5 8.5 8.5
91.8 96.0 29.0 68..2 99.9 97.3 99.4 93.0 35.4 57.3 94.0
7 4 11 8 1 3 2 6 10 9 5
0.08 0.13 0.45 0.55 0.04 1.23 0.03 0.13 0.82 1.50 0.38
3 4.5 7 8 2 10 1 4.5 9 11 6
2048 2048 1024 1024 1024 256 128 512 256 256 "256
Maximum inhibition 50% inhibition
Spearman Rank test statistic analysis Acid elution PEG rad. imm. Passive haem.
0.716 0.823
0.889
-
Acid elution
PEG rad. imm.
Passive haem.
Results from these 3 tests are summarized in tables 1 and 2, where the antisera are classified in rank orders according to immunoglobulin concentrations of eluates, diameters of precipitation circles and passive haemagglutination titers.
.5. Discussion The aim of this work was a classification of the antisera according to their strength in different types of reactions, either classical anti-aUotype reactions or interfering reactions. Such classification was expressed in rank orders, which can then be compared by non-parametric statistical analysis, such as the Spearman rank test, as 88
0.041 0.202 0.075
0.275 0.319 0.284
Maximum 150 Interfering activity
shown in tables 1 and 2. This test yields an r s parameter, that, like the ordinary correlation coefficient, r, can range from - 1 (complete discordance) to +1 (complete concordance), 0 corresponding to a random distribution of the series of observations studied. An r s > 0.602 corresponds (for series of 11 samples, as in our case) to a P < 0.05, an r s > 0.735 to a P < 0.01 [10]. First we can observe that the results obtained with series of anti-b4 antisera are similar to those obtained with anti-b9 antisera. Therefore we will examine them together. As expected the data obtained by the 3 different determinations of anti-allotype activity-acid elution, PEG radial immunodiffusion and passive haemagglutination, are closely related, with r~ ranging from
Table 2 Comparison between anti-aUotype activity and interfering activity of anti-b9 alloantisera Antisera (phenotype of recipient)
F-15-5(b5/b5) F-15-6(b5/b5) 743(b4/b4) E-98-5(b4/b4) K-136-6(b5/b5) D-124-1(b4/b4) F-196-4(b4/b4) S-61(b4/b4) 745(bS/bS) S-40(b4/b4) L-574(b6/b6)
Interfering activity
Anti-aUotype activity Passive haemagglutination
Maximum inhibition 50% inhibition
Acid
elution
PEG radial immunodiffusion
mg/ml antiserum
Rank order
diameters (mm)
Rank titer order
Rank order
per cent
Rank order
tzl of antisera
Rank order
5.68 4.86 3.75 2.86 2.06 1.75 1.07 1.00 0.80 0.73 0.31
1 2 3 4 5 6 7 8 9 10 11
9.7 8.5 6.5 8.0 6.5 5.5 6.7 5.0 4.5 4.7 4.0
1 2 5.5 3 5.5 7 4 8 10 9 11
1 3 3 3 9.5 6 6 6 9.5 9.5 9.5
75.0 61.0 80.0 83.0 70.0 64.5 57.5 75.7 88.4 67.3 66.7
5 10 3 2 6 9 11 4 1 7 8
0.08 0.07 0.21 0.20 0.15 0.34 0.13 0.36 0.44 0.14 0.15
2 1 8 7 5.5 9 3 10 11 4 5.5
2048 1024 1024 1(124 256 512 512 512 256 256 256
Spearman Rank test statistic analysis Acid elution PEG rad. imm. Passive haem.
0.911 0.868
0.841
-
Acid elution
PEG rad. imm.
Passive haem.
0.716 to 0.911, corresponding i n a l l cases except one, to P < 0.01. A complete concordance of the 3 tests was not expected since they are probably measuring somewhat different antibody populations. For instance, IgM antibodies should be comparatively more effective in the haemagglutination reactions than in the Agar-precipitation method and antibodies of either very low or very high avidities would not be detected by the acid elution method. Taken together, these tests give a plausible indication of the antiallotype activity of the antisera. Considering the interfering activity of the different antisera from each inhibition curve, two quantitative parameters were derived, i.e. the maximum inhibition reached, and the amount of antiserum required for inhibition. The latter is more conveniently referred to
0.089 0.105 0.105
0.391 0.600 0.330
Maximum 150 Interfering activity
as the point on the curve corresponding to 50% of the maximum inhibition. When the antisera were classified according to either of these criteria and the corresponding rank orders compared with those expressing general antiallotype activity, no significant correlation was found. As seen in table 1 and 2, only in one case was a value o f P close to 5% significance reached. The conclusion that can be drawn is that the ability of an anti-allotype antiserum to interfere in a n t i g e n - a n t i b o d y reaction is independent of the total amount of anti-allotype antibodies it contains. Still, since the interference is specific (Acuto et al. [ 1]), it is reasonable to think that it is due to anti-allotype antibodies. This contradiction can be solved only if we assume that n o t all anti-allotype antibodies possess 89
