Int. Archs Allergy appl. Inimun. 51: 416-428 (1976)
Characteristics of Guinea Pig IgE1 Z oltán O vary, B ruce K aplan and Somei K ojima2 Department of Pathology, New York University Medical School, New York, N.Y.
Abstract. Anti-dinitrophenyl (DNP) IgE antibody was produced in guinea pigs (GP) by immunization and repeated boosters with 1 ug DNP-Ascaris extract in A1 (OH)s. The isoelectric point was determined to be 6.48 and the molecular weight to be 185,000 daltons. In absorbtion chromatography, rabbit anti-GPIgG, could not absorb the anaphylactic activity of the IgE. The activity of IgE was destroyed by heating at 56 °C for 1 h. IgE persisted in the skin for at least 3 weeks.
Introduction In the guinea pig, homologous skin sensitizing antibodies belong to two separate classes: IgGi and IgE [1-5]. It has been shown that those of the IgG, class can be further subdivided into two subclasses, IgGla and IgGIb [5]. IgGlb has a slightly faster electrophoretic mobility and they can be differentiated in homologous passive cutaneous anaphylactic reactions because IgGlb persists significantly longer in the skin than IgG,a [5, 6]. Whereas IgG, antibodies are heat stable, IgE antibodies have been shown to be destroyed by heating at 56 °C for 1 h [4, 5]. Furthermore, IgE antibodies are able to persist in the skin much longer than either of the IgG, antibodies [5], These experiments were undertaken to further study IgE antibodies in the guinea pig.
Received: October 30, 1975.
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1 This investigation was supported by National Institutes of Health grant AI03075-17. 2 Recipient of a fellowship from the Cancer Research Institute, New York, N.Y.
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Materials and Methods Animals For immunization, female English Short Hair, Responder, Non-responder, and Hartley strain guinea pigs weighing 400 g were used. For passive cutaneous anaphy lactic reactions, female Hartley strain guinea pigs weighing 300 g were used. The Hartley strain guinea pigs were purchased from Russel Miller Farms, Cazenovia, N. Y. All other strains came from Camm Research Institute, Wayne, N.J.
Immunization For prodution of IgE anti-DNP antibodies, (schedule I) guinea pigs were immu nized intraperitoneally with 1 /¡g DNP-Asc plus 1 mg Al(OH)3 in 0.15 m NaCl and reinjected intraperitoneally at 30-day intervals with 1 ml of the same suspension. Seven days after each booster, the guinea pigs were bled from the retroorbital sinus and the titer of IgG, and IgE were measured by passive cutaneous anaphylaxis (PCA). Seven days after the fifth or sixth booster, the animals were exsanguinated. Those sera whose PCA activity persisted in the skin for 7 days and whose PCA ac tivity was destroyed by heating at 56 °C for 1 h were assumed to contain IgE. To raise IgG, anti-DNP antibodies (schedule II), a solution of DNP-BGG was emulsified with an equal volume of complete Freund’s adjuvant (CFA), to give a final protein concentration of 200 jUg/ml. 0.125 ml of this emulsion was injected into each of the four footpads, for a total of 0.5 ml per guinea pig. Each animal was then boosted 3 times at monthly intervals with four intradermal injections each time of O. 1 ml of 1 mg DNP-BGG/ml in 0.15 m NaCl. Seven days after the last booster, the animals were exsanguinated. Two sera (A and B) which showed a high PCA titer (8,000 and 5,000, respectively) were selected. The PCA activity of these sera was heat stable and if a sensitization period longer than 7 days was used, no PCA reaction could be obtained. Therefore, it was con cluded that these sera did not contain detectable IgE antibody. Sera of eight other guinea pigs were pooled and were used for absorption chromatography. All sera were used immediately or divided in small aliquots and stored at -70 °C until use. Different aliquots were used each time to avoid repeated freezing and thawing. Heating. Heating was carried out in a 56 °C water bath for 1 h using undiluted samples.
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Antigens Ascaris extract (Asc) [7] was obtained as described below. Ascaris suum adult worms were triturated in saline, lyophilized, and resuspended in saline. The suspen sion was then frozen and thawed several times, dialyzed against phosphate buffered saline (PBS) pH 7, and centrifuged. Aliquots were stored at -20 °C until use. Prote in content was determined by micro-Kjeldahl [8]. This protein was then coupled to dinitrophenyl (DNP) resulting in DNP3.4Asc. DNP was also coupled to bovine serum albumin (BSA), bovine /-globulin (BGG), and bovine fibrinogen (BF), (BSA, BGG, and BF were obtained from Armour Pharmaceuticals, Chicago, 111.) [9, 10], resulting in DNP5.,BSA (DNP38BSA), DNP2.6BGG (DNP30BGG), and DNP5BF (DNPI70BF). The subscripts refer to 10~7 mol DNP/mg protein (the number of groups of DNP/molecule of protein is shown in parentheses). Subscripts will be omitted in the text.
