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The effect of treatment with heat, 2-mercaptoethanol or cysteine on the antigenicity of IgE
lmmunvchemistry, 1972, Vol. 9, pp. 873-882. Pergamon Press. Printed in Great Britain
T H E EFFECT OF T R E A T M E N T W I T H H E A T , 2 - M E R C A P T O E T H A N O L OR CYSTEINE ON T H E A N T I G E N I C I T Y OF IgE* KOJI ITOt, KONRAD WICHER and CARL E. ARBESMAN Allergy Research Laboratory of the Buffalo General Hospital and the Departments of Medicine and Microbiology of the State University of New York at Buffalo (First received 21 December 1971; in revisedform 9 February 1972)
Abstract-The changes in the antigenicity of myeloma IgE (PS) heated at 56°C for various lengths of time or treated with 2ME or cysteine, were examined against antisera containing antibodies to native and/or to denatured IgE. The degree of denaturation of the heated IgE was time dependent. Antigenic changes have been already observed in samples heated for 30 min. Heating for more than 4 hr caused denaturation to such an extent that the antigenic characteristics for native IgE (PS) could no longer be recognized when examined by DDGP. The antigenicity of the Fc fragment of IgE heated for 24 hr changed almost completely and the antigenicity of Fab fragment completely. The Fc' and the F(ab')2 were heat resistant when heated at 56°C for 24 hr. It is suggested that the Fc' fragment is responsible for the heat resistancy of the F(ab')2. The 2ME treated IgE altered partially the antigenicity whereas the cysteine treated IgE failed to show any antigenic changes when examined by DDGP. INTRODUCTION The early work of Coca and Grove (1925) has shown that heating of reaginic antibodies at 56°C for 30 min diminishes the skin-fixing property. Loveless (1940) demonstrated that reaginic antibodies heated at 56°C for 5 hr or at 60°C for 2 min loses completely its skin-fixing property. Several authors (Gyenes et al., 1964; Ishizaka et al., 1966a; Leddy et al., 1962; Reid et al., 1966; Rockey and Kunkel, 1962) agreed that reduction and alkylation of reaginic antibodies (at that time not known to be associated with IgE) also influences the skin-fixing properties. In recent years, after it became known that reaginic antibodies are associated with IgE (Ishizaka et al., 1966b) and after the description of the first two myeloma IgE cases (Johannson and Bennich, 1967; Ogawa et al., 1969), the skin-fixing properties of the reaginic and myeloma IgE have been re-examined. Ishizaka and associates (Ishizaka et al., 1967) demonstrated that reaginic antibodies with specificity to ragweed antigen were not capable of fixing to tissue nor of precipitating with monospecific antiserum to IgE when exposed to 56°C for 2-4 hr, but did react with ragweed antigen in radioimmunodiffusion test. Ishizaka and Ishizaka (1969) also demonstrated that reduction and alkylation of reaginic antibodies resulted in a decrease in skin-sensitizing and antigen-binding *This study was supported in part by United States Public Health Service Research Grant 5-R01-AI-01303 and in part by United States Public Health Service Training Grant 5-T01-AI-00051 of the National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. tRecipient of the Dr. Henry C. Buswell and Bertha H. Buswell Fellowship. 873
874
KOJI ITO, KONRAD WICHER and CARL E. ARBESMAN
activity, whereas the IgE antigenic determinants of the molecules were not impaired, when examined against monospecific antiserum to IgE. Stanworth et al. (1970), using myeloma IgE (ND), demonstrated that reduction and alkylation of the IgE decreased the precipitability with monospecific antiserum to IgE when examined by a quantitative method. The physico-chemical properties of reaginic antibodies and of the myeloma IgE are influenced by the high temperature or chemical substances as was already reported. However, to our knowledge no reports are available demonstrating new antigenicity of the IgE acquired during heating or chemical treatment. This report presents such data. MATERIALS AND METHODS
Purification 0fIgE and preparation of IgE fragments IgE myeloma plasma from patient (PS) was obtained from Dr. M. Ogawa, through the courtesy of Dr. O. R. McIntyre, Dartmouth Medical School, Hanover, New Hampshire. Purified IgE, Fc, Fab, and F(ab')2 fragments of IgE were prepared in a similar way as described by Bennich and Johansson (1967). The Fc' fragment was prepared as previously described (Ito et al., 1971).
Antisera Rabbit and goat antisera to Fc fragment of IgE (PS) were produced as described in Ito et al. (1971). These antisera contained antibodies only to classspecific antigenic determinants when examined against native IgE (PS) and normal human plasma. Sheep antiserum (Lot. No. 8930) to Fc fragment of myeloma IgE (ND), containing antibodies to class-specific antigenic determinants of IgE was made by Drs. S. G. O. Johansson and H. Bennich, The Blood Center, University Hospital and Institute of Biochemistry, University of Uppsala, Uppsala, Sweden and was kindly provided by Dr. D. S. Rowe, WHO International Reference Center for Immunoglobulins, Lausanne, Switzerland. Antiserum to heated IgE (PS) was produced in a goat. Myeloma plasma, containing 1 mg/ml of IgE, was heated at 56°C for 24 hr. The goat was injected twice with 500 tzg of the heated IgE incorporated in complete Freund's adjuvant at a 2-week interval. The animal was bled 2 weeks after the second injection. After absorption with heated normal plasma this antiserum contained antibodies reacting with native and with heated IgE (PS). Antibodies to native IgE present in this antiserum could be absorbed with unheated IgE, leaving in the antiserum antibodies reacting only with heated IgE. Antiserum to h chain (IgG) was kindly supplied by Dr. Y. Yagi, Department of Biochemistry Research, Roswell Park Memorial Institute, Buffalo, New York.