the ability to interfere, but only a small minority of them do, so that their concentration in the antisera may not be strictly proportional to the overall antiallotypic activity of the antisera. In fact there is solid evidence that aUotypic determinants are multiple. Experiments by Mage et al. [11 ] have demonstrated that at least 3 Fab fragments of anti-b4 bind to each L-chain of b4 IgG. In addition amino acid sequences show differences at multiple sites [12-14]. If only some of the multiple allotypic determinants are involved in the interference phenomenon, the problem arises of whether they are located in the C- or the V-portion of the chain. Since antigen binding is a property of the V-region, its inhibition by anti-b allotypes antisera would be better explained by assuming the existence of b-locusassociated determinants in the V-region. Although the V L-portion of the molecule has not yet been separated in intact form to detect possible reactivity with antiallotype antisera, there is some evidence of allotyperelated substitutions in V k sequence [ 15-17]. An extensive analysis of 66 k-chain N-terminal sequences [18] has shown that no "allotype-specific" residues exist in this portion of the V-region, i.e. no amino acid residue was found that is unique to all L-chains having a given allotype. However, among the first 21 residues, 6 positions were found to possess 'allotype-associated" residues, i.e. residues occurring only in the chain of a given allotype, though not in all of them. It is conceivable that these allotype-associated structural differences may be immunogeneic, thus contributing to the set of multiple b-determinants. These structural differences involve only a subset of L-chains, and they would probably vary according to the individual rabbit and to the kind ofimmunoglobulin used for allo-immunization. Antibodies against such V-determinants would then be produced with widely different ratios with respect to the antibodies against C-determinants, since the latter are represented on all molecules. This would explain the observed lack of correlation between anti-aUotype activity and interfering activity of antisera. The possibility that there are b-locus determinant(s) in the variable part poses genetic problems for interpretation of b-locus aUotypes. If the 'two genes-one polypeptide chain hypothesis" is correct, the " b " determinants would be controlled by different genes, 90
i.e. the genes for the V L-pOrtiOn and the genes for the CL-portion. If V L and C L genes are separated by a distance similar to that found between V H and V C genes (0.3 map units) [19], the two sets of determinants should be reshuffled relatively rapidly, their association decreasing by a factor 1-0.003 = 0.997 at each generation [20] unless some special mechanism is assumed by which their linkage disequilibrium is maintained. On the other hand, even the "b" determinants which are certainly present on the constant portion require some ad hoc genetic hypothesis. In fact, at least two isotypic forms of k-chains are found in rabbits: K A possessing 5 cystein residues, and K B having two additional cystein residues [ 14,21 ]. Both forms carry as yet serologically indistinguishable b-locus determinants. It is hard to explain why they are never separated by crossing over yielding "mixed" b-phenotypes. An analogous problem is posed by the "a" locus allotypes, which are present on the V-region of most rabbit H-chains and hence should be shared by at least several different loci while behaving as simple Mendelian factors (although a special gene insertion mechanism has been proposed which may explain this fact [22]). The fixation in a given phase (cis or trans) of genetic information controlling different structures can be viewed as an extreme degree of linkage disequilibrium and it may represent, like allelic exclusion, a peculiarity of the immunoglobulin system.
Acknowledgement Thanks are due to Dr. Rose Mage for revising the manuscript and to Dr. Franco Celada for stimulating discussion. O-P. is the recipient of a fellowship from the Instituto Superiore di Sanit~ Viale Regina Elena 299, Rome.
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[3] Dubiski, S., Dudziak, Z., Skalba, D. and Dubiska, A. (1959) Immunology 2, 84. [4] Celada, F. (1966) J. Exp. Med. 124, 1. [5] Avrameas, S. and Ternynck, T. (1967) J. Biol. Chem. 242, 1651. [6] Tosi, R. and Landucci Tosi, S. (1973) in: Contemporary Topics in Molecular Immunology (Reisfeld, R. A. and Mandy, W. J. eds.) vol. 2 p. 79, Plenum Press, New York. [7] Little, J. R. and Donahue, H. (1968) in: Methods in Immunology and Immunochemistry, (Williams, C. A. and Chase, M. A. eds.) vol. II p. 171, Academic Press, New York and London. [8] Mancini, G., Carbonara, A. O. and Heremans, J. F. (1965) Immunochemistry 2,235. [9] Porter, R. R. (1959) Biochem. J. 73, 119. [ 10] Snedecor, G. W. and Co chram, W. G. (1969) in: Statistical Methods p. 194. Iowa State University Press, Ames, Iowa. [ 11 ] Mage, R. G., Reisfeld, R. A. and Dray, S. (1966) Immunochemistry 3,299.
[12] Appella, E., Rejnek, J. and Reisfeld, A. (1969) J. Mol. Biol. 41,473. [13] Frangione, B. (1969) FEBS Lett. 3,341. [14] Chen, K. C. S., Kindt, T. J. and Krause, R. M. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1995. [ 15] Waterfield, M. D., Morris, J. E., Hood, L. E. and Todd, C. W. (1973) J. Immunol. 110,227. [ 16] Thunberg, A. L., Lackland, H. and Kindt, T. J. (1973) J. Immunol. 111,1755. [17] Thunberg, A. L. and Kindt, T. J. (1975) Scand. J. Immunol. 4,197. [18] Cannon, E. L., Margolies, M. N., Strosberg, A. D., Chen, F. W., NeweU, J. and Haber, E. (1976) J. Immunol.
117,164. [ 19] Mage, R., Lieberman, R., Potter, M. and Terry, W. D. (1973) in: The Antigens (Sela, M.ed.) vol. 1 p. 299. Academic Press, New York. [20] Bodmer,W. F. (1973) Israel J. Med. Sci. 9, 1503. [21] Rejnek, J., Appella, E., Mage, R. G. and Reisfeld, R. A. (1969) Biochemistry 8, 2712. [22] Capra, D. and Kindt, T. J. (1975) Immunogenetics 1,5.
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