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Low molecular weight mercaptoethanol sensitive antibody in rabbits
lmmunochemistry. Pergamon Press 1969. Vol.6, pp. 649-658. Printed in Great Britain
LOW M O L E C U L A R W E I G H T M E R C A P T O E T H A N O L S E N S I T I V E A N T I B O D Y IN RABBITS* M. W. STEWARD?, C. W. TODD$, T. J. KINDT? and G. S. DAVID§ Department of Biology, City of Hope Medical Center, Duarte, Calif. 91010, U.S.A.
(First received 27 February 1969; in revisedform 27 March 1969) Abstract- Rabbit antibody prepared against the group a (heavy chain) ailotypic specificities of the rabbit has been shown to possess unexpected lability. Antibody activity is markedly reduced by treatment of antisera with mercaptoethanol or by dialysis against buffer of low ionic strength. By disc electrophoresis the mobility of the antibody is similar to that of IgG. Gradient centrifugation and gel fltration indicate that its molecular weight is appreciably lower than that of IgG. It is not inactivated by antisera directed against rabbit IgM or IgA but is inactivated by antisera directed against rabbit Fc7 or the group b (light chain) allotype specificity it possesses. This combination of properties suggests that these antibodies may constitute a new class of rabbit immunoglobulin. Antibodies of this type have been obtained against other antigenic determinants, but the conditions necessary to elicit a response consisting primarily of this component remain to be defined. INTRODUCTION T h e known i m m u n o g l o b u l i n classes in man n u m b e r at least five, lgG, IgA, IgM, IgD and IgE," o f which each o f the first three can be divided into welldefined subclasses. Such diversity has not yet been detected in the rabbit, only IgG, IgA and IgM are well established. Skin-fixing antibodies responsible for passive cutaneous anaphylaxis in the rabbit a p p e a r to constitute a f o u r t h class o f immunoglobulin, which may be the c o u n t e r p a r t o f IgE in m a n [ l ] . Evidence for an additional class or subclass o f rabbit immunoglobulin will be p r e s e n t e d here. This research began with an a t t e m p t to make fluorescent tagged antibody directed against allotypic specificity al.** Gammaglobulin precipitated f r o m an anti-al serum with 1.75M a m m o n i u m sulfate and passed t h r o u g h a diethylaminoethyl cellulose (DEAE) c o l u m n in 0.0175 M p H 6.5 p h o s p h a t e buffer was f o u n d to give no interfacial precipitin test with sera containing the al specificity. An examination o f the literature revealed that o t h e r authors had labelled the protein o f the a m m o n i u m sulfate precipitate to obtain fluorescent anti-allotype *Supported by a research Grant No. AI07995 and an animal care Grant No. FR00433 from the National Institutes of Health. ?Postdoctoral fellows of the National Institute of Allergy and Infectious Diseases. $Supported by a City of Hope fund established in the name of J. Gordon Roberts. §Postdoctoral fellow of the National Institute of General Medical Sciences. IrThe system of nomenclature recommended by WHO will be used for the immunoglobulin classes and fragments [2, 3]. **In the rabbit the group a specificities (al, a2 and a3) are present on the heavy chains. The group b specificities (b4, b5, b6 and b9) are present on the K light chain [4]. This subject has been reviewed by Oudin [5]. 649
650
M.W. STEWARD et al.
antibodies [6]. Examination of material obtained by ammonium sulfate precipitation showed that the anti-allotype activity was present but disappeared when the solution was dialyzed prior to chromatography on DEAE. This behavior prompted us to examine more fully the nature of this antibody. MATERIALS AND METHODS Antisera The anti-allotype sera were produced following the method of Oudin[7]. Rabbits lacking the allotype against which the antiserum was to be prepared were injected with ovalbumin immune precipitate from rabbit serum bearing that allotype, but no other known allotype absent in the injected rabbit. Antiserum against rabbit IgG was prepared by immunizing a goat with IgG isolated by two precipitations in 1"75 M ammonium sulfate, followed by chromatography over DEAE in 0-0175M pH 6"5 phosphate buffer. Antiserum against rabbit Fcy was prepared by absorbing anti-rabbit IgG serum with (Fab'),, obtained by pepsin hydrolysis of IgG[8]. Antiserum against rabbit IgA was prepared by immunizing a goat with IgA obtained from rabbit milk[9] in complete Freund's adjuvant. This serum was rendered specific for IgA by absorption with IgG. The method of M~ikel~i et al.[10] was used to obtain antiserum against rabbit IgM. A rabbit was injected intravenously with 1 ml of saline washed goat erythrocytes and bled five days later. Goat erythrocytes (6 ml) were added to the first peak from the Sephadex G200 chromatography of 7 ml of this serum. After incubation for 2 hr at 37 ° the cells were washed with saline. One-half portions of these cells were injected in complete Freund's adjuvant at a one month interval underneath the scapula of a goat. Two weeks following the second injection the goat was exsanguinated. On immunoelectrophoresis this serum showed antibodies to IgM, IgG, and three s-globulins. The antibodies against IgG were removed by absorption with IgG bound to carboxymethyl cellulose by the method of Dandliker et al.[11]. Agglutination techniques Type F[12] rabbit red blood cells were coated with rabbit IgG using the chromic chloride method of Gold and Fudenberg[13]. One-half ml of a saline solution containing 1.0 mg/ml of CrC13 • 6H20 was added to 1.0 ml of a saline solution containing 2"5 mg/ml of IgG. After mixing, 0-25 ml of a 50 per cent suspension of red blood cells in saline was added immediately. The mixture was shaken slowly for 30 min. The cells were washed three times with saline and resuspended to give a 2 per cent suspension. Such cells were readily agglutinated by homologous anti-allotype sera. Equal volumes of the dilution of antiserum to be tested and the 2 per cent cell suspension were shaken for 15 min at room temperature on a test plate. The plates were examined microscopically at low magnification for agglutination and scored 0-4+. Additional details on this method will be published elsewhere. R eduction and alkylation Serum samples were reduced by the addition of redistilled mercaptoethanol with rapid agitation to a final concentration of 0.1 M. The samples were then
Mercaptoethanol Sensitive Antibody
651
flushed with nitrogen and incubated for 30 min at room temperature. If a sample was to be alkylated, it was cooled to 0° in an ice bath and a 25 per cent molar excess of iodoacetamide added. After a minimum of 6 hr at 0°, excess reagents were removed by dialysis.