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Passive Cutaneous Anaphylaxis PCA was performed as described [11]. Sensitization periods varying between 4 h and 21 days were used. To determine IgE titers, unheated samples were used with a 7-day sensitization period (SP). For determination of IgG,a and IgG,b, heated sam ples were used with a SP of 4 and 48 h, respectively. The animals were challenged with 1 ml of 0.5°/o Evans blue dye (Eastman Organic Chemicals, Rochester, N.Y.) containing 500 «g of DNP-BSA in 0.15 m NaCl. The antibody titer was taken as the last dilution to give a threshold reaction (6 mm diameter blue spot) in 3 of 4 guinea pigs. Precipitations, chromatography, gel filtration, isoelectric focusing, pH determi nations, and absorptions were always done at 4 °C or in melting ice bath. Partial Purification and Determination of Molecular Weight 50 ml of serum with an IgE PCA titer of 1,000 and with an IgG, titer of 500 ob tained from English Short Hair guinea pigs after immunization according to sched ule I was pooled and precipitated at a final concentration of 50% ammonium sul fate, dissolved in 8 ml of 0.15 m NaCl, reprecipitated at 50% ammonium sulfate, re dissolved in 8 ml of 0.15 M NaCl and finally dialyzed extensively against 0.005 M phosphate bufer pH 8 (PB). DEAE. The dialyzed protein solution was first passed through a 40X2.5 cm column packed with DEAE-cellulose (Whatman DE52, Maidstone, Kent) which had been equilibrated with 0.005 m PB, pH 8.0. As it has been observed in preliminary studies that guinea pig reaginic antibody is best eluted with 0.035 M pH 8 bufer, stepwise elutions were performed with 0.005, 0.035, 0.05, and 0.1 m pH 8 PB. The fractions were eluted using an LKB peristaltic pump with an upward flow of 20 ml/ h. 2.5-ml fractions were collected by an LKB Uvicord II Absorbtiometer. All frac tions were titrated for IgE activity by PCA and the fractions showing reaginic activ ity were pooled. Sephadex G-200. The fractions from the DEAE column showing reaginic anti body activity were concentrated to a final volume of 5 ml by positive pressure di alysis. A pair of 2.5 X 90 cm columns were connected in series and packed with Se phadex G-200 (Pharmacia Fine Chemicals, Piscataway, N.J.) to form a single col umn with an effective length of 180 cm. The 5-ml sample was then layered on the column and eluted with borate buffered saline (0.01 M pH 8 borate buffer in 0.15 M NaCl) (BBS) with an upward flow of 12 ml/h. 2.0-ml fractions were collected and the OD280 was monitored by an LKB Uvicord Absorbtiometer. Individual fractions were assayed for reaginic antibody. After the peak of activity was found, all frac tions showing reaginic activity were pooled and concentrated to a final volume of 6 ml by positive pressure dialysis. This material shall be referred to as sample I. Molecular weight determination. The molecular weight of the reaginic antibody was determined using the same column. The column was calibrated by using dextran blue (Pharmacia Fine Chemicals) to measure the void volume, human IgG, la beled with 125I as a marker of 150,000 daltons, ovalbumin (Pharmacia Fine Chemi cals) as a marker of 45,000 daltons, and rabbit IgE as a marker of 215,000 daltons molecular weight. All of the above markers were a generous gift of Dr. Stephen K average is defined as (Ve-Vo)/(Vt-Vo). Ve is the elution volume of the mate
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Stux .
Characteristics of Guinea Pig IgE
419
rial whose molecular weight is to be measured, Vo is the void volume (as measured by dextran blue, and Vt is the total volume of the column. For comparison, serum A was passed on the same column with 12!I-labelled rab bit /-globulin (a generous gift of Dr. M orinobu T akahashi) as marker.