Heating of protein samples Purified IgE (PS) in a concentration of 1 mg/ml and the Fc, Fc', Fab, F(ab')~ fragments in a concentration of 500 lzg/ml in phosphate buffered saline of pH 7.2 were kept in glass tubes in a water bath of 56°C. Aliquots were taken at 30 rain. 1, 2, 4, 8, 24, 48 and 72 hr. All samples contained sodium azida in a concentration of 0.2 mg/ml as an antibacterial agent.
Antigenicity of IgE
875
Reduction and alkylation 0fIgE The procedure described by Ishizaka and Ishizaka (1969) was used with slight modifications. In brief, purified IgE (PS) in a concentration of 1 mg/ml in 0"3 M Tris-HCl buffer of pH 8.2 was mixed with 2-mercaptoethanol (2ME) in a final concentration of 0.1 M and incubated for 1 hr at room temperature. Thereafter, iodoacetamide in an amount corresponding to 10% molar excess of that of 2ME was added. The mixture was dialyzed overnight at 4°C against borate buffered saline, pH 8.0.
Aggregation 0fIgE with cysteine Purified IgE (PS) was treated with cysteine according to the method described by Ishizaka and Ishizaka (1969).
Immunoelectrophoresis (IEP) The micromethod of Scheidegger (1955) using LKB equipment and 1-5% special Noble agar* dissolved in barbital buffer of pH 8-6, ionic strength 0.05, was applied. Electrophoresis was carried out for 1 hr.
Double diffusion gel precipitation (DDGP) This test was performed using 1% special Noble agar* dissolved in the same barbital buffer as used for IEP.
Ultracentrifugation analysis Sedimentation velocity measurements were made in a model E Spinco Ultracentrifuge operated at 59,780 rev/min with 12 mm, 2° sector cells. The examined samples were adjusted to 10 mg/ml in 0-1 N Tris-HC1-0-2 M NaC1 pH 7.7. The measurements were made at 20°C and the $20. w of the corrected sedimentation coefficients was evaluated by extrapolation to zero protein concentration.
Biological activity of the heated I gE Heated and unheated IgE was used for inhibition of passive cutaneous anaphylaxis (PCA) in rhesus monkey skin. Serum of a patient allergic to ragweed and having a Prausnitz-Kiistner (PK) titer of 60 in monkey skin was used tor the test. Heat aggregated and native IgE (PS) in amounts from 10 to 0.03/zg (in 0.02 ml) were injected in duplicate into the skin of monkey. Twenty-four hours later the patient's serum in a dilution of 1 : 10 and 1 : 30 was injected into the same sites. On the third day the sites were challenged with ragweed extract. Prior to the challenge, 3 ml of 0.5% Evans blue was injected I.V. The results were read after 20 min. RES ULTS i. Double diffusion gel precipitation (DDGP) The IgE (PS) samples heated at 56°C for various lengths of time were examined in DDGP using goat, sheep and rabbit antisera to Fc fragment of IgE (PS). The results obtained with rabbit antiserum are seen in Fig. 1. The rabbit anti*Difco Laboratories, Detroit, Michigan 48201.
876
KOJI ITO, KONRAD WICHER and CARL E. ARBESMAN
serum to Fc fragment reacted with the unheated IgE (PS) as well as with IgE heated for 1 and 2 hr. A very broad, diffuse precipitation band (barely visible in this figure) was observed with the sample heated for 4 hr. No reaction was observed with samples heated for 8 and 24 hr. These results indicate that, in the described conditions, the structure of IgE heated for more than 4 hr at 56°C had changed to such an extent that the rabbit antiserum no longer recognized the antigenic determinants. A reaction of identity was observed between the native IgE (PS) and the samples heated for 1 and 2 hr and one may assume therefore that the reaction with the latter samples was due to the unaltered part of the IgE still present in the heated samples. The goat antiserum prepared with the Fc fragment of IgE (PS) could recognize the antigenic determinants of the native as well as of the heated IgE (PS). This reaction is demonstrated in Fig. 2. Two precipitation lines were observed with IgE (PS) heated for 1-4 hr (diffuse lines with 2 and 4 hr heated samples) and single precipitation lines with unheated IgE (PS), 8 and 24 hr heated IgE (PS). It appears that the samples heated for 1-4 hr contained antigenic determinants that are characteristic for both the unheated IgE (demonstrated by the reaction of identity with the native IgE) and the heat denatured IgE (demonstrated by the reaction of identity with IgE heated for 8-24 hr). Antigenic differences between native IgE (PS) and heated IgE (PS) were demonstrated by the reaction of non-identity. Immunoglobulin E heated for 30 min exhibited (not presented in this figure) similar immunodiffusion properties to that heated for 1 hr. It must be stressed that the goat antiserum reacting with both the unheated and heat altered IgE (PS) was obtained from an animal immunized with unheated Fc fragment of IgE (PS). Sheep antiserum to the Fc fragment of IgE (ND) gave reactions similar to those of the goat antiserum when tested against native and heated IgE (PS).
2. Immunoelectrophoresis (IEP) Immunoelectrophoretic mobility of heated and unheated IgE (PS) was examined using goat antiserum containing antibodies to native and heat denatured IgE. The results are seen in Fig. 3. A difference in the electrophoretic mobility was observed. The heated IgE barely moved from the place of application, demonstrating a very short precipitation line, in contrast with the line obtained with unheated IgE (PS).