Disc electrophoresis Disc electrophoresis was performed according to the method of Ornstein [14] and Davis[15] with several modifications. The stacking gel was omitted, riboflavin was used as the catalyst for polymerization of the running gel[16], and the acrylamide concentration was 4 per cent. Acrylamide and N,N'-methylene-b/s acrylamide were recrystallized from chloroform and acetone respectively.
Gradient centrifugation Gradient centrifugation was performed in a model L265B Spinco®centrifuge using an SW40 rotor. Samples were layered onto a linear gradient of 5-20 per cent sucrose in 0.1 M pH 6.8 phosphate buffer. Centrifugation was carried out at 4° for 19 hr at 35000 rev/min. Fractions of 0.5 ml were collected through a hole at the bottom of the centrifuge tube.
Gelfiltration Gel filtration was performed on a 90 × 2.5 cm column of Sephadex® G200 in 0.1 M pH 6.8 potassium phosphate buffer.
Inactivation by antisera Anti-al was mixed with rabbit anti-b4 or goat antiserum to rabbit Fcy, IgA or IgM in proportions of 1 : 1, 1:2, 1:4, 1:8 and 1 : 16 to determine the optimum ratio. The mixtures were incubated two hours at 37° and overnight at 4°. After centrifugation the supernatant was tested for ami-al activity by hemagglutination. The lowest hemagglutination titer is reported. After the anti-Fcy precipitations, the excess anti-Fcy activity in the supernatant was removed by absorption with rabbit serum lacking the al determinant. This was done to prevent hemagglutination of the IgG-coated cells by anti-Fcy. RESULTS
Loss of anti-allotype activity on isolation of lgG Our initial observation that IgG, isolated from anti-al serum by the usual technique of ammonium sulfate precipitation followed by DEAE chromatography, lacked ability to react with serum containing the al specificity lead us to examine other anti-allotype sera. As shown in Table 1 the IgG obtained from five of six anti-allotype sera showed similar inactivity. Activity was found to persist in the solution obtained by dissolving the material precipitated by ammonium sulfate or sodium sulfate but disappeared when this solution was dialyzed against 0.0175M pH 6"5 phosphate buffer preparatory to DEAE chromatography.
Effect of mercaptoethanol treatment The loss of activity by dialysis against low ionic strength buffer suggested that
652
M.W. STEWARD et al. Table 1. Loss of anti-aliotype activity on isolation of IgG from anti-allotype sera
Rabbit
Anti
451 953 418 502 856
al al a3 b4 b5
Anti-allotype activity* Salt Serum precipitation? IgG$ + + + + +
+ +
-
+
+
*Measured by interfacial precipitin test and by the diffusion in tube method of Oudin. ?Obtained by precipitating sera with (NH4)2SO4, 23 per cent, or NazSO4, 18 per cent, (w/v). $Isolated from salt precipitates by passage through a DEAE cellulose column in 0-0175M phosph:lte buffer, pH 6"5. the anti-allotype antibody might be IgM. Accordingly its stability in the presence o f m e r c a p t o e t h a n o l was examined. T h e presence o f anti-allotype antibody was quantitated using an agglutination m e t h o d e m p l o y i n g rabbit erythrocytes coated with I g G o f allotype al using chromic chloride as a b i n d e r [13]. Table 2 presents the results obtained with an anti-al serum. T h e original s e r u m possessed a titer o f 2"106; t r e a t m e n t with 0.1 M m e r c a p t o e t h a n o l r e d u c e d this titer to 512. T h e I g G isolated f r o m this serum had a residual titer o f 256. O n m e r c a p t o e t h a n o l treatment this titer was r e d u c e d to 32. Using the interfacial precipitation test as a rapid screening m e t h o d the effect o f m e r c a p t o e t h a n o l on a n u m b e r o f anti-allotype sera was examined. As shown Table 2. Effect of mercaptoethanol treatment on agglutination titers of an anti-al serum and its isolated IgG
Treatment
Agglutination titer* Serum Isolated IgG
None Reduction?
2 × l0 t 512:~
256 32
*Assayed using rabbit red blood cells coated with al, b4 IgG by the chromic chloride method. For the isolated IgG the titer is given for a solution of 8 mg/ml. ?Mercaptoethanol 0"1 M for 1 hr at room temperature. ~The serum sample was also alkylated after reduction using a 25 per cent molar excess of iodoacetamide for 6 hr at 0°"
Mercaptoethanol Sensitive Antibody
653
in T a b l e 3 all antisera d i r e c t e d against the g r o u p a (heavy chain) specificities w e r e r e n d e r e d inactive, a l t h o u g h s o m e activity occasionally persisted in antisera to t h e g r o u p b (light chain) specificities. Since these antisera w e r e p r e p a r e d by injection o f o v a l b u m i n specific precipitates[7], the antisera also c o n t a i n e d a n t i - o v a l b u m i n activity. T h e a n t i - o v a l b u m i n activity in these sera was d e s t r o y e d o r r e d u c e d by m e r c a p t o e t h a n o l t r e a t m e n t . I n contrast, a n t i - o v a l b u m i n activity in a n t i s e r a p r e p a r e d against o v a l b u m i n alone persisted. Table 3. Mercaptoethanol sensitivity of anti-allotype and anti-ovalbumin sera*
Rabbit
Antigen
953 1029 1059 1151 1163 1247 840 1171
al-oval a2-oval a3-oval b4-oval
1171
859 1274 1274 546 1160
b5-oval b6-oval
1160 1041
1337 1337 1212
1218
b9-oval oval
oval
Time elapsed since first immunizing injection (weeks) 28 40 46 32 25 17 Pooled sera 6 26 Pooled sera 13 33 Pooled sera 17 43 25 11 31 1 3 7 21 26 39 6
Reaction after treatment with 0' 1 M mercaptoethanolt Antiallotype ------÷ -
Antiovalbumin
4-4h
±
+ -± -
+~ -4-
-
÷ -+
-2 + ++ ++ ++ ++ +
*Anti-allotype sera were prepared by injection of rabbits with ovalbumin-anti-ovalbumin precipitates in complete Freund's adjuvant according to the method o f Oudin [7]. Anti-ovalbumin sera were prepared by monthly injections of 10 mg ovalbumin in complete Freund's adjuvant. tSera were allowed to react for 30 rain at room temperature with 0-1 M mercaptoethanol. Results are expressed as strength of reaction with antigen by interfacial precipitin test relative to that of untreated antiserum. The significance o f the symbols is - , activity completely destroyed; ±, partial loss o f activity; ÷, slight loss; + + , no noticeable loss. SReactions of untreated antisera were extremely weak.