Absorption Chromatography Specific guinea pig (GP) anti-DNP antibodies were obtained by the method of E isen [12]. Briefly, 140 ml of the GP anti-DNP-BGG pool was precipitated by DNP-BF at the equivalence point. The precipitate was then dissolved using 20 ml of 0.1 m DNP-OH. Then 700 mg of streptomycin (Mann Research Laboratories, New York, N.Y.) was added and the mixture was left overnight at 4 °C. The mixture was then centrifuged and the supernatant was dialyzed extensively against 0.005 m PB pH 8.0, layered on a DEAE (Whatman A-52) column and eluted with 0.005 M PB pH 8.0. It has been shown that at this molarity only IgG2 anti-DNP is eluted [13]. The material eluted from the column was concentrated to 13.6 ml (4 mg protein/ml) by positive pressure dialysis, and contained no detectable IgG, (as checked by PCA). This material was coupled to Sepharose 4B beads (Pharmacia Fine Chemicals) by the cyanogen bromide method [14] with an efficiency of 97°/o. The final column measured 25.6X0.6 cm and contained 39 mg total protein. 12 ml of a rabbit anti-GPIgG, (a generous gift of Dr. V ictor N ussenzweig ) was precipitated at a final concentration of 33°/o ammonium sulfate, redissolved in 7 ml of 0.15 m NaCl and extensively dialyzed against BBS. The antiserum was then pas sed through the Sepharose GPIgG, column. The rabbit anti-GPIgG, serum that came through the Sepharose anti-GPIgG2 column was concentrated by positive pressure dialysis to 5 ml (8.2 mg protein/ml) and coupled to Sepharose 4B beads (Pharmacia Fine Chemicals) by the above meth od with an efficiency of 88%. The final rabbit anti-GPIgG, column had a volume of 16X0.6 cm and a total of 36.35 mg protein. 0.5 ml of the purified GPIgE sample I (the material concentrated after Sephadex G-200 filtration) was passed through the Sepharose rabbit anti-GPIgG, column. The material that came through the column was concentrated to a volume of 2.4 ml by positive pressure dialysis and tested for reaginic antibody activity by PCA. The ma terial that remained on the column was eluted using 5 m guanidine HC1 (Eastman Kodak, Rochester, N.Y.) which had been adjusted to pH 7 with Tris (hydroxyme thyl) Aminomethane (Fischer Scientific, Fairlawn, N.J.). This material was dialyzed against BBS, concentrated to a volume of 2.0 ml, and titrated for PCA activity.
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Isoelectric Focusing Isoelectric focusing was performed on 0.5 ml of sample I or on 0.5 ml of serum A or serum B. Ampholine with a range of pH 5-10 (LKB Produkter, Brommal, Sweden) was used. An LKB 3371E power supply was adjusted to a constant 600 V, and the amperage was allowed to decrease until no further change was seen. After 72 h, 2.5-ml fractions were removed by an LKB peristaltic pump with a flow rate of 17 ml/h, and their OD280 was determined by an LKB Uvicord II Absortiometer. The pH of each fraction was measured using a radiometer titrator type TTT1 (Radiome ter, Copenhagen). Each fraction was individually dialyzed against BSS and titrated by PCA for IgE and IgG,.
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Table I. IgE and IgEi titers of English Short Hair guinea pigs 7 days after boosters Guinea pig No.1
1 2 3 4 5 6 7 8 9 10
Boost second2 IgE
IgGi
fifth2 IgE
IgGi
0 100 100 0 100 100 0 0 0 100
20 20 20 0 20 20 0 0 0 20
0 8,000 4,000 1,000 8,000 8,000 0 0 0 0
100 500 2,000 500 500 500 0 100 0 0
1 Guinea pigs immunized according to schedule I. 2 Figures in table refer to reciprocal of last dilution giving reaction (6 mm blue spot) in 3 of 4 guinea pigs.
0.5 ml of serum A and serum B were also passed through the column before and after the reaginic antibody to test the effectiveness of the column.
Of all the strains tested, the best results were obtained with the English Short Hair strain. Generally, about half of these animals produced IgE antibody. In table I, results from one experiment are summarized. The worst response was obtained with the Hartley strain. Only 1 of 10 animals responded, and the PCA titer of IgE antibody was never higher than ten. In the other two strains (Camm Responder and Non-responder), 2-4 of 10 animals produced IgE antibody, but the titers were lower than in the English Short Hair strain. In every case, heated and unheated sera from individual animals were titrated using a SP of 4 h, 2 and 7 days. Though generally IgE and IgG, were detected in the same sera, there was no correlation between titers of IgE and IgG,. It must be emphasized that in some cases a high titer of IgE was associated with a low IgG, antibody titer (table I).
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Results
Characteristics of Guinea Pig IgE
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Table II. Effect of different SP on heated and unheated serum from guinea pig No. 25, scored as reaction size (mm diameter) performed in four guinea pigs Serum, days after immuni zation
Dilution
Heating at 56°C 1h
Sensitization period 4h 2 days
7 days
1:100 1:400 1:800 1:1,600
-
18 20 20 18 13 14 13 14 10 13 12 10 0 12 8 8
20 17 15 10
1:10 1:100 1:400 1:800 1:1,600
+ + + + +
11 10 12 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
9 0 0 0 0
9 0 0 0
1:100 1:400 1:800 1:1,600
-
20 20 20 18 14 15 14 14 12 10 12 12 8 8 10 10
30 22 17 15
30 25 19 18
ND 12 6 0 0
ND 12 0 0 0
0 0 0 0
1:10 1:100 1:400 1:800 1:1,600
-
+ + + + +
10 12 0 6 0 0 0 0
6 0 0 0
22 23 20 22 18 22 13 17 13 18 12 8 8 12 0 6
0
0 0 0 0 0
8 0 0 0 0
25 20 14 11
18 18 16 14
0 14 0 0 0 0 0 0
18 18 16 12 13 12 11 6 12 10 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
30 20 20 20 22 13 13 13 20 12 13 10 12 8 8 6 ND 0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
For each SP, all dilutions of heated and unheated samples of both sera were injected into the same guinea pig; every vertical column represents one guinea pig.