3. The antigenicity of the 2ME and cysteine-treated IgE The antigenicity of purified IgE treated with 2ME or with cysteine or heated for 24 hr at 56°C were compared in DDGP. The antisera used were an absorbed rabbit antiserum containing antibodies only to native IgE and an absorbed goat antiserum containing antibodies only to denatured IgE. The results are presented in Figs. 4 and 5. A strong precipitation line of the heated IgE, extending (spur) over the weaker precipitation line obtained with the 2ME treated sample is seen in Fig. 4. No precipitation reaction was observed with the cysteine treated IgE. This goat antiserum did not react with native IgE (PS) and one may assume that the cysteine treated IgE is not sufficiently denatured and behaves like the native IgE. This was confirmed (Fig. 5) by using rabbit antiserum reacting only
Antigenicity of IgE
877
with native IgE. A precipitation line of identity with native IgE, cysteine and 2ME treated samples was observed. In view of the results presented in Figs. 4 and 5 one may assume that 2ME only partially alters the IgE (PS) which still reacts with antibodies to native IgE (PS). The altered antigenicity of the 2ME treated IgE differs from that of the heat treated IgE as demonstrated by the spur and the difference in the intensities of the precipitation lines as seen in Fig. 4.
4. Ultracentrifugation analysis Purified, unheated IgE (PS) was examined by analytical ultracentrifugation (Spinco Model E). Only one sharp peak was obtained proving the purity of this sample (Fig. 6a). The same material was heated at 56°C for 24 hr and re-examined in the analytical ultracentrifuge (Fig. 6b). Three different peaks were observed, the sedimentation coefficient o f the first peak (1) was 10"2 S; the next one (2) 11.9 S and the fastest peak (3) was approximately 15 S. The molecular weight of the aggregated IgE because of the heterogeneity of this material could not be calculated. The different aggregates overlapped and, in addition, in the slower moving part (x) there was some smaller molecular weight material. During the 24 hr heating small molecular peptides were probably split off.
5. Biological activity of the heat treated IgE The results of the inhibition reaction of the PCA in monkey skin are presented in Table 1. The reaginic titer of the ragweed allergic patient serum when examined in monkey skin was 60. When diluted 1 : 10 the reaction of the patient's serum was inhibited by 3 ~g of native IgE (PS), and when diluted 1 : 30 it was inhibited by 0.3 ~g of the IgE (PS). The 24 hr heated IgE did not exhibit any inhibitory activity even at a concentration of 10 ~g.
6. Examination of heated IgE fragments Heated and unheated Fc, Fc' F(ab')2 fragments were examined in DDGP with a goat antiserum containing antibodies to both native and heat-aggregated IgE. The heated and unheated F(ab')2 and Fab fragments also were examined with a rabbit antiserum to X chain of IgG. The results of the examination of the Fc and Fc' fragments are presented in Fig. 7. Precipitation line of identity beTable 1. PCA Reaction in a monkey preinjected intradermally with native and heated IgE Dilution of patient serum a
Preinjected IgE (PS)
Concentrations in ~g 3 1 0-3 0-1 0"03
10
1:10
Unheated Heated
+
1 : 30
Unheated Heated
. +
+ .
. +
+ +
+ +
+ +
+ +
. +
-1-
+ +
+ +
algE level of this serum was 800 ng/ml and PCA titer in monkey was 60.
878
KOJI ITO, KONRAD WICHER and CARL E. ARBESMAN
tween the heated and unheated Fc' fragments was observed; the heated Fc fragment has been altered to such an extent that only a very faint precipitation line (barely visible in this figure) could be seen, whereas the non-heated Fc reacted very strongly. These results indicate that the isolated Fc fragment is quite susceptible to heating whereas the Fc' fragment seems to be heat resistant as was demonstrated by the line of identity with the unheated Fc'. The precipitation reaction of the unheated and heated F(ab')2 fragments with the same goat antiserum is presented in Fig. 8. A precipitation line of identity with the unheated and heated F(ab')2 fragments was observed, indicating a resistance to heat, similar to the Fc' fragment. The unheated and heated Fab fragments of IgE were examined against rabbit antiserum to X light chain of IgG (the IgE (PS) is a ~, type myeloma protein). The results are seen in Fig. 9. The heated Fab fragment did not react with the antiserum, whereas the unheated reacted very strongly. This indicates that the ~ chain of Fab fragment is altered completely by heating or that the determinants on the ~, chains may have been masked through architectural alterations during heating. Unheated and heated samples of F(ab')2 were tested against the same rabbit antiserum to ~ chain and against a rabbit antiserum containing antibodies only to native IgE (PS). The results are in Fig. 10. Reaction of identity was observed between the heated and unheated F(ab')2 fragments with the antiserum to native IgE. The unheated F(ab')2 merged into a line of identity with the unheated Fc' fragment. The unheated and heated F(ab')2 fragments also reacted with the antiserum to ~, chain showing a spur extending over the unheated F(ab')2. It is possible that the rabbit antiserum could contain antibodies to denatured L chain and this spur could be due to denatured L chain in the heated F(ab')2. DISCUSSION Two different antisera in goats were produced. One goat was immunized with Fc fragment of IgE (PS). The purified Fc fragment was kept for approximately 10 weeks at -20°C prior to the immunization. This antiserum, after absorption with normal human plasma contained antibodies reacting with both native and heat denatured IgE (PS). The character of the latter antibody has not been sufficiently studied. We may only speculate about this antibody. It is possible that the Fc preparation contained a very small amount of aggregated Fc fragment responsible for the production of this antibody. That IgE easily undergoes spontaneous aggregation was observed by Ishizaka et al. (1970) who reported that IgE kept in the lyophilized state produced wheal and flare reactions when injected into human skin. This reaction was most likely due to spontaneously aggregated IgE. A second goat was immunized with a preparation of IgE (PS)-rich plasma heated for 24 hr. This antiserum, after absorption with normal human plasma, contained antibodies to denatured and native IgE (PS). The antibodies to native IgE could be absorbed by native IgE leaving only antibodies to denatured IgE and vice versa. We have demonstrated that IgE heated for more than 4 hr does not exhibit any antigenic determinants characteristic for native IgE when examined in DDGP. However, even after 24 hr heating some heat resistant antigenic determinants characteristic for the native antigenicity are still present, and cause the production of antibodies in an immun-
Antigenicity of IgE
879
ized goat. The antibody to the native antigenic determinants in the serum of the goat immunized with heated IgE (PS)-rich plasma might be an antibody to the idiotype-specific antigenic determinants. It cannot be excluded however that this antibody could be produced by the heat resistant Fc' fragment, which, during or after the heating of IgE sample, could spontaneously be dissociated. Such spontaneous dissociation has been observed with native IgE (PS) in our laboratories, but we have not yet explored this phenomenon. The antibody to the heat denatured IgE may be an antibody to aggregates of whole molecules and not an antibody to aggregates of IgE (PS) fragments. Therefore this antibody might not be identical with the antibody to denatured IgE of the first goat immunized with Fc preparation. Rabbits were immunized with freshly prepared Fc fragment of the native IgE (PS). This most probably explains why the rabbits did not produce antibodies to altered IgE. The antiserum examined under the experimental conditions described demonstrated only antibody to native IgE. We have, however, in addition examined the rabbit antiserum in DDGP versus heat denatured IgE, using wells of 1 cm in diameter for the antiserum, which were filled up twice. In such conditions a very faint precipitation line was observed with the heated IgE. We may conclude therefore, that the rabbit can also recognize altered antigenic determinants of the IgE. The production of the antibodies to altered protein by the rabbit would depend on the degree of denaturation and the number of altered IgE molecules. However, it may also be that the rabbit is less capable of recognizing the denatured antigens than the goat. The heated IgE has been examined by ultracentrifugal analysis. It was found that the 24 hr heated protein consisted of" heterogenous molecules of sedimentation coefficients in the range from 10 to 15 S. Because of the heterogeneity we may assume that the temperature of 56°C has aggregated the IgE and that because of that the molecular weight of this material could not be determined. The degree of denaturation of IgE at 56°C is time dependent. Antigenic changes already have been observed in the sample heated for 30 rain. Heating for more than 4 hr causes aggregation to such an extent that the antigenic characteristics for native IgE can no longer be recognized when examined in DDGP. The question arose whether all fragments of IgE are susceptible to heat in the same degree. From the experiments performed with isolated Fc, Fc', F(ab')2 and Fab fragments we have observed that the Fc' and the F(ab')., resisted the 24 hr heating at 56°C and did not change their antigenicity when examined in the DDGP against goat antiserum containing antibodies to native and heated lgE. The Fc fragment heated for 24 hr reacted very weakly with the same goat antiserum. The heated Fc fragment was also examined against antisera containing only antibodies to native or only to heat aggregated IgE; a very weak precipitation reaction was seen only with the antibodies to native IgE. This suggests that the heated Fc fragment contained most probably a very small amount of Fc' which is heat resistant. The antigenicity of the heated Fab fragment of IgE was changed to such an extent that it could not react with anti Xchain antiserum, however, the F(ab')._, fragment, heated for 24 hr, reacted with this antiserum. The most likely explanation for the variation of the heat susceptibility of the different IgE fragments is that the Fc' does not contain intra chain S-S bonds,
880
KOJI ITO, KONRAD WlCHER and CARL E. ARBESMAN
as it was reported by Bennich and Johansson (1967) and, as such, may resist the heat aggregation. Since the F(ab')2 contains a portion antigenically identical with the Fc', it is likely that the Fc' is a part of the F(ab')2 and serves as a supporting structure for the Fab portions. We might therefore conclude that the heat resistant Fc' f r a g m e n t is responsible for the heat resistance of the F(ab')2 f r a g m e n t of IgE (PS). T h e aggregation of IgE (PS) by 2ME changes partially the structure of this protein, T h e new antigenicity of the IgE acquired after 2ME treatment differs in part f r o m that acquired after heating at 56°C. T h e cysteine, in our experimental conditions did not alter the IgE (PS) in a sufficient degree to be able to detect it in DDGP. Acknowledgement-The authors wish to acknowledge the competent help of Mr. Paul M. Bronson of the Immunochemistry Laboratory, Department of Microbiology, School of Medicine, State University of New York at Buffalo, Buffalo, New York, who performed the ultracentrifugal analysis.
REFERENCES Bennich H. and Johansson S. G. O. (1967) Nobel Symposium 3, Ganm~a Globulins (Edited by KillanderJ.), p. 199. Almqvist & Wiksell, Stockholm (1967). Coca A. F. and Grove E. F. (1925)J. Immun., 10,445. Gyenes, L., Sehon, A. H., Freedman, S. O. and Ovary, Z. (1964) Int. Archs Allergy 24, 106. Ishizaka K. and Ishizaka T. (1969)J. Immun. 10'2, 69. Ishizaka K., Ishizaka T. and Richter M. (1966a)J. Allergy 37, 135. Ishizaka K., Ishizaka T. and Hornbrook M. M. (1966b)J. Immun. 97, 75. Ishizaka K., Ishizaka T. and Menzel A. E. O. (1967)J. Immun. 99, 610. Ishizaka T., Ishizaka K., Bennich H. andJohansson S. G. O. (1970)J. Immun. 104, 854. Ito K., Wicher K. and Arbesman C. E. (1971) Int. ArchsAUergy, 41,477. Johansson S. G. O. and Bennich H. (1967) Immunology 13,381. Leddy J. P., Freeman G. L., Luz A. and Todd R. H. (1962) Proc. Soc. exp. Biol. Med. 111, 7. Loveless M. H. (1940)J. Immun. 38, 25. Ogawa M., Kochwa S., Smith C., Ishizaka K. and Mclntyre O. R. (1969) New Engl. J. Med. 281, 1217. Reid R. T., Minden P. and Farr R. S. (1966)J. exp. Med. 123,845. RockeyJ. H. and Kunkel H. G. (1962) Proc. Soc. exp. Biol. Med. 110, 101. Scheidegger J. J. (1955) Int. Archs Allergy 7, 103. Stanworth D. R., Housley J., Bennich H. and Johansson S. G. O. (1970)Immunochemistry 7,321.
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T H E EFFECT OF T R E A T M E N T W I T H H E A T , 2 - M E R C A P T O E T H A N O L OR CYSTEINE ON T H E A N T I G E N I C I T Y OF IgE* KOJI ITOt, KONRAD WICHER and CARL E. ARBESMAN Allergy Research Laboratory of the Buffalo General Hospital and the Departments of Medicine and Microbiology of the State University of New York at Buffalo (First received 21 December 1971; in revisedform 9 February 1972)
Abstract-The changes in the antigenicity of myeloma IgE (PS) heated at 56°C for various lengths of time or treated with 2ME or cysteine, were examined against antisera containing antibodies to native and/or to denatured IgE. The degree of denaturation of the heated IgE was time dependent. Antigenic changes have been already observed in samples heated for 30 min. Heating for more than 4 hr caused denaturation to such an extent that the antigenic characteristics for native IgE (PS) could no longer be recognized when examined by DDGP. The antigenicity of the Fc fragment of IgE heated for 24 hr changed almost completely and the antigenicity of Fab fragment completely. The Fc' and the F(ab')2 were heat resistant when heated at 56°C for 24 hr. It is suggested that the Fc' fragment is responsible for the heat resistancy of the F(ab')2. The 2ME treated IgE altered partially the antigenicity whereas the cysteine treated IgE failed to show any antigenic changes when examined by DDGP. INTRODUCTION The early work of Coca and Grove (1925) has shown that heating of reaginic antibodies at 56°C for 30 min diminishes the skin-fixing property. Loveless (1940) demonstrated that reaginic antibodies heated at 56°C for 5 hr or at 60°C for 2 min loses completely its skin-fixing property. Several authors (Gyenes et al., 1964; Ishizaka et al., 1966a; Leddy et al., 1962; Reid et al., 1966; Rockey and Kunkel, 1962) agreed that reduction and alkylation of reaginic antibodies (at that time not known to be associated with IgE) also influences the skin-fixing properties. In recent years, after it became known that reaginic antibodies are associated with IgE (Ishizaka et al., 1966b) and after the description of the first two myeloma IgE cases (Johannson and Bennich, 1967; Ogawa et al., 1969), the skin-fixing properties of the reaginic and myeloma IgE have been re-examined. Ishizaka and associates (Ishizaka et al., 1967) demonstrated that reaginic antibodies with specificity to ragweed antigen were not capable of fixing to tissue nor of precipitating with monospecific antiserum to IgE when exposed to 56°C for 2-4 hr, but did react with ragweed antigen in radioimmunodiffusion test. Ishizaka and Ishizaka (1969) also demonstrated that reduction and alkylation of reaginic antibodies resulted in a decrease in skin-sensitizing and antigen-binding *This study was supported in part by United States Public Health Service Research Grant 5-R01-AI-01303 and in part by United States Public Health Service Training Grant 5-T01-AI-00051 of the National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. tRecipient of the Dr. Henry C. Buswell and Bertha H. Buswell Fellowship. 873
874
KOJI ITO, KONRAD WICHER and CARL E. ARBESMAN
activity, whereas the IgE antigenic determinants of the molecules were not impaired, when examined against monospecific antiserum to IgE. Stanworth et al. (1970), using myeloma IgE (ND), demonstrated that reduction and alkylation of the IgE decreased the precipitability with monospecific antiserum to IgE when examined by a quantitative method. The physico-chemical properties of reaginic antibodies and of the myeloma IgE are influenced by the high temperature or chemical substances as was already reported. However, to our knowledge no reports are available demonstrating new antigenicity of the IgE acquired during heating or chemical treatment. This report presents such data. MATERIALS AND METHODS
Purification 0fIgE and preparation of IgE fragments IgE myeloma plasma from patient (PS) was obtained from Dr. M. Ogawa, through the courtesy of Dr. O. R. McIntyre, Dartmouth Medical School, Hanover, New Hampshire. Purified IgE, Fc, Fab, and F(ab')2 fragments of IgE were prepared in a similar way as described by Bennich and Johansson (1967). The Fc' fragment was prepared as previously described (Ito et al., 1971).
Antisera Rabbit and goat antisera to Fc fragment of IgE (PS) were produced as described in Ito et al. (1971). These antisera contained antibodies only to classspecific antigenic determinants when examined against native IgE (PS) and normal human plasma. Sheep antiserum (Lot. No. 8930) to Fc fragment of myeloma IgE (ND), containing antibodies to class-specific antigenic determinants of IgE was made by Drs. S. G. O. Johansson and H. Bennich, The Blood Center, University Hospital and Institute of Biochemistry, University of Uppsala, Uppsala, Sweden and was kindly provided by Dr. D. S. Rowe, WHO International Reference Center for Immunoglobulins, Lausanne, Switzerland. Antiserum to heated IgE (PS) was produced in a goat. Myeloma plasma, containing 1 mg/ml of IgE, was heated at 56°C for 24 hr. The goat was injected twice with 500 tzg of the heated IgE incorporated in complete Freund's adjuvant at a 2-week interval. The animal was bled 2 weeks after the second injection. After absorption with heated normal plasma this antiserum contained antibodies reacting with native and with heated IgE (PS). Antibodies to native IgE present in this antiserum could be absorbed with unheated IgE, leaving in the antiserum antibodies reacting only with heated IgE. Antiserum to h chain (IgG) was kindly supplied by Dr. Y. Yagi, Department of Biochemistry Research, Roswell Park Memorial Institute, Buffalo, New York.
Heating of protein samples Purified IgE (PS) in a concentration of 1 mg/ml and the Fc, Fc', Fab, F(ab')~ fragments in a concentration of 500 lzg/ml in phosphate buffered saline of pH 7.2 were kept in glass tubes in a water bath of 56°C. Aliquots were taken at 30 rain. 1, 2, 4, 8, 24, 48 and 72 hr. All samples contained sodium azida in a concentration of 0.2 mg/ml as an antibacterial agent.
Antigenicity of IgE
875
Reduction and alkylation 0fIgE The procedure described by Ishizaka and Ishizaka (1969) was used with slight modifications. In brief, purified IgE (PS) in a concentration of 1 mg/ml in 0"3 M Tris-HCl buffer of pH 8.2 was mixed with 2-mercaptoethanol (2ME) in a final concentration of 0.1 M and incubated for 1 hr at room temperature. Thereafter, iodoacetamide in an amount corresponding to 10% molar excess of that of 2ME was added. The mixture was dialyzed overnight at 4°C against borate buffered saline, pH 8.0.
Aggregation 0fIgE with cysteine Purified IgE (PS) was treated with cysteine according to the method described by Ishizaka and Ishizaka (1969).
Immunoelectrophoresis (IEP) The micromethod of Scheidegger (1955) using LKB equipment and 1-5% special Noble agar* dissolved in barbital buffer of pH 8-6, ionic strength 0.05, was applied. Electrophoresis was carried out for 1 hr.
Double diffusion gel precipitation (DDGP) This test was performed using 1% special Noble agar* dissolved in the same barbital buffer as used for IEP.
Ultracentrifugation analysis Sedimentation velocity measurements were made in a model E Spinco Ultracentrifuge operated at 59,780 rev/min with 12 mm, 2° sector cells. The examined samples were adjusted to 10 mg/ml in 0-1 N Tris-HC1-0-2 M NaC1 pH 7.7. The measurements were made at 20°C and the $20. w of the corrected sedimentation coefficients was evaluated by extrapolation to zero protein concentration.
Biological activity of the heated I gE Heated and unheated IgE was used for inhibition of passive cutaneous anaphylaxis (PCA) in rhesus monkey skin. Serum of a patient allergic to ragweed and having a Prausnitz-Kiistner (PK) titer of 60 in monkey skin was used tor the test. Heat aggregated and native IgE (PS) in amounts from 10 to 0.03/zg (in 0.02 ml) were injected in duplicate into the skin of monkey. Twenty-four hours later the patient's serum in a dilution of 1 : 10 and 1 : 30 was injected into the same sites. On the third day the sites were challenged with ragweed extract. Prior to the challenge, 3 ml of 0.5% Evans blue was injected I.V. The results were read after 20 min. RES ULTS i. Double diffusion gel precipitation (DDGP) The IgE (PS) samples heated at 56°C for various lengths of time were examined in DDGP using goat, sheep and rabbit antisera to Fc fragment of IgE (PS). The results obtained with rabbit antiserum are seen in Fig. 1. The rabbit anti*Difco Laboratories, Detroit, Michigan 48201.
876
KOJI ITO, KONRAD WICHER and CARL E. ARBESMAN
serum to Fc fragment reacted with the unheated IgE (PS) as well as with IgE heated for 1 and 2 hr. A very broad, diffuse precipitation band (barely visible in this figure) was observed with the sample heated for 4 hr. No reaction was observed with samples heated for 8 and 24 hr. These results indicate that, in the described conditions, the structure of IgE heated for more than 4 hr at 56°C had changed to such an extent that the rabbit antiserum no longer recognized the antigenic determinants. A reaction of identity was observed between the native IgE (PS) and the samples heated for 1 and 2 hr and one may assume therefore that the reaction with the latter samples was due to the unaltered part of the IgE still present in the heated samples. The goat antiserum prepared with the Fc fragment of IgE (PS) could recognize the antigenic determinants of the native as well as of the heated IgE (PS). This reaction is demonstrated in Fig. 2. Two precipitation lines were observed with IgE (PS) heated for 1-4 hr (diffuse lines with 2 and 4 hr heated samples) and single precipitation lines with unheated IgE (PS), 8 and 24 hr heated IgE (PS). It appears that the samples heated for 1-4 hr contained antigenic determinants that are characteristic for both the unheated IgE (demonstrated by the reaction of identity with the native IgE) and the heat denatured IgE (demonstrated by the reaction of identity with IgE heated for 8-24 hr). Antigenic differences between native IgE (PS) and heated IgE (PS) were demonstrated by the reaction of non-identity. Immunoglobulin E heated for 30 min exhibited (not presented in this figure) similar immunodiffusion properties to that heated for 1 hr. It must be stressed that the goat antiserum reacting with both the unheated and heat altered IgE (PS) was obtained from an animal immunized with unheated Fc fragment of IgE (PS). Sheep antiserum to the Fc fragment of IgE (ND) gave reactions similar to those of the goat antiserum when tested against native and heated IgE (PS).
2. Immunoelectrophoresis (IEP) Immunoelectrophoretic mobility of heated and unheated IgE (PS) was examined using goat antiserum containing antibodies to native and heat denatured IgE. The results are seen in Fig. 3. A difference in the electrophoretic mobility was observed. The heated IgE barely moved from the place of application, demonstrating a very short precipitation line, in contrast with the line obtained with unheated IgE (PS).
3. The antigenicity of the 2ME and cysteine-treated IgE The antigenicity of purified IgE treated with 2ME or with cysteine or heated for 24 hr at 56°C were compared in DDGP. The antisera used were an absorbed rabbit antiserum containing antibodies only to native IgE and an absorbed goat antiserum containing antibodies only to denatured IgE. The results are presented in Figs. 4 and 5. A strong precipitation line of the heated IgE, extending (spur) over the weaker precipitation line obtained with the 2ME treated sample is seen in Fig. 4. No precipitation reaction was observed with the cysteine treated IgE. This goat antiserum did not react with native IgE (PS) and one may assume that the cysteine treated IgE is not sufficiently denatured and behaves like the native IgE. This was confirmed (Fig. 5) by using rabbit antiserum reacting only
Antigenicity of IgE
877
with native IgE. A precipitation line of identity with native IgE, cysteine and 2ME treated samples was observed. In view of the results presented in Figs. 4 and 5 one may assume that 2ME only partially alters the IgE (PS) which still reacts with antibodies to native IgE (PS). The altered antigenicity of the 2ME treated IgE differs from that of the heat treated IgE as demonstrated by the spur and the difference in the intensities of the precipitation lines as seen in Fig. 4.
4. Ultracentrifugation analysis Purified, unheated IgE (PS) was examined by analytical ultracentrifugation (Spinco Model E). Only one sharp peak was obtained proving the purity of this sample (Fig. 6a). The same material was heated at 56°C for 24 hr and re-examined in the analytical ultracentrifuge (Fig. 6b). Three different peaks were observed, the sedimentation coefficient o f the first peak (1) was 10"2 S; the next one (2) 11.9 S and the fastest peak (3) was approximately 15 S. The molecular weight of the aggregated IgE because of the heterogeneity of this material could not be calculated. The different aggregates overlapped and, in addition, in the slower moving part (x) there was some smaller molecular weight material. During the 24 hr heating small molecular peptides were probably split off.
5. Biological activity of the heat treated IgE The results of the inhibition reaction of the PCA in monkey skin are presented in Table 1. The reaginic titer of the ragweed allergic patient serum when examined in monkey skin was 60. When diluted 1 : 10 the reaction of the patient's serum was inhibited by 3 ~g of native IgE (PS), and when diluted 1 : 30 it was inhibited by 0.3 ~g of the IgE (PS). The 24 hr heated IgE did not exhibit any inhibitory activity even at a concentration of 10 ~g.
6. Examination of heated IgE fragments Heated and unheated Fc, Fc' F(ab')2 fragments were examined in DDGP with a goat antiserum containing antibodies to both native and heat-aggregated IgE. The heated and unheated F(ab')2 and Fab fragments also were examined with a rabbit antiserum to X chain of IgG. The results of the examination of the Fc and Fc' fragments are presented in Fig. 7. Precipitation line of identity beTable 1. PCA Reaction in a monkey preinjected intradermally with native and heated IgE Dilution of patient serum a
Preinjected IgE (PS)
Concentrations in ~g 3 1 0-3 0-1 0"03
10
1:10
Unheated Heated
+
1 : 30
Unheated Heated
. +
+ .
. +
+ +
+ +
+ +
+ +
. +
-1-
+ +
+ +
algE level of this serum was 800 ng/ml and PCA titer in monkey was 60.
878
KOJI ITO, KONRAD WICHER and CARL E. ARBESMAN
tween the heated and unheated Fc' fragments was observed; the heated Fc fragment has been altered to such an extent that only a very faint precipitation line (barely visible in this figure) could be seen, whereas the non-heated Fc reacted very strongly. These results indicate that the isolated Fc fragment is quite susceptible to heating whereas the Fc' fragment seems to be heat resistant as was demonstrated by the line of identity with the unheated Fc'. The precipitation reaction of the unheated and heated F(ab')2 fragments with the same goat antiserum is presented in Fig. 8. A precipitation line of identity with the unheated and heated F(ab')2 fragments was observed, indicating a resistance to heat, similar to the Fc' fragment. The unheated and heated Fab fragments of IgE were examined against rabbit antiserum to X light chain of IgG (the IgE (PS) is a ~, type myeloma protein). The results are seen in Fig. 9. The heated Fab fragment did not react with the antiserum, whereas the unheated reacted very strongly. This indicates that the ~ chain of Fab fragment is altered completely by heating or that the determinants on the ~, chains may have been masked through architectural alterations during heating. Unheated and heated samples of F(ab')2 were tested against the same rabbit antiserum to ~ chain and against a rabbit antiserum containing antibodies only to native IgE (PS). The results are in Fig. 10. Reaction of identity was observed between the heated and unheated F(ab')2 fragments with the antiserum to native IgE. The unheated F(ab')2 merged into a line of identity with the unheated Fc' fragment. The unheated and heated F(ab')2 fragments also reacted with the antiserum to ~, chain showing a spur extending over the unheated F(ab')2. It is possible that the rabbit antiserum could contain antibodies to denatured L chain and this spur could be due to denatured L chain in the heated F(ab')2. DISCUSSION Two different antisera in goats were produced. One goat was immunized with Fc fragment of IgE (PS). The purified Fc fragment was kept for approximately 10 weeks at -20°C prior to the immunization. This antiserum, after absorption with normal human plasma contained antibodies reacting with both native and heat denatured IgE (PS). The character of the latter antibody has not been sufficiently studied. We may only speculate about this antibody. It is possible that the Fc preparation contained a very small amount of aggregated Fc fragment responsible for the production of this antibody. That IgE easily undergoes spontaneous aggregation was observed by Ishizaka et al. (1970) who reported that IgE kept in the lyophilized state produced wheal and flare reactions when injected into human skin. This reaction was most likely due to spontaneously aggregated IgE. A second goat was immunized with a preparation of IgE (PS)-rich plasma heated for 24 hr. This antiserum, after absorption with normal human plasma, contained antibodies to denatured and native IgE (PS). The antibodies to native IgE could be absorbed by native IgE leaving only antibodies to denatured IgE and vice versa. We have demonstrated that IgE heated for more than 4 hr does not exhibit any antigenic determinants characteristic for native IgE when examined in DDGP. However, even after 24 hr heating some heat resistant antigenic determinants characteristic for the native antigenicity are still present, and cause the production of antibodies in an immun-
Antigenicity of IgE
879
ized goat. The antibody to the native antigenic determinants in the serum of the goat immunized with heated IgE (PS)-rich plasma might be an antibody to the idiotype-specific antigenic determinants. It cannot be excluded however that this antibody could be produced by the heat resistant Fc' fragment, which, during or after the heating of IgE sample, could spontaneously be dissociated. Such spontaneous dissociation has been observed with native IgE (PS) in our laboratories, but we have not yet explored this phenomenon. The antibody to the heat denatured IgE may be an antibody to aggregates of whole molecules and not an antibody to aggregates of IgE (PS) fragments. Therefore this antibody might not be identical with the antibody to denatured IgE of the first goat immunized with Fc preparation. Rabbits were immunized with freshly prepared Fc fragment of the native IgE (PS). This most probably explains why the rabbits did not produce antibodies to altered IgE. The antiserum examined under the experimental conditions described demonstrated only antibody to native IgE. We have, however, in addition examined the rabbit antiserum in DDGP versus heat denatured IgE, using wells of 1 cm in diameter for the antiserum, which were filled up twice. In such conditions a very faint precipitation line was observed with the heated IgE. We may conclude therefore, that the rabbit can also recognize altered antigenic determinants of the IgE. The production of the antibodies to altered protein by the rabbit would depend on the degree of denaturation and the number of altered IgE molecules. However, it may also be that the rabbit is less capable of recognizing the denatured antigens than the goat. The heated IgE has been examined by ultracentrifugal analysis. It was found that the 24 hr heated protein consisted of" heterogenous molecules of sedimentation coefficients in the range from 10 to 15 S. Because of the heterogeneity we may assume that the temperature of 56°C has aggregated the IgE and that because of that the molecular weight of this material could not be determined. The degree of denaturation of IgE at 56°C is time dependent. Antigenic changes already have been observed in the sample heated for 30 rain. Heating for more than 4 hr causes aggregation to such an extent that the antigenic characteristics for native IgE can no longer be recognized when examined in DDGP. The question arose whether all fragments of IgE are susceptible to heat in the same degree. From the experiments performed with isolated Fc, Fc', F(ab')2 and Fab fragments we have observed that the Fc' and the F(ab')., resisted the 24 hr heating at 56°C and did not change their antigenicity when examined in the DDGP against goat antiserum containing antibodies to native and heated lgE. The Fc fragment heated for 24 hr reacted very weakly with the same goat antiserum. The heated Fc fragment was also examined against antisera containing only antibodies to native or only to heat aggregated IgE; a very weak precipitation reaction was seen only with the antibodies to native IgE. This suggests that the heated Fc fragment contained most probably a very small amount of Fc' which is heat resistant. The antigenicity of the heated Fab fragment of IgE was changed to such an extent that it could not react with anti Xchain antiserum, however, the F(ab')._, fragment, heated for 24 hr, reacted with this antiserum. The most likely explanation for the variation of the heat susceptibility of the different IgE fragments is that the Fc' does not contain intra chain S-S bonds,
880
KOJI ITO, KONRAD WlCHER and CARL E. ARBESMAN
as it was reported by Bennich and Johansson (1967) and, as such, may resist the heat aggregation. Since the F(ab')2 contains a portion antigenically identical with the Fc', it is likely that the Fc' is a part of the F(ab')2 and serves as a supporting structure for the Fab portions. We might therefore conclude that the heat resistant Fc' f r a g m e n t is responsible for the heat resistance of the F(ab')2 f r a g m e n t of IgE (PS). T h e aggregation of IgE (PS) by 2ME changes partially the structure of this protein, T h e new antigenicity of the IgE acquired after 2ME treatment differs in part f r o m that acquired after heating at 56°C. T h e cysteine, in our experimental conditions did not alter the IgE (PS) in a sufficient degree to be able to detect it in DDGP. Acknowledgement-The authors wish to acknowledge the competent help of Mr. Paul M. Bronson of the Immunochemistry Laboratory, Department of Microbiology, School of Medicine, State University of New York at Buffalo, Buffalo, New York, who performed the ultracentrifugal analysis.
REFERENCES Bennich H. and Johansson S. G. O. (1967) Nobel Symposium 3, Ganm~a Globulins (Edited by KillanderJ.), p. 199. Almqvist & Wiksell, Stockholm (1967). Coca A. F. and Grove E. F. (1925)J. Immun., 10,445. Gyenes, L., Sehon, A. H., Freedman, S. O. and Ovary, Z. (1964) Int. Archs Allergy 24, 106. Ishizaka K. and Ishizaka T. (1969)J. Immun. 10'2, 69. Ishizaka K., Ishizaka T. and Richter M. (1966a)J. Allergy 37, 135. Ishizaka K., Ishizaka T. and Hornbrook M. M. (1966b)J. Immun. 97, 75. Ishizaka K., Ishizaka T. and Menzel A. E. O. (1967)J. Immun. 99, 610. Ishizaka T., Ishizaka K., Bennich H. andJohansson S. G. O. (1970)J. Immun. 104, 854. Ito K., Wicher K. and Arbesman C. E. (1971) Int. ArchsAUergy, 41,477. Johansson S. G. O. and Bennich H. (1967) Immunology 13,381. Leddy J. P., Freeman G. L., Luz A. and Todd R. H. (1962) Proc. Soc. exp. Biol. Med. 111, 7. Loveless M. H. (1940)J. Immun. 38, 25. Ogawa M., Kochwa S., Smith C., Ishizaka K. and Mclntyre O. R. (1969) New Engl. J. Med. 281, 1217. Reid R. T., Minden P. and Farr R. S. (1966)J. exp. Med. 123,845. RockeyJ. H. and Kunkel H. G. (1962) Proc. Soc. exp. Biol. Med. 110, 101. Scheidegger J. J. (1955) Int. Archs Allergy 7, 103. Stanworth D. R., Housley J., Bennich H. and Johansson S. G. O. (1970)Immunochemistry 7,321.
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