M.W. STEWARD et al.
654
Gel filtration Despite the loss o f activity by dialysis against buffer o f low ionic strength and by t r e a t m e n t with mercaptoethanol, gel filtration clearly indicated the activity was not d u e to macroglobulin. As shown in Fig. 1, activity as m e a s u r e d by the hemagglutination assay was eluted with the fractions o f the descending arm of the second peak, intermediate to the positions o f 125I labelled IgG and (Fab')2 used as markers. GEL
FILTRATION ?'KI
1'50] 5"0-
1.25~ 2"5i i
1"00- 2"O-
g
.i03
c~
:K
c:)
E 0.754
1"5-
C) I i
-lO 2
05
0.50- 1'0]
0,25-
O-
-101
o~-~,r~ "/
,
,
0 2 20
24
28
I
,1111 32
36
40
i
44
48
FRACTION NOI
Fig. 1. Elution profile of 4 ml anti-al serum (e~---Q) on Sephadex® G200. The peaks shown for 12'~I labelled IgG (Q---C)) and (Fab')2 (El---D) give elution positions for protein molecules of mol. wt. ca. 160,000 and 100,000 respectively. The (Fab')~ data is from a second run on the same column, and anti-al serum was included to confirm the relative positions of the peaks. Bars show anti-allotype activity by the hemagglutination technique.
Gradient centrifugation In confirmation o f the results obtained by gel filtration, centrifugation in a 5-20 per cent sucrose gradient indicated the molecular weight to be less than that of IgG for both anti-al and anti-b4 antibody (Fig. 2). However, for the anti-b4 m o r e activity is seen at the position o f the ascending a r m o f the IgG m a r k e r curve. This may reflect the t e n d e n c y noted above for some antibody to the g r o u p b allotypes to be m o r e resistant to m e r c a p t o e t h a n o l treatment. This confirmation is considered particularly significant in view o f the fact that an increase in the specific volume o f a protein would decrease the sedimentation velocity but would lead to m o r e rapid elution in gel filtration.
Mercaptoethanol Sensitive Antibody
655
DENSITY GRADIENT CENT R I F UGAT ION 2.0
2"O1
Anti- o 1
,/;,
/t
i o
E
I"0
l'O-J a
l
104 "
,/I[ k/
103 10z
101
o
OJ 0
4"0
2"0
3"0
1"5
2
4
6
8
10 12
, 14
18 20
o
o
"Z
m
~
&h r \
104
Yl/I lil \
0"5
o
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Anti- b4 •
u
o
'1 16
102
101
"-'-..
I L ~ I I I I ) I ~LL~:_~
...... : . ~ - ~ ' l l l l l l l
0 2 BOTTOM
4
6
B
10 12
FF 14
16
o
18 20 TOP
FRACTION
NO.
Fig. 2. Centrifugation of 0.2 ml anti-al and anti-b4 serum (O------Q) on 5-20 per cent sucrose density gradients. A radiolabelled IgG marker (O---O) included in the samples gives the position of a 7S molecule. Antiallotype activity measured by hemagglutination is shown by bars.
E lectrophor etic mobility Using disc electrophoresis the agglutinating activity was found across the region occupied by IgG with a peak in the center (Fig. 3). Effects of antisera on anti-a1 activity With the hope of obtaining information on the immunoglobulin class, the effect of various antisera on anti-al activity was examined. As seen in Table 4, the effect of anti-IgA and anti-IgM was minimal, but anti-Fcy showed marked reduction of activity. Confirming the validity of the test, the activity of the anti-al, which carried b4 allotypic specificity, was also reduced by anti-b4 serum. DISCUSSION
Mercaptoethanol sensitive, slowly sedimenting antibody has been reported for goldfish and frogs[17], and mercaptoethanol sensitive 7S antibody has
IMM-- Vol. 6 No. 5 - - B
656
M.W. STEWARD
et al.
Table 4. Effect of various sera on anti-al activity Treatment
Hemagglutination titer
Normal goat serum* Anti-Fcy* Anti-IgA* Anti-IgM* Normal rabbit serum? Anti-M?
30,000 500 26,000 13,000 21,000 600
*IgG used as coat was al,b4. ?IgG used as coat was al,a3,b5. been reported in the chicken [18]. In the duck[19], a mercaptoethanol sensitive antibody to bovine serum albumin slightly larger than 7S has been described and contrasted to antibody to bovine serum albumin obtained in rabbits. The Farr technique[20] was used to measure retention of antibody activity. Presumably this technique would not detect a change in precipitability of the antigen-antibody complex, if combining capacity were unchanged as described below for the mouse. An IgA mouse myeloma protein which binds 2,4-dinitrophenyl groups and has a molecular weight of approx. 120,000 has been described by Eisen et al.[21]. Also mercaptoethanol sensitive 7S antibody has been reported tot the mouse [22, 23]. Treatment of mouse antisera to ferritin with mercaptoethanol destroyed its ability to agglutinate ferritin-coated erythrocytes. In contrast, treatment of antisera to ovalbumin with mercaptoethanol had no detectable effect on the agglutination of ovalbumin-coated cells. The ability of both antisera to precipitate their respective antigens was reduced. This reduction resulted front inability to precipitate rather than from inability to combine with antigen. The changes were reversible if treated antisera were allowed to reoxidize without alkylation by iodoacetamide. Our current studies indicate that the mercaptoethanol sensitive antibody in rabbits may follow this pattern. The properties noted for the anti-allotype antibodies examined here are not consistent with those of known rabbit immunoglobulin classes, although a nonprecipitating antibody to ovalbumin with a sedimentation constant of 4.4S has been described by Sutherland and Campbell[24]. In man the human agglutinators tk~r detection of the Gm allotypes are nearly always IgM[25j. While the lack of activity at low ionic strength and sensitivity to mercaptoethanol are suggestive of IgM, the behavior in gel filtration and gradient centrifugation, as well as the failure to react significantly with anti-IgM precludes this possibility. The IgM subunit in the rabbit [26] and in man [27] has a molecular weight greater than IgG. In those instances in which 7S IgM has been noted in man [28-31 ], its molecular weight was similar to that of the IgM subunit. Similarly both rabbit IgA [32] and rabbit homocytotropic antibody[1 I, which may be the rabbit counterpart of human IgE, possess higher molecular weights than IgG. While the inactivation by anti-Fc y suggests IgG, the result might equally be attributed to a cross-reacting class or to the unrecognized presence of a distinct
f 200 ~
too o
Fig. 3. Disc electrophoresis of anti-al serum (upper photograph). The graph indicates the anti-al activity of extruded fractions of the gel assayed by hemagglutination. The lower photograph indicates the position of an isolated IgG included for reference.
(Facingpage 656)
Mercaptoethanol Sensitive Antibody
657
class in the immunizing antigen believed to be pure IgG. O u r present data indicate that the molecular weight may be in the vicinity of 125,000. A h u m a n myeloma protein of mol. wt. 125,000 has been reported [33]. This protein had y type determinants on the heavy chain and K light chains. Both chains appeared to possess lower than normal molecular weights. Molecular weight is obviously of critical importance in characterizing this antibody. T h e antibody responsible for the effects noted here must be isolated from other serum components before definite assignment of an immunoglobulin class can be made. A second consideration concerns the nature of the antigen and the conditions responsible for this type of antibody response. While most of the results reported above were obtained with antisera directed against the group a allotypic specificities, limited data with group b specificities and ovalbumin indicate that this response is not limited to the group a specificities. In contrast to most immunizations, preparation of antisera to group a specificities by injection of ovalbumin specific precipitates is a slow process. Precipitating antibodies are rarely obtained before three to four months of immunization. Precipitating antibody to group b specificities normally appears d u r i n g the second month. Possibly the response is influenced in part by the fact that the immunization is being provoked by an i m m u n e precipitate. T h e nature of i m m u n e complexes used as antigens can markedly influence the nature of the resulting response [34]. It should be noted that in the case of antibody directed against group a specificities and provoked by ovalbumin specific precipitates, the antigen is the antibody of the i m m u n e complex. Studies to define the role of antigen in determining the nature of the response are in progress. Acknowledgement--The authors thank Mrs. Karen Feintuch for competent technical
assistance in this research.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
REFERENCES Lindqvist K. J., Immunochemistry 5, 525 (1968). W.H.O. Committee, Bull. Wld Hlth Org. 30,447 (1964). W.H.O. Committee,J. Immun. 100, 1143 (1968). Dray S., Dubiski S., Kelus A., Lennox E. S. and Oudin J., Nature, Lond. 195, 785 (1962). OudinJ.,J. CeUPhysiol. 67, Suppl. 1, 77 (1966). Pernis B., Chiappino G., Kelus A. S. and Gell P. G. H.,J. exp. Med. 122, 853 (1965). OudinJ.,J. exp. Med. 112, 107 (1960). Nisonoff A., Wissler F. C., Lipman L. N. and Woernley D. L., Archs Biochem. Biophys. 89, 230 (1960). Kindt T. J., Steward M. W. and Todd C. W., Biochem. biophys. Res. Commun. 31, 9 (1968). M~ikel~iO., Kostiainen E., Koponen T. and Ruoslahti E., Nobel Symposium 3 (Edited by Killander J.), p. 505. Interscience, New York (1967). Dandliker W. B., De Saussure V. A. and Levandoski N., Immunochemistry 5, 357 (1968). Cohen C., Ann. N.Y. Acad. Sci. 97, 26 (1962). Gold E. R. and Fudenberg H. H.,J. Immun. 99, 859 (1967). Ornstein L., Ann. N.Y. Acad. Sci. 121,321 (1964). Davis B.J., Ann. N.Y. Acad. Sci. 121,404 (1964). BrewerJ. M., Science 156, 256 (1967).
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17. Uhr J. W., Finkelstein M. S. and Franklin E. C., Proc. Soc. exp. Biol. Med. 111, 13 (1962). 18. Rosenquist G. L. and Gilden R. V., Biochim. Biophys. Acta 78,543 (1963). 19. Grey H. M., Proc. Soc. exp. Biol. Med. 113, 963 (1963). 20. Farr R. S.,J. infect. Dis. 103,239 (1958). 21. Eisen H. N., Simms E. S. and Potter M.,Biochem. 7, 4126 (1968). 22. Adler F. L.,J. Immun. 95, 26 (1965). 23. Adler F. L.,J. Immun. 95, 39 (1965). 24. Sutherland G. B. and Campbell D. H.,J. Immun. 80, 294 (1958). 25. Steinberg A. G., Prog. med. Genet. 2, 1 (1962). 26. Lamm M. E. and Small P. A.,Jr., Biochem. 5, 267 (1966). 27. Miller F. and Metzger H.,J. biol. Chem. 240, 3325 (1965). 28. Solomon A. and Kunkel H. G., Am.J, Med. 42, 958 (1967). 29. Cooper A. G., Science 157, 933 (1967). 30. StoboJ. D. and Tomasi T. B.,Jr.,J. clin. Invest. 46, 1329 (1967). 31. Swedlund H. A., Gleich G.J. and Chodirker W. B.,J. Immun. 100, 1296 (1968). 32. CebraJ.J. and Small P. A.,Jr., Biochem. 6, 503 (1967). 33. Lewis A. F., Bergsagel D. E., Bruce-Robertson A., Schachter R. K. and Connell G. E., Blood32, 189 (1968). 34. UhrJ. W. and M611er G., Adv. Immunol. 8, 81 (1968).
LOW M O L E C U L A R W E I G H T M E R C A P T O E T H A N O L S E N S I T I V E A N T I B O D Y IN RABBITS* M. W. STEWARD?, C. W. TODD$, T. J. KINDT? and G. S. DAVID§ Department of Biology, City of Hope Medical Center, Duarte, Calif. 91010, U.S.A.
(First received 27 February 1969; in revisedform 27 March 1969) Abstract- Rabbit antibody prepared against the group a (heavy chain) ailotypic specificities of the rabbit has been shown to possess unexpected lability. Antibody activity is markedly reduced by treatment of antisera with mercaptoethanol or by dialysis against buffer of low ionic strength. By disc electrophoresis the mobility of the antibody is similar to that of IgG. Gradient centrifugation and gel fltration indicate that its molecular weight is appreciably lower than that of IgG. It is not inactivated by antisera directed against rabbit IgM or IgA but is inactivated by antisera directed against rabbit Fc7 or the group b (light chain) allotype specificity it possesses. This combination of properties suggests that these antibodies may constitute a new class of rabbit immunoglobulin. Antibodies of this type have been obtained against other antigenic determinants, but the conditions necessary to elicit a response consisting primarily of this component remain to be defined. INTRODUCTION T h e known i m m u n o g l o b u l i n classes in man n u m b e r at least five, lgG, IgA, IgM, IgD and IgE," o f which each o f the first three can be divided into welldefined subclasses. Such diversity has not yet been detected in the rabbit, only IgG, IgA and IgM are well established. Skin-fixing antibodies responsible for passive cutaneous anaphylaxis in the rabbit a p p e a r to constitute a f o u r t h class o f immunoglobulin, which may be the c o u n t e r p a r t o f IgE in m a n [ l ] . Evidence for an additional class or subclass o f rabbit immunoglobulin will be p r e s e n t e d here. This research began with an a t t e m p t to make fluorescent tagged antibody directed against allotypic specificity al.** Gammaglobulin precipitated f r o m an anti-al serum with 1.75M a m m o n i u m sulfate and passed t h r o u g h a diethylaminoethyl cellulose (DEAE) c o l u m n in 0.0175 M p H 6.5 p h o s p h a t e buffer was f o u n d to give no interfacial precipitin test with sera containing the al specificity. An examination o f the literature revealed that o t h e r authors had labelled the protein o f the a m m o n i u m sulfate precipitate to obtain fluorescent anti-allotype *Supported by a research Grant No. AI07995 and an animal care Grant No. FR00433 from the National Institutes of Health. ?Postdoctoral fellows of the National Institute of Allergy and Infectious Diseases. $Supported by a City of Hope fund established in the name of J. Gordon Roberts. §Postdoctoral fellow of the National Institute of General Medical Sciences. IrThe system of nomenclature recommended by WHO will be used for the immunoglobulin classes and fragments [2, 3]. **In the rabbit the group a specificities (al, a2 and a3) are present on the heavy chains. The group b specificities (b4, b5, b6 and b9) are present on the K light chain [4]. This subject has been reviewed by Oudin [5]. 649
650
M.W. STEWARD et al.
antibodies [6]. Examination of material obtained by ammonium sulfate precipitation showed that the anti-allotype activity was present but disappeared when the solution was dialyzed prior to chromatography on DEAE. This behavior prompted us to examine more fully the nature of this antibody. MATERIALS AND METHODS Antisera The anti-allotype sera were produced following the method of Oudin[7]. Rabbits lacking the allotype against which the antiserum was to be prepared were injected with ovalbumin immune precipitate from rabbit serum bearing that allotype, but no other known allotype absent in the injected rabbit. Antiserum against rabbit IgG was prepared by immunizing a goat with IgG isolated by two precipitations in 1"75 M ammonium sulfate, followed by chromatography over DEAE in 0-0175M pH 6"5 phosphate buffer. Antiserum against rabbit Fcy was prepared by absorbing anti-rabbit IgG serum with (Fab'),, obtained by pepsin hydrolysis of IgG[8]. Antiserum against rabbit IgA was prepared by immunizing a goat with IgA obtained from rabbit milk[9] in complete Freund's adjuvant. This serum was rendered specific for IgA by absorption with IgG. The method of M~ikel~i et al.[10] was used to obtain antiserum against rabbit IgM. A rabbit was injected intravenously with 1 ml of saline washed goat erythrocytes and bled five days later. Goat erythrocytes (6 ml) were added to the first peak from the Sephadex G200 chromatography of 7 ml of this serum. After incubation for 2 hr at 37 ° the cells were washed with saline. One-half portions of these cells were injected in complete Freund's adjuvant at a one month interval underneath the scapula of a goat. Two weeks following the second injection the goat was exsanguinated. On immunoelectrophoresis this serum showed antibodies to IgM, IgG, and three s-globulins. The antibodies against IgG were removed by absorption with IgG bound to carboxymethyl cellulose by the method of Dandliker et al.[11]. Agglutination techniques Type F[12] rabbit red blood cells were coated with rabbit IgG using the chromic chloride method of Gold and Fudenberg[13]. One-half ml of a saline solution containing 1.0 mg/ml of CrC13 • 6H20 was added to 1.0 ml of a saline solution containing 2"5 mg/ml of IgG. After mixing, 0-25 ml of a 50 per cent suspension of red blood cells in saline was added immediately. The mixture was shaken slowly for 30 min. The cells were washed three times with saline and resuspended to give a 2 per cent suspension. Such cells were readily agglutinated by homologous anti-allotype sera. Equal volumes of the dilution of antiserum to be tested and the 2 per cent cell suspension were shaken for 15 min at room temperature on a test plate. The plates were examined microscopically at low magnification for agglutination and scored 0-4+. Additional details on this method will be published elsewhere. R eduction and alkylation Serum samples were reduced by the addition of redistilled mercaptoethanol with rapid agitation to a final concentration of 0.1 M. The samples were then
Mercaptoethanol Sensitive Antibody
651
flushed with nitrogen and incubated for 30 min at room temperature. If a sample was to be alkylated, it was cooled to 0° in an ice bath and a 25 per cent molar excess of iodoacetamide added. After a minimum of 6 hr at 0°, excess reagents were removed by dialysis.
Disc electrophoresis Disc electrophoresis was performed according to the method of Ornstein [14] and Davis[15] with several modifications. The stacking gel was omitted, riboflavin was used as the catalyst for polymerization of the running gel[16], and the acrylamide concentration was 4 per cent. Acrylamide and N,N'-methylene-b/s acrylamide were recrystallized from chloroform and acetone respectively.
Gradient centrifugation Gradient centrifugation was performed in a model L265B Spinco®centrifuge using an SW40 rotor. Samples were layered onto a linear gradient of 5-20 per cent sucrose in 0.1 M pH 6.8 phosphate buffer. Centrifugation was carried out at 4° for 19 hr at 35000 rev/min. Fractions of 0.5 ml were collected through a hole at the bottom of the centrifuge tube.
Gelfiltration Gel filtration was performed on a 90 × 2.5 cm column of Sephadex® G200 in 0.1 M pH 6.8 potassium phosphate buffer.
Inactivation by antisera Anti-al was mixed with rabbit anti-b4 or goat antiserum to rabbit Fcy, IgA or IgM in proportions of 1 : 1, 1:2, 1:4, 1:8 and 1 : 16 to determine the optimum ratio. The mixtures were incubated two hours at 37° and overnight at 4°. After centrifugation the supernatant was tested for ami-al activity by hemagglutination. The lowest hemagglutination titer is reported. After the anti-Fcy precipitations, the excess anti-Fcy activity in the supernatant was removed by absorption with rabbit serum lacking the al determinant. This was done to prevent hemagglutination of the IgG-coated cells by anti-Fcy. RESULTS
Loss of anti-allotype activity on isolation of lgG Our initial observation that IgG, isolated from anti-al serum by the usual technique of ammonium sulfate precipitation followed by DEAE chromatography, lacked ability to react with serum containing the al specificity lead us to examine other anti-allotype sera. As shown in Table 1 the IgG obtained from five of six anti-allotype sera showed similar inactivity. Activity was found to persist in the solution obtained by dissolving the material precipitated by ammonium sulfate or sodium sulfate but disappeared when this solution was dialyzed against 0.0175M pH 6"5 phosphate buffer preparatory to DEAE chromatography.
Effect of mercaptoethanol treatment The loss of activity by dialysis against low ionic strength buffer suggested that
652
M.W. STEWARD et al. Table 1. Loss of anti-aliotype activity on isolation of IgG from anti-allotype sera
Rabbit
Anti
451 953 418 502 856
al al a3 b4 b5
Anti-allotype activity* Salt Serum precipitation? IgG$ + + + + +
+ +
-
+
+
*Measured by interfacial precipitin test and by the diffusion in tube method of Oudin. ?Obtained by precipitating sera with (NH4)2SO4, 23 per cent, or NazSO4, 18 per cent, (w/v). $Isolated from salt precipitates by passage through a DEAE cellulose column in 0-0175M phosph:lte buffer, pH 6"5. the anti-allotype antibody might be IgM. Accordingly its stability in the presence o f m e r c a p t o e t h a n o l was examined. T h e presence o f anti-allotype antibody was quantitated using an agglutination m e t h o d e m p l o y i n g rabbit erythrocytes coated with I g G o f allotype al using chromic chloride as a b i n d e r [13]. Table 2 presents the results obtained with an anti-al serum. T h e original s e r u m possessed a titer o f 2"106; t r e a t m e n t with 0.1 M m e r c a p t o e t h a n o l r e d u c e d this titer to 512. T h e I g G isolated f r o m this serum had a residual titer o f 256. O n m e r c a p t o e t h a n o l treatment this titer was r e d u c e d to 32. Using the interfacial precipitation test as a rapid screening m e t h o d the effect o f m e r c a p t o e t h a n o l on a n u m b e r o f anti-allotype sera was examined. As shown Table 2. Effect of mercaptoethanol treatment on agglutination titers of an anti-al serum and its isolated IgG
Treatment
Agglutination titer* Serum Isolated IgG
None Reduction?
2 × l0 t 512:~
256 32
*Assayed using rabbit red blood cells coated with al, b4 IgG by the chromic chloride method. For the isolated IgG the titer is given for a solution of 8 mg/ml. ?Mercaptoethanol 0"1 M for 1 hr at room temperature. ~The serum sample was also alkylated after reduction using a 25 per cent molar excess of iodoacetamide for 6 hr at 0°"
Mercaptoethanol Sensitive Antibody
653
in T a b l e 3 all antisera d i r e c t e d against the g r o u p a (heavy chain) specificities w e r e r e n d e r e d inactive, a l t h o u g h s o m e activity occasionally persisted in antisera to t h e g r o u p b (light chain) specificities. Since these antisera w e r e p r e p a r e d by injection o f o v a l b u m i n specific precipitates[7], the antisera also c o n t a i n e d a n t i - o v a l b u m i n activity. T h e a n t i - o v a l b u m i n activity in these sera was d e s t r o y e d o r r e d u c e d by m e r c a p t o e t h a n o l t r e a t m e n t . I n contrast, a n t i - o v a l b u m i n activity in a n t i s e r a p r e p a r e d against o v a l b u m i n alone persisted. Table 3. Mercaptoethanol sensitivity of anti-allotype and anti-ovalbumin sera*
Rabbit
Antigen
953 1029 1059 1151 1163 1247 840 1171
al-oval a2-oval a3-oval b4-oval
1171
859 1274 1274 546 1160
b5-oval b6-oval
1160 1041
1337 1337 1212
1218
b9-oval oval
oval
Time elapsed since first immunizing injection (weeks) 28 40 46 32 25 17 Pooled sera 6 26 Pooled sera 13 33 Pooled sera 17 43 25 11 31 1 3 7 21 26 39 6
Reaction after treatment with 0' 1 M mercaptoethanolt Antiallotype ------÷ -
Antiovalbumin
4-4h
±
+ -± -
+~ -4-
-
÷ -+
-2 + ++ ++ ++ ++ +
*Anti-allotype sera were prepared by injection of rabbits with ovalbumin-anti-ovalbumin precipitates in complete Freund's adjuvant according to the method o f Oudin [7]. Anti-ovalbumin sera were prepared by monthly injections of 10 mg ovalbumin in complete Freund's adjuvant. tSera were allowed to react for 30 rain at room temperature with 0-1 M mercaptoethanol. Results are expressed as strength of reaction with antigen by interfacial precipitin test relative to that of untreated antiserum. The significance o f the symbols is - , activity completely destroyed; ±, partial loss o f activity; ÷, slight loss; + + , no noticeable loss. SReactions of untreated antisera were extremely weak.
M.W. STEWARD et al.
654
Gel filtration Despite the loss o f activity by dialysis against buffer o f low ionic strength and by t r e a t m e n t with mercaptoethanol, gel filtration clearly indicated the activity was not d u e to macroglobulin. As shown in Fig. 1, activity as m e a s u r e d by the hemagglutination assay was eluted with the fractions o f the descending arm of the second peak, intermediate to the positions o f 125I labelled IgG and (Fab')2 used as markers. GEL
FILTRATION ?'KI
1'50] 5"0-
1.25~ 2"5i i
1"00- 2"O-
g
.i03
c~
:K
c:)
E 0.754
1"5-
C) I i
-lO 2
05
0.50- 1'0]
0,25-
O-
-101
o~-~,r~ "/
,
,
0 2 20
24
28
I
,1111 32
36
40
i
44
48
FRACTION NOI
Fig. 1. Elution profile of 4 ml anti-al serum (e~---Q) on Sephadex® G200. The peaks shown for 12'~I labelled IgG (Q---C)) and (Fab')2 (El---D) give elution positions for protein molecules of mol. wt. ca. 160,000 and 100,000 respectively. The (Fab')~ data is from a second run on the same column, and anti-al serum was included to confirm the relative positions of the peaks. Bars show anti-allotype activity by the hemagglutination technique.
Gradient centrifugation In confirmation o f the results obtained by gel filtration, centrifugation in a 5-20 per cent sucrose gradient indicated the molecular weight to be less than that of IgG for both anti-al and anti-b4 antibody (Fig. 2). However, for the anti-b4 m o r e activity is seen at the position o f the ascending a r m o f the IgG m a r k e r curve. This may reflect the t e n d e n c y noted above for some antibody to the g r o u p b allotypes to be m o r e resistant to m e r c a p t o e t h a n o l treatment. This confirmation is considered particularly significant in view o f the fact that an increase in the specific volume o f a protein would decrease the sedimentation velocity but would lead to m o r e rapid elution in gel filtration.
Mercaptoethanol Sensitive Antibody
655
DENSITY GRADIENT CENT R I F UGAT ION 2.0
2"O1
Anti- o 1
,/;,
/t
i o
E
I"0
l'O-J a
l
104 "
,/I[ k/
103 10z
101
o
OJ 0
4"0
2"0
3"0
1"5
2
4
6
8
10 12
, 14
18 20
o
o
"Z
m
~
&h r \
104
Yl/I lil \
0"5
o
o
=
t ~°~
0
I'0
g
Anti- b4 •
u
o
'1 16
102
101
"-'-..
I L ~ I I I I ) I ~LL~:_~
...... : . ~ - ~ ' l l l l l l l
0 2 BOTTOM
4
6
B
10 12
FF 14
16
o
18 20 TOP
FRACTION
NO.
Fig. 2. Centrifugation of 0.2 ml anti-al and anti-b4 serum (O------Q) on 5-20 per cent sucrose density gradients. A radiolabelled IgG marker (O---O) included in the samples gives the position of a 7S molecule. Antiallotype activity measured by hemagglutination is shown by bars.
E lectrophor etic mobility Using disc electrophoresis the agglutinating activity was found across the region occupied by IgG with a peak in the center (Fig. 3). Effects of antisera on anti-a1 activity With the hope of obtaining information on the immunoglobulin class, the effect of various antisera on anti-al activity was examined. As seen in Table 4, the effect of anti-IgA and anti-IgM was minimal, but anti-Fcy showed marked reduction of activity. Confirming the validity of the test, the activity of the anti-al, which carried b4 allotypic specificity, was also reduced by anti-b4 serum. DISCUSSION
Mercaptoethanol sensitive, slowly sedimenting antibody has been reported for goldfish and frogs[17], and mercaptoethanol sensitive 7S antibody has
IMM-- Vol. 6 No. 5 - - B
656
M.W. STEWARD
et al.
Table 4. Effect of various sera on anti-al activity Treatment
Hemagglutination titer
Normal goat serum* Anti-Fcy* Anti-IgA* Anti-IgM* Normal rabbit serum? Anti-M?
30,000 500 26,000 13,000 21,000 600
*IgG used as coat was al,b4. ?IgG used as coat was al,a3,b5. been reported in the chicken [18]. In the duck[19], a mercaptoethanol sensitive antibody to bovine serum albumin slightly larger than 7S has been described and contrasted to antibody to bovine serum albumin obtained in rabbits. The Farr technique[20] was used to measure retention of antibody activity. Presumably this technique would not detect a change in precipitability of the antigen-antibody complex, if combining capacity were unchanged as described below for the mouse. An IgA mouse myeloma protein which binds 2,4-dinitrophenyl groups and has a molecular weight of approx. 120,000 has been described by Eisen et al.[21]. Also mercaptoethanol sensitive 7S antibody has been reported tot the mouse [22, 23]. Treatment of mouse antisera to ferritin with mercaptoethanol destroyed its ability to agglutinate ferritin-coated erythrocytes. In contrast, treatment of antisera to ovalbumin with mercaptoethanol had no detectable effect on the agglutination of ovalbumin-coated cells. The ability of both antisera to precipitate their respective antigens was reduced. This reduction resulted front inability to precipitate rather than from inability to combine with antigen. The changes were reversible if treated antisera were allowed to reoxidize without alkylation by iodoacetamide. Our current studies indicate that the mercaptoethanol sensitive antibody in rabbits may follow this pattern. The properties noted for the anti-allotype antibodies examined here are not consistent with those of known rabbit immunoglobulin classes, although a nonprecipitating antibody to ovalbumin with a sedimentation constant of 4.4S has been described by Sutherland and Campbell[24]. In man the human agglutinators tk~r detection of the Gm allotypes are nearly always IgM[25j. While the lack of activity at low ionic strength and sensitivity to mercaptoethanol are suggestive of IgM, the behavior in gel filtration and gradient centrifugation, as well as the failure to react significantly with anti-IgM precludes this possibility. The IgM subunit in the rabbit [26] and in man [27] has a molecular weight greater than IgG. In those instances in which 7S IgM has been noted in man [28-31 ], its molecular weight was similar to that of the IgM subunit. Similarly both rabbit IgA [32] and rabbit homocytotropic antibody[1 I, which may be the rabbit counterpart of human IgE, possess higher molecular weights than IgG. While the inactivation by anti-Fc y suggests IgG, the result might equally be attributed to a cross-reacting class or to the unrecognized presence of a distinct
f 200 ~
too o
Fig. 3. Disc electrophoresis of anti-al serum (upper photograph). The graph indicates the anti-al activity of extruded fractions of the gel assayed by hemagglutination. The lower photograph indicates the position of an isolated IgG included for reference.
(Facingpage 656)
Mercaptoethanol Sensitive Antibody
657
class in the immunizing antigen believed to be pure IgG. O u r present data indicate that the molecular weight may be in the vicinity of 125,000. A h u m a n myeloma protein of mol. wt. 125,000 has been reported [33]. This protein had y type determinants on the heavy chain and K light chains. Both chains appeared to possess lower than normal molecular weights. Molecular weight is obviously of critical importance in characterizing this antibody. T h e antibody responsible for the effects noted here must be isolated from other serum components before definite assignment of an immunoglobulin class can be made. A second consideration concerns the nature of the antigen and the conditions responsible for this type of antibody response. While most of the results reported above were obtained with antisera directed against the group a allotypic specificities, limited data with group b specificities and ovalbumin indicate that this response is not limited to the group a specificities. In contrast to most immunizations, preparation of antisera to group a specificities by injection of ovalbumin specific precipitates is a slow process. Precipitating antibodies are rarely obtained before three to four months of immunization. Precipitating antibody to group b specificities normally appears d u r i n g the second month. Possibly the response is influenced in part by the fact that the immunization is being provoked by an i m m u n e precipitate. T h e nature of i m m u n e complexes used as antigens can markedly influence the nature of the resulting response [34]. It should be noted that in the case of antibody directed against group a specificities and provoked by ovalbumin specific precipitates, the antigen is the antibody of the i m m u n e complex. Studies to define the role of antigen in determining the nature of the response are in progress. Acknowledgement--The authors thank Mrs. Karen Feintuch for competent technical
assistance in this research.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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17. Uhr J. W., Finkelstein M. S. and Franklin E. C., Proc. Soc. exp. Biol. Med. 111, 13 (1962). 18. Rosenquist G. L. and Gilden R. V., Biochim. Biophys. Acta 78,543 (1963). 19. Grey H. M., Proc. Soc. exp. Biol. Med. 113, 963 (1963). 20. Farr R. S.,J. infect. Dis. 103,239 (1958). 21. Eisen H. N., Simms E. S. and Potter M.,Biochem. 7, 4126 (1968). 22. Adler F. L.,J. Immun. 95, 26 (1965). 23. Adler F. L.,J. Immun. 95, 39 (1965). 24. Sutherland G. B. and Campbell D. H.,J. Immun. 80, 294 (1958). 25. Steinberg A. G., Prog. med. Genet. 2, 1 (1962). 26. Lamm M. E. and Small P. A.,Jr., Biochem. 5, 267 (1966). 27. Miller F. and Metzger H.,J. biol. Chem. 240, 3325 (1965). 28. Solomon A. and Kunkel H. G., Am.J, Med. 42, 958 (1967). 29. Cooper A. G., Science 157, 933 (1967). 30. StoboJ. D. and Tomasi T. B.,Jr.,J. clin. Invest. 46, 1329 (1967). 31. Swedlund H. A., Gleich G.J. and Chodirker W. B.,J. Immun. 100, 1296 (1968). 32. CebraJ.J. and Small P. A.,Jr., Biochem. 6, 503 (1967). 33. Lewis A. F., Bergsagel D. E., Bruce-Robertson A., Schachter R. K. and Connell G. E., Blood32, 189 (1968). 34. UhrJ. W. and M611er G., Adv. Immunol. 8, 81 (1968).