Persistence in the Skin A typical experiment of PCA reaction is shown in table II, using indi vidual sera from an English Short Hair guinea pig (after the second and
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For those experiments measuring the persistance in the skin, individu al sera were used. As stated above, for DEAE chromatography, molecu lar weight determination, electrofocusing, and absorption experiments, the serum pooled from English Short Hair guinea pigs was used. The IgE produced by these animals was very unstable, and its activity was de creased even when stored at -70 °C. After 3 months, 50-75% of the IgE activity was lost as shown by PCA [K ojima et al., to be published].
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Fraction number
Fig. 1. Sephadex G-200 gel filtration of GPIgE. 2.0-ml fractions were individu ally checked for reaginic antibody activity by PCA (vertical bars).
third boosters). Very similar results were obtained with individual sam ples of sera containing IgE even from other strains taken 7 days after any of the boosters. The only variable was the titer of the sera. Heating for 1 h at 56 °C consistently reduced the titer. As can be seen from table II, the heated serum with a 7-day SP had a lower PCA titer than with a 4-hour SP (91 day bleeding). But even with 4 h SP, the titer was reduced by 75% or more. End dilution titers of heated sera A and B were the same as that of the unheated sera. The persistence in the skin was assayed using individual high titer sera (titer of 1,600). Even with a 21-day SP, the unheated sample gave a PCA reaction at a dilution of 1,600. Identical results were obtained with a 14day SP (not shown).
Sephadex G-200 Chromatography The fraction from the DEAE chromatography was concentrated from 77.5 to 5 ml and put on a Sephadex G-200 column. The peak of PCA ac tivity ran faster than the bulk of the protein (fig. 1). That this PCA activ-
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DEAE Chromatography The heat-labile PCA activity was eluted by the 0.035 and the 0.05 m PB. The pooled fractions, with a volume of 77.5 ml, showed a PCA titer of 20, but upon heating only the undiluted sample gave a PCA reaction. It is well known that IgG! is also eluted at 0.035-0.05 M [13].
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423
Fig. 2. Estimation of the molecular weight of GPIgE by Sephadex G-200 gel fil tration markers. Ovalbumin = 45,000 daltons; 125I human IgG, = 150,000 daltons; rabbit IgE = 215,000 daltons.
ity was actually due to IgE, was shown by the fact that the pooled frac tions, which had a PCA titer of 200 unheated with a 7-day SP, had a PCA titer of only 10 upon heating for 1 h at 56 °C with a 4-hour or 7-day SP. The Sephadex G-200 column was also used for determination of the molecular weight. The K average of the markers and the unknown prote in were determined, as seen in figure 2. The molecular weight of the GPIgE was found to be 185,000 daltons. The peak of IgG, PCA activity coincided exactly with the peak of the t25I-labelled rabbit y-globulin which was taken as 140,000 daltons. Isoelectric Focusing Isoelectric focusing was also performed on two sera with high titer IgGi. When serum A was fractionated, the PCA activity was found in fractions ranging from pH 7.01-6.3, with a peak at 6.6. When serum B was used, the PCA activity was found in the range pH 6.87-6.20, with a peak at 6.87. When sample I was electrofocused, the IgE titer was found between pH 6.2 and pH 7.1 with a peak at pH 6.53. Another trial gave a range of pH 6.08-6.5, with a peak at 6.48. These results indicate that the anti-DNP IgE is only slightly more electronegative than the IgG,.
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Affinity Chromatography Samples of both IgG, and IgE containing antisera were passed through the column containing rabbit anti-GPIgG, coupled to Sepharose 4B
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beads. Serum A was first passed through the column. After passage through the column, the serum which had an original PCA titer of 8,000 had a titer less than 5 when normalized to the same volume. After elution with 5 m guanidine HC1, the fraction eluted showed a titer of 650 when normalized. Sample I, at the time used for affinity chromatography, had an IgE ti ter of 200. After passage through the same anti-GPIgG, column, the normalized sample had an IgE titer of 100, a reduction of only 50%. Whereas a line was observed in double diffusion in agar against the rabbit anti-GPIgG! before passage through the column, no line was observed af ter passage. After elution with guanidine HC1, 5 M, no PCA activity was detected. Finally, serum B was passed through the same rabbit anti-GPIgG, col umn to ascertain that the column was still active. After passage through the column, the serum, when normalized, had an IgG, activity of only 8% as compared to the original sample. The fraction eluted from the column by 5 m guanidine HC1 had an IgG, titer by PCA of 500 when normalized.
It is well known that high amounts of antigen and CFA produce IgG, and IgG, antibodies in guinea pigs [1-3]. It is remarkable to note that low amounts of antigen and Al(OH)3 as adjuvant (schedule I) produces high titer IgE antibodies in most of those animals that are producing IgE anti bodies. The influence of low doses of antigen and Al(OH)3 as adjuvant for preferential production of IgE over IgG was already observed in other species such as the rabbit [15] and the mouse [16], The IgG, content us ing schedule I for immunization is generally much less than that observed in animals immunized with large doses of antigen and CFA. Moreover, some animals did not produce antibodies using schedule I for immuniza tion (table I). It is also interesting to note that the IgE antibody content of the sera in those animals that responded, progressively increased after the boosters. The first to observe heat labile (reaginic or IgE) antibodies in guinea pigs was C atty [4] in animals infested with Trichinella spiralis. D obson et al. [17, 18] used A. suum infestation for IgE production in guinea pigs. P arish [5] was the first to obtain IgE antibodies against a natural protein and also against a haptenic determinant. He was also the first to use alum
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Discussion
Characteristics of Guinea Pig IgE
425
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as an adjuvant. Others could also obtain IgE antibodies in guinea pigs against haptenic determinants [19-21] or against natural proteins [19, 21-23] using as adjuvant alum [20] pertussis vaccine [19], lipopolysaccharides [22, 23], or synthetic double stranded RNA [23]. The original observations of C atty [4] and P arish [5] showed that heating at 56 °C for 1 h destroyed reaginic guinea pig antibodies. Other species also exhibit this general property of reaginic antibodies, including the mouse [24], the rat [25], the rabbit [26, 27] and man [28]. We also observed that heating at 56 °C for 1 h drastically reduced reaginic anti body activity. With a sensitization period of 7 days, the titer of the heated serum was consistently less than that seen at 4 h. The small residual ac tivity after heating might be due to some IgGlb contamination, especially as the titer seems to drop with longer sensitization periods. However, it is not impossible that a heat-stable subpopulation of the IgE exists, similar to what has been suggested in the rabbit [27]. L indquist [29] described a heat-stable rabbit IgE antibody. However, he titrated his sera by the diameter of the reaction, and not by end-dilu tion titer. It has been pointed out that end-dilution titer is more reliable and more sensitive than the measurement of the size of the reaction [27]. It is remarkable that IgE activity can persist in the skin for up to 21 days. This is similar to what is seen in the case of human IgE [30]. Samples of pooled and individual sera gave similar results when heat ed and unheated specimens were titrated for PCA activity. Because of this similarity, and because there was not enough IgE containing serum from a single guinea pig, it was necessary to pool several sera for molecular weight determination, electrofocusing, and absorbtion experiments. The molecular weight of GPIgE in these experiments was shown to be 185,000 daltons. This confirms and extends the observations of D obson et al. [17, 18]. These authors, however, did not give precise figures and they also found that a fraction of IgE had an S rate of 8, whereas another fraction had an S rate of 11 and concluded that the 11 S fraction is probably a polymer or an aggregate of the 8 S fraction. Contrary to rabbit IgE [27], electrofocusing showed very little differ ence between GPIgE and IgGj. The molecular weight of 185,000 daltons is substantially higher than that of IgG,. The higher molecular weight of IgE is consistent with data from man [31] and rabbits [27, 29, 32]. In addition to heat lability and long persistance in the skin, affinity chromatography was used to definitely differentiate between IgG, and IgE.
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The Sepharose 4B specific rabbit anti-GPIgG, column absorbed all the IgG, content of the sera which were passed through the column and after elution with 5 M guanidine a part of the IgG, content was recovered. There was only 50% absorption of IgE by the column. Elution with 5 M guanidine HC1 is a very drastic treatment, and it is not surprising that only a fraction of the IgG, antibody could be re covered. Keeping in mind the fragility of IgE, a reduction in titer from 200 to 100 after passage through this column is not surprising. However, it is also possible that our rabbit antiserum contained traces of anti-IgE. These results show that the rabbit anti-GPIgG, antiserum can distinguish between IgG, and IgE, and does not absorb the latter. Therefore, cross reacting determinants were not demonstrated between these two classes of immunoglobulins. Of course, this does not eliminate the possibility of their existence. However, with carefully selected anti-GPIgG, antisera, it is possible to differentiate between IgG, and IgE. Until sufficient amounts of purified GPIgE antibodies can be isolated for sequencing or until an IgE guinea pig myeloma is obtained for com plete physicochemical and immunological characterization, IgE antibod ies in this species can be defined as those heat-labile antibodies which persist in the injected skin sites for weeks and which are not absorbed by anti-IgG, antibodies. Acknowledgements
de
The authors wish to thank Mr. C saba de Szalay and Mrs. Z suzsanna F ekete N agyivany for their excellent technical help, and Mr. F rank R eilly for his effi
cacious secretarial assistance.
1 Benacerraf, B.; O vary, Z.; Bloch , K. J., and F ranklin, E. C.: Properties of guinea pig 7 S antibodies. I. Electrophoretic separation of two types of guinea pig 7 S antibodies. J. exp. Med. 117: 937-949 (1963). 2 O vary, Z.; Benacerraf, B., and B loch, K. J.: Properties of guinea pig 7 S anti bodies. II. Identification of antibodies involved in passive cutaneous and system ic anaphylaxis. J. exp. Med. 117: 951-964 (1963). 3 B loch , K. J.; K ourilsky, F . M.; O vary, Z., and Benacerraf, B.: Properties of guinea pig 7 S antibodies. III. Identification of antibodies involved in comple ment fixation and hemolysis. J. exp. Med. 117: 965-981 (1963).
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References
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4 C atty, D.: The immunology of nematode infections Trichinosis in guinea pigs as a model. Monogr. Allergy, vol. 5, p. 1 (Karger, Basel 1969).
and guinea pig anaphylactic antibodies. J. Immun. 108: 1055-1062 (1972). 7 Strejan , G. and C ampbell, D. H.: Hypersensitivity to Ascaris antigens. I. Skinsensitizing activity of serum fractions from guinea pigs sensitized to crude ex tracts. J. Immun. 98: 893-900 (1967). 8 M ayer, M. M.: Kjeldhal nitrogen determination; in K abat and M ayer Experi mental immunology, p. 476-483 (Thomas, Springfield 1971). 9 E isen , H. N.; Belman, S., and C arsten, M. E.: The reaction of 2,4-dinitrobenzenesulfonic acid with free amino groups of proteins. J. Am. chem. Soc. 75: 4583-4585 (1953). 10 O vary, Z. and Benacerraf, B.: Immunological specificity of the secondary re sponse with dinitrophenylated proteins. Proc. Soc. exp. Biol. Med. 114: 72-76 (1963). 11 O vary, Z.: Passive cutaneous anaphylaxis; in A ckroyd Immunological methods. CIOMS symposium, p. 259-283 (Blackwell, London 1964). 12 E isen , H. N.: Preparation of purified anti-2,4-dinitrophenyl antibodies; in E isen Methods in medical research, p. 94-102 (Year Book, Chicago 1964). 13 Spitz , E. and O vary, Z.: Q uantitative studies on y2 anti-dinitrophenyl antibod ies. Proc. Soc. exp. Biol. Med. 122: 253-257 (1966). 14 A xen , R.; P orath, J., and E rnback, S.: Chemical coupling of peptides and pro teins to polysaccharides by means of cyanogen halides. Nature, Lond. 214: 1302-1304(1967). 15 R evoltella , R. and O vary, Z.: Preferential production of rabbit reaginic anti bodies. Int. Archs Allergy appl. Immun. 36: 282-289 (1969). 16 R evoltella , R. and O vary, Z.: Reaginic antibody production in different mouse strains. Immunology 17: 45-54 (1969). 17 D obson, C.; M orseth , D. J. and Soulsby, E. J. L.: Immunoglobulin E-type anti bodies induced by Ascaris suum infections in guinea pigs. J. Immun. 106: 128-133 (1971). 18 D o bso n , C.; R o c k e y , J. H., and S o u l s b y , E. J. L.: Immunoglobulin E antibod ies in guinea pigs. Characterization of monomeric and polymeric components. J. Immun. 107: 1431-1439 (1971). 19 M ota, I. and P erini, A.: A heat labile mercaptoethanol susceptible homocytotropic antibody in the guinea pig. Life Sci. 9: 923-930 (1970). 20 L evine , B. B.; C hang, H., jr., and V az, N. M.: The production of hapten-specif ic reaginic antibodies in the guinea pig. J. Immun. 106: 29-33 (1971). 21 P erini, A. and M ota, I.: Heterogeneity of guinea-pig homocytotropic antibodies. Immunology 22: 915-923 (1972). 22 P erini, A. and M ota, I.: The production of IgE and IgG, antibodies in guineapigs immunized with antigen and bacterial lipopolysaccharides. Immunology 25: 297-305 (1973).
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5 P arish, W. E.: Hom ologous serum passive cutaneous anaphylaxis in guinea pigs m ediated by two y, o r y,-type heat-stable globulins and a non-y, heat-labile reagin. J. Immun. 105: 1296-1298 (1970). 6 O vary, Z. and W arner, N. L.: Electrophoretic and antigenic analysis of mouse
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23 M ota, I. and P erini, A.: The effect of a synthetic double-stranded RNA on IgG, and IgE production by guinea-pigs. A comparative study with lipopolysac charide. Immunology 29: 319-323 (1975). 24 M ota, I. and P eixoto , J. M.: A skin sensitizing and thermolabile antibody in the mouse. Life Sci. 5: 1723-1728 (1966). 25 Binaghi, R. A. and Benacerraf, B.: The production of anaphylactic antibody in the rat. J. Immun. 92: 920-933 (1964). 26 Z vaifler , N. J. and Becker , E. L.: Rabbit anaphylactic antibody. J. exp. Med. 123: 935-941 (1966). 27 Stux , S. and O vry, Z.: IgE and a new subclass, lgGa-homologous skin sensitiz ing antibodies in the rabbit. Fed. Proc. Fed. Am. Socs exp. Biol. 34: 1000 (1975).
28 I shizaka, K.; I shizaka, T., and M enzel , A. E. O.: Physicochemical properties of reaginic antibody. VI. Effect of heat on -/E-, yG- and yA-antibodies in the sera of ragweed sensitive patients. J. Immun. 99: 610-618 (1967). 29 L indqvist , K. J.: A unique class of rabbit immunoglobulins eliciting passive cu taneous anaphylaxis in homologous skin. Immunochemistry 5: 525-542 (1968). 30 A ugustin , R.: Techniques for the study and assay of reagins in allergic sub jects; in W eir Handbook of experimental immunology, p. 42.1-42.42 (Blackwell, Oxford 1973). 31 J ohansson, S. G. O. and Bennich , H.: Immunological studies of an atypical (myeloma) immunoglobulin. Immunology 13: 381-394 (1967). 32 Z vaifler , N. J. and R obinson , J. O.: Rabbit homocytotropic antibody. A unique rabbit immunoglobulin analogous to human IgE. J. exp. Med. 130: 907-930 (1969).
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Correspondence to: Dr. Z oltán O vary, Department of Pathology, New York Uni versity Medical Center, 550 First Avenue, New York, N Y 10016 (USA)
Characteristics of Guinea Pig IgE1 Z oltán O vary, B ruce K aplan and Somei K ojima2 Department of Pathology, New York University Medical School, New York, N.Y.
Abstract. Anti-dinitrophenyl (DNP) IgE antibody was produced in guinea pigs (GP) by immunization and repeated boosters with 1 ug DNP-Ascaris extract in A1 (OH)s. The isoelectric point was determined to be 6.48 and the molecular weight to be 185,000 daltons. In absorbtion chromatography, rabbit anti-GPIgG, could not absorb the anaphylactic activity of the IgE. The activity of IgE was destroyed by heating at 56 °C for 1 h. IgE persisted in the skin for at least 3 weeks.
Introduction In the guinea pig, homologous skin sensitizing antibodies belong to two separate classes: IgGi and IgE [1-5]. It has been shown that those of the IgG, class can be further subdivided into two subclasses, IgGla and IgGIb [5]. IgGlb has a slightly faster electrophoretic mobility and they can be differentiated in homologous passive cutaneous anaphylactic reactions because IgGlb persists significantly longer in the skin than IgG,a [5, 6]. Whereas IgG, antibodies are heat stable, IgE antibodies have been shown to be destroyed by heating at 56 °C for 1 h [4, 5]. Furthermore, IgE antibodies are able to persist in the skin much longer than either of the IgG, antibodies [5], These experiments were undertaken to further study IgE antibodies in the guinea pig.
Received: October 30, 1975.
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1 This investigation was supported by National Institutes of Health grant AI03075-17. 2 Recipient of a fellowship from the Cancer Research Institute, New York, N.Y.
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Materials and Methods Animals For immunization, female English Short Hair, Responder, Non-responder, and Hartley strain guinea pigs weighing 400 g were used. For passive cutaneous anaphy lactic reactions, female Hartley strain guinea pigs weighing 300 g were used. The Hartley strain guinea pigs were purchased from Russel Miller Farms, Cazenovia, N. Y. All other strains came from Camm Research Institute, Wayne, N.J.
Immunization For prodution of IgE anti-DNP antibodies, (schedule I) guinea pigs were immu nized intraperitoneally with 1 /¡g DNP-Asc plus 1 mg Al(OH)3 in 0.15 m NaCl and reinjected intraperitoneally at 30-day intervals with 1 ml of the same suspension. Seven days after each booster, the guinea pigs were bled from the retroorbital sinus and the titer of IgG, and IgE were measured by passive cutaneous anaphylaxis (PCA). Seven days after the fifth or sixth booster, the animals were exsanguinated. Those sera whose PCA activity persisted in the skin for 7 days and whose PCA ac tivity was destroyed by heating at 56 °C for 1 h were assumed to contain IgE. To raise IgG, anti-DNP antibodies (schedule II), a solution of DNP-BGG was emulsified with an equal volume of complete Freund’s adjuvant (CFA), to give a final protein concentration of 200 jUg/ml. 0.125 ml of this emulsion was injected into each of the four footpads, for a total of 0.5 ml per guinea pig. Each animal was then boosted 3 times at monthly intervals with four intradermal injections each time of O. 1 ml of 1 mg DNP-BGG/ml in 0.15 m NaCl. Seven days after the last booster, the animals were exsanguinated. Two sera (A and B) which showed a high PCA titer (8,000 and 5,000, respectively) were selected. The PCA activity of these sera was heat stable and if a sensitization period longer than 7 days was used, no PCA reaction could be obtained. Therefore, it was con cluded that these sera did not contain detectable IgE antibody. Sera of eight other guinea pigs were pooled and were used for absorption chromatography. All sera were used immediately or divided in small aliquots and stored at -70 °C until use. Different aliquots were used each time to avoid repeated freezing and thawing. Heating. Heating was carried out in a 56 °C water bath for 1 h using undiluted samples.
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Antigens Ascaris extract (Asc) [7] was obtained as described below. Ascaris suum adult worms were triturated in saline, lyophilized, and resuspended in saline. The suspen sion was then frozen and thawed several times, dialyzed against phosphate buffered saline (PBS) pH 7, and centrifuged. Aliquots were stored at -20 °C until use. Prote in content was determined by micro-Kjeldahl [8]. This protein was then coupled to dinitrophenyl (DNP) resulting in DNP3.4Asc. DNP was also coupled to bovine serum albumin (BSA), bovine /-globulin (BGG), and bovine fibrinogen (BF), (BSA, BGG, and BF were obtained from Armour Pharmaceuticals, Chicago, 111.) [9, 10], resulting in DNP5.,BSA (DNP38BSA), DNP2.6BGG (DNP30BGG), and DNP5BF (DNPI70BF). The subscripts refer to 10~7 mol DNP/mg protein (the number of groups of DNP/molecule of protein is shown in parentheses). Subscripts will be omitted in the text.
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Passive Cutaneous Anaphylaxis PCA was performed as described [11]. Sensitization periods varying between 4 h and 21 days were used. To determine IgE titers, unheated samples were used with a 7-day sensitization period (SP). For determination of IgG,a and IgG,b, heated sam ples were used with a SP of 4 and 48 h, respectively. The animals were challenged with 1 ml of 0.5°/o Evans blue dye (Eastman Organic Chemicals, Rochester, N.Y.) containing 500 «g of DNP-BSA in 0.15 m NaCl. The antibody titer was taken as the last dilution to give a threshold reaction (6 mm diameter blue spot) in 3 of 4 guinea pigs. Precipitations, chromatography, gel filtration, isoelectric focusing, pH determi nations, and absorptions were always done at 4 °C or in melting ice bath. Partial Purification and Determination of Molecular Weight 50 ml of serum with an IgE PCA titer of 1,000 and with an IgG, titer of 500 ob tained from English Short Hair guinea pigs after immunization according to sched ule I was pooled and precipitated at a final concentration of 50% ammonium sul fate, dissolved in 8 ml of 0.15 m NaCl, reprecipitated at 50% ammonium sulfate, re dissolved in 8 ml of 0.15 M NaCl and finally dialyzed extensively against 0.005 M phosphate bufer pH 8 (PB). DEAE. The dialyzed protein solution was first passed through a 40X2.5 cm column packed with DEAE-cellulose (Whatman DE52, Maidstone, Kent) which had been equilibrated with 0.005 m PB, pH 8.0. As it has been observed in preliminary studies that guinea pig reaginic antibody is best eluted with 0.035 M pH 8 bufer, stepwise elutions were performed with 0.005, 0.035, 0.05, and 0.1 m pH 8 PB. The fractions were eluted using an LKB peristaltic pump with an upward flow of 20 ml/ h. 2.5-ml fractions were collected by an LKB Uvicord II Absorbtiometer. All frac tions were titrated for IgE activity by PCA and the fractions showing reaginic activ ity were pooled. Sephadex G-200. The fractions from the DEAE column showing reaginic anti body activity were concentrated to a final volume of 5 ml by positive pressure di alysis. A pair of 2.5 X 90 cm columns were connected in series and packed with Se phadex G-200 (Pharmacia Fine Chemicals, Piscataway, N.J.) to form a single col umn with an effective length of 180 cm. The 5-ml sample was then layered on the column and eluted with borate buffered saline (0.01 M pH 8 borate buffer in 0.15 M NaCl) (BBS) with an upward flow of 12 ml/h. 2.0-ml fractions were collected and the OD280 was monitored by an LKB Uvicord Absorbtiometer. