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The immunoglobulins of the dog—I. Identification of canine immunoglobulins homologous to human IgA and IgM
Immunochemistry. Pergamon Press 1968. Vol. 5, pp. 425-432. Printed in Great Britain
THE IMMUNOGLOBULINS OF THE DOG--I. IDENTIFICATION OF CANINE IMMUNOGLOBULINS HOMOLOGOUS TO HUMAN IgA AND IgM JEAN-PIERREVAERMAN and JosEPH F. HEREMANS Research Department of Internal Pathology, University of Louvain, Cliniques Universitaires Saint-Pierre, 69, rue de Bruxelles, Louvain (Leuven), Belgium
(Received22 December t967; in revisedform 12 February 1968) AbstraetDSelected antisera against human IgA were found to cross-react with a protein from canine serum and milk, which had the physicochemical properties attributed to 'intermediate-sedimenting yx' immunoglobulin. It is concluded that this protein may be called the canine homologue of IgA. Similarly, extensive cross-reactlon was observed between human IgM and a protein from canine serum having properties typical of '~,M'. A RECENT study of the dog's immunoglobulins [1, 2] led to the individualization of six classes of components with antibody activity, which were called '7Sy~,', '7Sy2b', '7S~o', '7S71', 'intermediate Syt', and 'yM', respectively. The homology between canine 'yM' and human IgM was assumed on the basis of their similar behavior during zone electrophoresis, immunoelectrophoresis, chromatography on DEAE-cellulose and Sephadex G-200 columns, and ultracentrifugation in a sucrose density gradient. The canine homologue of human IgA was however not so convincingly identified, but was tentatively assumed to be the 'intermediate Syl'. The designation 'intermediate S ( = sedimentation coefficient)' alludes to the behavior of this component in the ultracentrifuge and upon gel-filtration, and these properties, which suggest a molecular weight higher than that of IgG, are to some extent reminiscent of those of human IgA. A more convincing argument in favor of the homology was drawn from the preferential secretion of the 'intermediate $71' in canine milk, saliva and bronchial mucus, a property shared by h u m a n IgA. Besides, the secretory type of 'intermediate STt' was shown to possess antigenic determinants not found in the 'intermediate $7~' from canine serum, which is again a property typical for IgA[3]. The present work is concerned with the demonstration that antisera specific for human IgA and human IgM are able to recognize antigenic determinants in canine 'intermediate $9,1' and canine 'yM', respectively. MATERIALS AND METHODS
1. Serum and milk serum A pool of canine sera and defibrinated canine plasmas was used as the starting material for the various fractionation studies hereafter described. The normal human serum used was likewise a pool from many individual samples. Samples of human and canine milk were obtained at various times after delivery. Milk serum was prepared by acidification at p H 4-6 by means of acetic acid followed by centrifugation at 90,000 × g for 30 rain, in order to remove fat and precipitated casein. The clear intermediate layer was recovered and neutralised by adding solid tris. 425
426
JEAN-PIERREVAERg~AZ~and JosEPH F. HEREMANS
2. Antisera (a) Anti-human IgA. Raised in 30 rabbits injected either with purified IgA-myeloma proteins, IgA from normal human serum, milk, bronchial fluid, saliva or intestinal secretions, or with isolated a-chain material from an IgA-myeloma protein; after absorption with human IgG or the serum from a patient completely lacking IgA these antisera were shown to be specific for human IgA. (b) Anti.human IgM. Obtained from 17 rabbits immunized with normal human IgM, and made specific for this antigen by means of absorption with human IgG. (e) Anti-canine '7Sys,' -b '7S~,2b'. Prepared by injecting 2 rabbits with heavy chain material from those immunoglobulins of canine serum that were eluted from a DEAE-eeUulose column by means of an 0-01 M sodium phosphate buffer o f p H 8.0 containing 0.03 M NaCI. Transferrin was removed by salting out the y2,.~ components by means of 1.6 M ammonium sulfate. The heavy polypeptide chains from ~,,, were obtained by the classical procedure of reduction and alkylation followed by gel-filtration on Sephadex G-200 in 1 N acetic acid[4]. The antisera were absorbed with light chains and transferrln obtained as by-products from the above-dcscribed fractionation. (d) Anti-canine 7Syso. Obtained from 3 rabbits immunized intravenously with heatkilled Salmonella organisms agglutinated by sera from dogs that had been immunized against the same bacterial antigen. Prior to injection into the rabbits, the bacterial agglutinates were washed carefully with buffered saline. It was found that such rabbit antisera could be made specific for the dog's 7Syso by judicious absorption with canine 7Sys,,b and 18S~M. (e) Anti-canine ~M. Obtained from the sera of the same rabbits injected with Salmonella agglutinates. To this effect their antisera were absorbed with a slow-moving electrophoretic fraction from normal canine serum known to contain only 7S~2,, 7Sysb, and 7S72c globulins. (f) Anti-canine 7S~1. Obtained from a rabbit injected with the fraction of canine serum which was eluted from a DEAE-cellulose column with pH 8.0 sodium phosphate buffers between 0.02 and 0.04 molar concentrations of the eluant. The antiserum was made specific for the aimed antigen by means of absorption with transferrin and 7Sy2,,b. (g) Anti-canine 'int.-Syl' (anti-canine IgA). Obtained by immunizing a rabbit with dialyzed canine saliva. After absorption with canine 7Sy2,.b, this antiserum revealed only one strong precipitin line in canine milk-serum, and one weak line of fl,mobility in canine serum. This antigen was found to have the physicochemical properties attributed to canine 'Intermediate-sedimenting 7~'[1]. (h) Anti-canine albumin. Prepared by injecting a rabbit with the albumin fraction isolated by preparative eleetrophoresis of canine serum on a Pevikon ® block. (i) Anti-canine transferrin. Obtained by absorbing the serum from one of the rabbits used to prepare the anti-7Sy,,, b, by means of 7Sy2,. b. (j) Anti-canine as-macroglobulin. Obtained by injecting 2 rabbits with the first peak eluted from a Sephadex G-200 column to which had been applied a sample of canine serum proteins precipitated by 1.6-2.4 M ammonium sulfate at pH 6.8. This antiserum was absorbed with euglobulins precipitated from diluted canine serum by dialysis against 0.01 M phosphate buffer of pH 6.0.
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(k) Anti-horse ferritin. Obtained from a rabbit immunized with a commercial sample of crystalline horse spleen ferrritin. 3. Fractionation methods (a) Sephadex G-200 gelfiltration. A 50 ml sample of canine serum was applied on a 7 × 90 cm column of Sephadex G-200 and eluted with 2 ~o NaC1 buffered at p H 7.5 with 0.01 M sodium phosphate and containing 0"1~o sodium azide. The eluates were collected in fractions of 15 ml, whose extinction at 280 m/z was recorded and whose composition was analyzed in Ouchterlony plates, using the various antisera described. (b) Density gradient ultracentrifugation. This procedure was carried out in a Beckman Model L ultracentrifuge using the SW-39 rotor. The tubes received 4.5 ml of a linear-gradient solution of 4 5 ~ o - 1 0 ~ sucrose in saline buffered at p H 8.4 (9 vol. 0"9~o NaCI -}- 1 vol. 0-2 M sodium borate buffer). On top of this gradient were layered, successively, (1) 0.2 ml of a mixture of 1 vol. canine serum and 2 vol. buffered 1 0 ~ sucrose; (2) 0.2 ml of a mixture of 2 vol. canine serum and 1 vol. buffered 1 0 ~ sucrose; and (3) 0.2 ml of canine serum. The preparations were centrifuged for 17 hours at 110,000 × g, at 4°C. The rotor was stabilized by hand at the start as well as the end of the run. Each tube was punctured at the bottom and 27 two-drop fractions were collected. From each fraction a 50 tzl aliquot was mixed with 1 • 3 ml of saline and its extinction measured at 280 m/z. The remainder of each fraction was tested against the various specific antisera by means of diffusion in Ouchterlony plates. (c) DEAE-cellulose chromatography. DEAE-cellulose (Macherey & Nagel, Dtiren, Germany; 0.67 m-equiv./g) was washed successively with 0.25 M N a O H , distilled water, 0.1 M HCI, distilled water, and 0.01 M N a , H P O , , prior to being packed in a 5 × 30 cm column, to which 60 ml of canine serum dialysed against the same equilibrating buffer was applied. The sample was protected by overlaying it with 50 ml of the same buffer. Elution was started with a gradient of increasing ionic strength and decreasing p H obtained by a serial arrangement of two l-liter flasks containing respectively 0.01 M NazHPO4 and 0.1 M NaH,PO4. When the 0.1 M N a H 2 P O , flask was emptied, its contents were replaced with 1 liter of 0.4 M Nail2 PO,. Fractions were collected in 15 ml aliquots and analyzed as previously described. (d) Preparative zone electrophoresis. A 27 ml sample of canine serum was concentrated threefold by pressure dialysis against 0.033 M sodium barbital-HCl buffer o f p H 8.6, and applied to a 50 × 30 × 1 cm Pevikon block prepared with the same buffer. Electrophoresis was carried out for 24 hours at room temperature, under a potential gradient of 6 V.cm-1, after which 23 fractions were cut out and eluted by means of 25 ml of saline. After centrifugation, the protein contents of the fractions were measured on 0- 1 ml aliquots, by means of the Lowry method, and each fraction was tested against the different antisera.
4. Antigenic analyses Standard micromethods were used for the antigenic analyses in Ouchterlony plates and immunoelectrophoresis. Antigens were quantitated by means of the single radial immunodiffusion method of Mancini et al. [5].
428
JEAH-PXElU~VAERMANand JOSEPH F.
HEREMANS
RESULTS
I. Antigenic analyses of whole canine serum and milk serum (a) Studies with antisera directed against human IgA. When canine serum was analysed by immunoeleetrophoresis, a single precipltin line, similar in shape and position to the IgA line of human serum, though much weaker than the latter, was called forth by four (~317-8-9, ~ HA 4-5, ~ / H A 3 and ~¢ L 341) ofthe thirty antisera directed against human IgA (Fig. 1 A). Three of these antisera had been raised against purified c~-chains and the fourth against purified salivary IgA. A similar precipitin llne was obtained with canine milk (Fig. 1 A). Absorption of these antisera with canine serum or milk did not abolish their reactivity towards human serum- or milk-IgA but removed all antibodies against the corresponding canine antigens (Fig. 1 B). All reactivity with human as well as canine antigens disappeared after absorption with IgA isolated from human serum or milk. When human and canine serum or milk proteins were compared side by side in Ouchterlony plates, with either of the four above cited anti-human IgA antisera in the central wells (Fig. 2), the lines given by the human antigens formed conspicuous spurs over those of their canine counterparts. Cross-absorption experiments reproduced the results already described for the immunoelectrophoresis assays. Two radial immunodiffusion plates were set up, one containing 1.0 ml of antihuman IgA ( ~ L 317-8-9), 0. I ml of anti-ferritin and 3.4 ml of buffered saline, and the other containing the same mixture of antisera but with 3-4 ml of canine serum as a substitute for the buffer. It had previously been verified that this amount of canine antigen would suffice to absorb all cross-reacting antibodies out of the antiserum. The anti-ferritin antiserum was added in order to provide an internal marker allowing to verify that the concentration of the total antibody mixture employed in both plates was the same. In each plate were punched out, in triplicate, two series of five wells, one series receiving a set of dilutions of human serum, and the other series a set of dilutions of horse spleen ferritin. After completion of the diffusion (4 days at 37°C), the precipitin areas from each triplicate series were used to construct linear regression lines. The slope of the ferritin : antiferridn system was the same in both plates, indicating that no significant methodological error could be invoked to explain the 15 per cent rise of the slope of the IgA: anti-IgA calibration curve after absorption. (b) Studies with antiserum against canine salivary 'int.-S71'. When assayed by immunoelectrophoresis, this antiserum developed a single strong llne of t32 mobility with canine milk-serum, and a similar, though much weaker line with canine serum. No reaction was obtained with human serum. When canine serum or milk serum was tested against this antiserum and the antiserum specific for human IgA ( # L 317-8-9), diffusing from adjoining wells, complete fusion of the precipitin lines was observed (Fig. 3). (c) Studies with antisera directed against human IgM. The two antisera against human IgM developed single, clear, precipitin lines when used in immunoelectrophoretic analyses of canine serum. These lines were similar to human IgM lines in shape and position (Fig. 4), and failed to appear when the antisera had been absorbed by human serum.
FIG. 1. Immunoelectrophoretic analyses of h u m a n (h) and canine (c) serum and milk proteins. Antiserum used for the top half of the figure (A) was rabbit # 317-8-9 a n t i - h u m a n - I g A ; for the bottom half (B), the same antiserum, absorbed with canine serum.
FIG. 2. Comparative Ouchterlony analyses of h u m a n and canine serum and milk proteins. Centre well (A): rabbit ~ 317-8-9 anti-human-IgA. Peripheral wells: 1 and 5, canine serum; 2 and 6, canine milk serum; 3 and 7, h u m a n milk serum; 4 and 8, h u m a n serum. (Facing p. 428)
Fro. 3. Comparative Ouchterlony analyses of rabbit ~ 317-8-9 anti-human-IgA and rabbit anti-canine-int.-S71 antisera. Top wells: left, canine milk serum (C.M.); right, canine serum (C.S.). Bottom wells: 1, rabbit anti-canine-int.-Syl;fl, rabbit ~ 317-8-9 anti-human-IgA,
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FIo. 4. Immunoelectrophoretic analyses of human (H.S.) and canine (C.S.) serum, as developed with rabbit anti-human-IgM.
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FIG. 5. Comparative Ouchterlony analyses of human (H.S.) and canine (C.S.) serum, tested against rabbit anti-human-IgM.
The Immunoglobulins of the D o g - - I
429
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Fzo. 6. Sephadex G-200 gel-filtration pattern of canine serum. The localization of the various ~'nmunoglobulins, a2M, albumin and transferrin was revealed by means of appropriate specific antisera; ~,A indicates reaction with both rabbit # 317-8-9 anti-human-IgA and rabbit anti-canine-int.-Syt; 7M indicates reaction with both anti-canine-~,M and antihuman-IgM.
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FzG. 7. Sucrose density gradient ultracentrifugal pattern of canine serum. Same indications as in Fig. 7.
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The Immunoglobulins of the Dog--I
431
In Ouchterlony plates a reaction of partial antigenic identity was obtained when human and canine serum were tested side by side by means of the anti-human IgM antisera (Fig. 5). Quantitative immunodiffusion plates were set up with one of these antisera ( ~ L 231), following the experimental model described for IgA. The slope of the IgM calibration curve was found to have risen by 28 per cent as a result of the absorption of antibodies cross-reacting with canine IgM.
2. Antigenic analyses of different canine serum fractions The distribution of canine proteins reactive with antisera against human IgA and IgM among the different fractions obtained by filtration through Sephadex G-200, density gradient ultracentrifugation, chromatography on DEAE-cellulose, and electrophoresis on Pevikon are illustrated in Figs. 6-9. The distributions of canine proteins reacting with anti-canine int. S~'x and anti-canine yM corresponded exactly to the distributions of canine proteins reacting with anti-human IgA and anti-human IgM, respectively. For comparison, the distribution of canine a2-macroglobulin, transferrin, albumin, and the immunoglobulins 72,, b, 9',o, and 7S~,x is also indicated in these diagrams. DISCUSSION The ultimate criterion for establishing homologies between proteins of different animal species will certainly have to be derived from comparative studies on their amino acid sequences. Pending the achievement of such a goal, the best indirect approach to the problem may probably be sought in criteria based on antigenic properties, since the latter often constitute sensitive and reliable indices of relevant features pertaining to primary and secondary macromolecular structures. Gross data concerning the amino acid or carbohydrate content, or physical properties such as electrophoretic mobility or sedimentation in the ultracentrifuge represent a third order of criteria for comparison. The recent example of the misidentification of the horse's IgT as being the equine version of human IgA, sufficiently stresses the subordinate quality of the last-mentioned class of arguments[6]. In their recent study on the dog's immunoglobulins, Johnson and Vaughan[1] suggest that the protein called by them 'intermediate-Syl', may be the canine counterpart to human IgA, on the basis of arguments drawn from its behavior in the ultracentrifuge and during gel-filtration, its fast electrophoretic mobility, its preferential excretion in milk, saliva and bronchial mucus, as well as from the presence, in secretory 'int.-S71', of antigenic determinants not found in the 'int.-Syl' from the serum. The present study shows that a protein, present in dog's serum and milk, and endowed with the physicochemical properties described by Johnson and Vaughan[ 1] as typical for 'int.-Sy,', does indeed cross-react with certain antisera specific for human IgA. No other canine protein was found to display such an immunological cross-reaction with anti-human IgA. It therefore seems justified henceforth to use the name of 'IgA' or '~A' in place of the designation 'int.-S~,~', and this new terminology has been used in constructing the diagrams of Figs. 6-9. It has likewise been shown in the present study that the canine protein having the molecular size, electrophoretic mobility and immunoelectrophoretic properties typical for human IgM, is able to cross-react with antisera specific for this protein,
432
J~AN-PmmuZV A S ~
and JOSEPHF. I-lzRz~s
and that this property is not shared by any other canine protein. Hence the homology between canine '7M' and human IgM seems open to no doubt. Absorption of the anti-human IgM antiserum by addition of canine serum resulted in the removal of about 28 per cent of the antibodies reacting with the homologous antigen. In a similar experiment with anti-human IgA, the loss of antibodies by absorption with canine serum was about 15 per cent. These data suggest that the antigenic relationship between human and canine IgM is more intimate than the antigenic relationship between human and canine IgA. This conclusion is supported by the finding that only 4 out of 30 antisera against human IgA were able to react with canine IgA, whereas 13 out of 17 antisera against human IgM cross-reacted with the homologous antigen from the dog. Since completion of these experiments, exchange of reagents has confirmed the congruence of our classification of canine immunoglobulins with that proposed by Drs. Johnson and Vaughan. Acknowledgement--Financial support from the European Community for Coal and Steel, 6240-11/035 and from the Fonds de la Recherche Scientifique M~dicale, Brussels (Grant # 803) is gratefully acknowledged.
REFERENCES 1. JoHNSO~J.S. and VAUO~ANJ.H., jr. Imraun. 98, 923 (1967). 2. JOHNSONJ. S., VAUOHANJ. H. and Swisher S. N., jr. Immun. 95~ 935 (1967). 3. TOMASIT. B., TAN E. M., SOLOMONA. and PRENDEROASTR. A., jr. exp. Med. 121~ I01 (1965). 4. FLEISCH~NJ. B., PORTFRR. R. and PRESSE. M., BiochemJ. 88, 220 (1963). 5. M~NCI~ G., ~ o N . ~ a ~ A. O. and HEm~tANSJ. F., Immunochemistcy2~ 235 (1965). 6. WEIRR., GrVOLD. and PORa'ERR. R., Nature, Lond. 212, 205 (1966).
THE IMMUNOGLOBULINS OF THE DOG--I. IDENTIFICATION OF CANINE IMMUNOGLOBULINS HOMOLOGOUS TO HUMAN IgA AND IgM JEAN-PIERREVAERMAN and JosEPH F. HEREMANS Research Department of Internal Pathology, University of Louvain, Cliniques Universitaires Saint-Pierre, 69, rue de Bruxelles, Louvain (Leuven), Belgium
(Received22 December t967; in revisedform 12 February 1968) AbstraetDSelected antisera against human IgA were found to cross-react with a protein from canine serum and milk, which had the physicochemical properties attributed to 'intermediate-sedimenting yx' immunoglobulin. It is concluded that this protein may be called the canine homologue of IgA. Similarly, extensive cross-reactlon was observed between human IgM and a protein from canine serum having properties typical of '~,M'. A RECENT study of the dog's immunoglobulins [1, 2] led to the individualization of six classes of components with antibody activity, which were called '7Sy~,', '7Sy2b', '7S~o', '7S71', 'intermediate Syt', and 'yM', respectively. The homology between canine 'yM' and human IgM was assumed on the basis of their similar behavior during zone electrophoresis, immunoelectrophoresis, chromatography on DEAE-cellulose and Sephadex G-200 columns, and ultracentrifugation in a sucrose density gradient. The canine homologue of human IgA was however not so convincingly identified, but was tentatively assumed to be the 'intermediate Syl'. The designation 'intermediate S ( = sedimentation coefficient)' alludes to the behavior of this component in the ultracentrifuge and upon gel-filtration, and these properties, which suggest a molecular weight higher than that of IgG, are to some extent reminiscent of those of human IgA. A more convincing argument in favor of the homology was drawn from the preferential secretion of the 'intermediate $71' in canine milk, saliva and bronchial mucus, a property shared by h u m a n IgA. Besides, the secretory type of 'intermediate STt' was shown to possess antigenic determinants not found in the 'intermediate $7~' from canine serum, which is again a property typical for IgA[3]. The present work is concerned with the demonstration that antisera specific for human IgA and human IgM are able to recognize antigenic determinants in canine 'intermediate $9,1' and canine 'yM', respectively. MATERIALS AND METHODS
1. Serum and milk serum A pool of canine sera and defibrinated canine plasmas was used as the starting material for the various fractionation studies hereafter described. The normal human serum used was likewise a pool from many individual samples. Samples of human and canine milk were obtained at various times after delivery. Milk serum was prepared by acidification at p H 4-6 by means of acetic acid followed by centrifugation at 90,000 × g for 30 rain, in order to remove fat and precipitated casein. The clear intermediate layer was recovered and neutralised by adding solid tris. 425
426
JEAN-PIERREVAERg~AZ~and JosEPH F. HEREMANS
2. Antisera (a) Anti-human IgA. Raised in 30 rabbits injected either with purified IgA-myeloma proteins, IgA from normal human serum, milk, bronchial fluid, saliva or intestinal secretions, or with isolated a-chain material from an IgA-myeloma protein; after absorption with human IgG or the serum from a patient completely lacking IgA these antisera were shown to be specific for human IgA. (b) Anti.human IgM. Obtained from 17 rabbits immunized with normal human IgM, and made specific for this antigen by means of absorption with human IgG. (e) Anti-canine '7Sys,' -b '7S~,2b'. Prepared by injecting 2 rabbits with heavy chain material from those immunoglobulins of canine serum that were eluted from a DEAE-eeUulose column by means of an 0-01 M sodium phosphate buffer o f p H 8.0 containing 0.03 M NaCI. Transferrin was removed by salting out the y2,.~ components by means of 1.6 M ammonium sulfate. The heavy polypeptide chains from ~,,, were obtained by the classical procedure of reduction and alkylation followed by gel-filtration on Sephadex G-200 in 1 N acetic acid[4]. The antisera were absorbed with light chains and transferrln obtained as by-products from the above-dcscribed fractionation. (d) Anti-canine 7Syso. Obtained from 3 rabbits immunized intravenously with heatkilled Salmonella organisms agglutinated by sera from dogs that had been immunized against the same bacterial antigen. Prior to injection into the rabbits, the bacterial agglutinates were washed carefully with buffered saline. It was found that such rabbit antisera could be made specific for the dog's 7Syso by judicious absorption with canine 7Sys,,b and 18S~M. (e) Anti-canine ~M. Obtained from the sera of the same rabbits injected with Salmonella agglutinates. To this effect their antisera were absorbed with a slow-moving electrophoretic fraction from normal canine serum known to contain only 7S~2,, 7Sysb, and 7S72c globulins. (f) Anti-canine 7S~1. Obtained from a rabbit injected with the fraction of canine serum which was eluted from a DEAE-cellulose column with pH 8.0 sodium phosphate buffers between 0.02 and 0.04 molar concentrations of the eluant. The antiserum was made specific for the aimed antigen by means of absorption with transferrin and 7Sy2,,b. (g) Anti-canine 'int.-Syl' (anti-canine IgA). Obtained by immunizing a rabbit with dialyzed canine saliva. After absorption with canine 7Sy2,.b, this antiserum revealed only one strong precipitin line in canine milk-serum, and one weak line of fl,mobility in canine serum. This antigen was found to have the physicochemical properties attributed to canine 'Intermediate-sedimenting 7~'[1]. (h) Anti-canine albumin. Prepared by injecting a rabbit with the albumin fraction isolated by preparative eleetrophoresis of canine serum on a Pevikon ® block. (i) Anti-canine transferrin. Obtained by absorbing the serum from one of the rabbits used to prepare the anti-7Sy,,, b, by means of 7Sy2,. b. (j) Anti-canine as-macroglobulin. Obtained by injecting 2 rabbits with the first peak eluted from a Sephadex G-200 column to which had been applied a sample of canine serum proteins precipitated by 1.6-2.4 M ammonium sulfate at pH 6.8. This antiserum was absorbed with euglobulins precipitated from diluted canine serum by dialysis against 0.01 M phosphate buffer of pH 6.0.
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(k) Anti-horse ferritin. Obtained from a rabbit immunized with a commercial sample of crystalline horse spleen ferrritin. 3. Fractionation methods (a) Sephadex G-200 gelfiltration. A 50 ml sample of canine serum was applied on a 7 × 90 cm column of Sephadex G-200 and eluted with 2 ~o NaC1 buffered at p H 7.5 with 0.01 M sodium phosphate and containing 0"1~o sodium azide. The eluates were collected in fractions of 15 ml, whose extinction at 280 m/z was recorded and whose composition was analyzed in Ouchterlony plates, using the various antisera described. (b) Density gradient ultracentrifugation. This procedure was carried out in a Beckman Model L ultracentrifuge using the SW-39 rotor. The tubes received 4.5 ml of a linear-gradient solution of 4 5 ~ o - 1 0 ~ sucrose in saline buffered at p H 8.4 (9 vol. 0"9~o NaCI -}- 1 vol. 0-2 M sodium borate buffer). On top of this gradient were layered, successively, (1) 0.2 ml of a mixture of 1 vol. canine serum and 2 vol. buffered 1 0 ~ sucrose; (2) 0.2 ml of a mixture of 2 vol. canine serum and 1 vol. buffered 1 0 ~ sucrose; and (3) 0.2 ml of canine serum. The preparations were centrifuged for 17 hours at 110,000 × g, at 4°C. The rotor was stabilized by hand at the start as well as the end of the run. Each tube was punctured at the bottom and 27 two-drop fractions were collected. From each fraction a 50 tzl aliquot was mixed with 1 • 3 ml of saline and its extinction measured at 280 m/z. The remainder of each fraction was tested against the various specific antisera by means of diffusion in Ouchterlony plates. (c) DEAE-cellulose chromatography. DEAE-cellulose (Macherey & Nagel, Dtiren, Germany; 0.67 m-equiv./g) was washed successively with 0.25 M N a O H , distilled water, 0.1 M HCI, distilled water, and 0.01 M N a , H P O , , prior to being packed in a 5 × 30 cm column, to which 60 ml of canine serum dialysed against the same equilibrating buffer was applied. The sample was protected by overlaying it with 50 ml of the same buffer. Elution was started with a gradient of increasing ionic strength and decreasing p H obtained by a serial arrangement of two l-liter flasks containing respectively 0.01 M NazHPO4 and 0.1 M NaH,PO4. When the 0.1 M N a H 2 P O , flask was emptied, its contents were replaced with 1 liter of 0.4 M Nail2 PO,. Fractions were collected in 15 ml aliquots and analyzed as previously described. (d) Preparative zone electrophoresis. A 27 ml sample of canine serum was concentrated threefold by pressure dialysis against 0.033 M sodium barbital-HCl buffer o f p H 8.6, and applied to a 50 × 30 × 1 cm Pevikon block prepared with the same buffer. Electrophoresis was carried out for 24 hours at room temperature, under a potential gradient of 6 V.cm-1, after which 23 fractions were cut out and eluted by means of 25 ml of saline. After centrifugation, the protein contents of the fractions were measured on 0- 1 ml aliquots, by means of the Lowry method, and each fraction was tested against the different antisera.
4. Antigenic analyses Standard micromethods were used for the antigenic analyses in Ouchterlony plates and immunoelectrophoresis. Antigens were quantitated by means of the single radial immunodiffusion method of Mancini et al. [5].
428
JEAH-PXElU~VAERMANand JOSEPH F.
HEREMANS
RESULTS
I. Antigenic analyses of whole canine serum and milk serum (a) Studies with antisera directed against human IgA. When canine serum was analysed by immunoeleetrophoresis, a single precipltin line, similar in shape and position to the IgA line of human serum, though much weaker than the latter, was called forth by four (~317-8-9, ~ HA 4-5, ~ / H A 3 and ~¢ L 341) ofthe thirty antisera directed against human IgA (Fig. 1 A). Three of these antisera had been raised against purified c~-chains and the fourth against purified salivary IgA. A similar precipitin llne was obtained with canine milk (Fig. 1 A). Absorption of these antisera with canine serum or milk did not abolish their reactivity towards human serum- or milk-IgA but removed all antibodies against the corresponding canine antigens (Fig. 1 B). All reactivity with human as well as canine antigens disappeared after absorption with IgA isolated from human serum or milk. When human and canine serum or milk proteins were compared side by side in Ouchterlony plates, with either of the four above cited anti-human IgA antisera in the central wells (Fig. 2), the lines given by the human antigens formed conspicuous spurs over those of their canine counterparts. Cross-absorption experiments reproduced the results already described for the immunoelectrophoresis assays. Two radial immunodiffusion plates were set up, one containing 1.0 ml of antihuman IgA ( ~ L 317-8-9), 0. I ml of anti-ferritin and 3.4 ml of buffered saline, and the other containing the same mixture of antisera but with 3-4 ml of canine serum as a substitute for the buffer. It had previously been verified that this amount of canine antigen would suffice to absorb all cross-reacting antibodies out of the antiserum. The anti-ferritin antiserum was added in order to provide an internal marker allowing to verify that the concentration of the total antibody mixture employed in both plates was the same. In each plate were punched out, in triplicate, two series of five wells, one series receiving a set of dilutions of human serum, and the other series a set of dilutions of horse spleen ferritin. After completion of the diffusion (4 days at 37°C), the precipitin areas from each triplicate series were used to construct linear regression lines. The slope of the ferritin : antiferridn system was the same in both plates, indicating that no significant methodological error could be invoked to explain the 15 per cent rise of the slope of the IgA: anti-IgA calibration curve after absorption. (b) Studies with antiserum against canine salivary 'int.-S71'. When assayed by immunoelectrophoresis, this antiserum developed a single strong llne of t32 mobility with canine milk-serum, and a similar, though much weaker line with canine serum. No reaction was obtained with human serum. When canine serum or milk serum was tested against this antiserum and the antiserum specific for human IgA ( # L 317-8-9), diffusing from adjoining wells, complete fusion of the precipitin lines was observed (Fig. 3). (c) Studies with antisera directed against human IgM. The two antisera against human IgM developed single, clear, precipitin lines when used in immunoelectrophoretic analyses of canine serum. These lines were similar to human IgM lines in shape and position (Fig. 4), and failed to appear when the antisera had been absorbed by human serum.
FIG. 1. Immunoelectrophoretic analyses of h u m a n (h) and canine (c) serum and milk proteins. Antiserum used for the top half of the figure (A) was rabbit # 317-8-9 a n t i - h u m a n - I g A ; for the bottom half (B), the same antiserum, absorbed with canine serum.
FIG. 2. Comparative Ouchterlony analyses of h u m a n and canine serum and milk proteins. Centre well (A): rabbit ~ 317-8-9 anti-human-IgA. Peripheral wells: 1 and 5, canine serum; 2 and 6, canine milk serum; 3 and 7, h u m a n milk serum; 4 and 8, h u m a n serum. (Facing p. 428)
Fro. 3. Comparative Ouchterlony analyses of rabbit ~ 317-8-9 anti-human-IgA and rabbit anti-canine-int.-S71 antisera. Top wells: left, canine milk serum (C.M.); right, canine serum (C.S.). Bottom wells: 1, rabbit anti-canine-int.-Syl;fl, rabbit ~ 317-8-9 anti-human-IgA,
bt.S.
C.S.
FIo. 4. Immunoelectrophoretic analyses of human (H.S.) and canine (C.S.) serum, as developed with rabbit anti-human-IgM.
Q
FIG. 5. Comparative Ouchterlony analyses of human (H.S.) and canine (C.S.) serum, tested against rabbit anti-human-IgM.
The Immunoglobulins of the D o g - - I
429
O.D. 280 mp
Z.O
1.0
60
~ZM
1.A 7zc
~'Za,b 7S~'1
Fzo. 6. Sephadex G-200 gel-filtration pattern of canine serum. The localization of the various ~'nmunoglobulins, a2M, albumin and transferrin was revealed by means of appropriate specific antisera; ~,A indicates reaction with both rabbit # 317-8-9 anti-human-IgA and rabbit anti-canine-int.-Syt; 7M indicates reaction with both anti-canine-~,M and antihuman-IgM.
O.D.280rap
?
0.2
Oft
21
Fraction n° ~-M ~ZM ~2c ~,2a,b -7S ~-1 • Transferrin Albumin
FzG. 7. Sucrose density gradient ultracentrifugal pattern of canine serum. Same indications as in Fig. 7.
430
JBAN-Pmm~ VAsa~ua~ and JossPH F. I-Izm~M~s
O.D.ZlOmp
1.0
0"5
,•,Fraction R °
~{2M
t^
~za,b
7s~, v
FIo. 8. DEAE-cellulose chromatographic pattern of canine serum. Same indications as in Fig. 7. O.D.s~omp
0.6
I
0.4
O-Z
~
tion
?M ~2M 7A
v
?zc ?za,b F
A
v
v
7s?~ Transf.
Fro. 9. Pevikon block zonal electrophoretic pattern of canine serum. Same indications as in Fig. 7.
The Immunoglobulins of the Dog--I
431
In Ouchterlony plates a reaction of partial antigenic identity was obtained when human and canine serum were tested side by side by means of the anti-human IgM antisera (Fig. 5). Quantitative immunodiffusion plates were set up with one of these antisera ( ~ L 231), following the experimental model described for IgA. The slope of the IgM calibration curve was found to have risen by 28 per cent as a result of the absorption of antibodies cross-reacting with canine IgM.
2. Antigenic analyses of different canine serum fractions The distribution of canine proteins reactive with antisera against human IgA and IgM among the different fractions obtained by filtration through Sephadex G-200, density gradient ultracentrifugation, chromatography on DEAE-cellulose, and electrophoresis on Pevikon are illustrated in Figs. 6-9. The distributions of canine proteins reacting with anti-canine int. S~'x and anti-canine yM corresponded exactly to the distributions of canine proteins reacting with anti-human IgA and anti-human IgM, respectively. For comparison, the distribution of canine a2-macroglobulin, transferrin, albumin, and the immunoglobulins 72,, b, 9',o, and 7S~,x is also indicated in these diagrams. DISCUSSION The ultimate criterion for establishing homologies between proteins of different animal species will certainly have to be derived from comparative studies on their amino acid sequences. Pending the achievement of such a goal, the best indirect approach to the problem may probably be sought in criteria based on antigenic properties, since the latter often constitute sensitive and reliable indices of relevant features pertaining to primary and secondary macromolecular structures. Gross data concerning the amino acid or carbohydrate content, or physical properties such as electrophoretic mobility or sedimentation in the ultracentrifuge represent a third order of criteria for comparison. The recent example of the misidentification of the horse's IgT as being the equine version of human IgA, sufficiently stresses the subordinate quality of the last-mentioned class of arguments[6]. In their recent study on the dog's immunoglobulins, Johnson and Vaughan[1] suggest that the protein called by them 'intermediate-Syl', may be the canine counterpart to human IgA, on the basis of arguments drawn from its behavior in the ultracentrifuge and during gel-filtration, its fast electrophoretic mobility, its preferential excretion in milk, saliva and bronchial mucus, as well as from the presence, in secretory 'int.-S71', of antigenic determinants not found in the 'int.-Syl' from the serum. The present study shows that a protein, present in dog's serum and milk, and endowed with the physicochemical properties described by Johnson and Vaughan[ 1] as typical for 'int.-Sy,', does indeed cross-react with certain antisera specific for human IgA. No other canine protein was found to display such an immunological cross-reaction with anti-human IgA. It therefore seems justified henceforth to use the name of 'IgA' or '~A' in place of the designation 'int.-S~,~', and this new terminology has been used in constructing the diagrams of Figs. 6-9. It has likewise been shown in the present study that the canine protein having the molecular size, electrophoretic mobility and immunoelectrophoretic properties typical for human IgM, is able to cross-react with antisera specific for this protein,
432
J~AN-PmmuZV A S ~
and JOSEPHF. I-lzRz~s
and that this property is not shared by any other canine protein. Hence the homology between canine '7M' and human IgM seems open to no doubt. Absorption of the anti-human IgM antiserum by addition of canine serum resulted in the removal of about 28 per cent of the antibodies reacting with the homologous antigen. In a similar experiment with anti-human IgA, the loss of antibodies by absorption with canine serum was about 15 per cent. These data suggest that the antigenic relationship between human and canine IgM is more intimate than the antigenic relationship between human and canine IgA. This conclusion is supported by the finding that only 4 out of 30 antisera against human IgA were able to react with canine IgA, whereas 13 out of 17 antisera against human IgM cross-reacted with the homologous antigen from the dog. Since completion of these experiments, exchange of reagents has confirmed the congruence of our classification of canine immunoglobulins with that proposed by Drs. Johnson and Vaughan. Acknowledgement--Financial support from the European Community for Coal and Steel, 6240-11/035 and from the Fonds de la Recherche Scientifique M~dicale, Brussels (Grant # 803) is gratefully acknowledged.
REFERENCES 1. JoHNSO~J.S. and VAUO~ANJ.H., jr. Imraun. 98, 923 (1967). 2. JOHNSONJ. S., VAUOHANJ. H. and Swisher S. N., jr. Immun. 95~ 935 (1967). 3. TOMASIT. B., TAN E. M., SOLOMONA. and PRENDEROASTR. A., jr. exp. Med. 121~ I01 (1965). 4. FLEISCH~NJ. B., PORTFRR. R. and PRESSE. M., BiochemJ. 88, 220 (1963). 5. M~NCI~ G., ~ o N . ~ a ~ A. O. and HEm~tANSJ. F., Immunochemistcy2~ 235 (1965). 6. WEIRR., GrVOLD. and PORa'ERR. R., Nature, Lond. 212, 205 (1966).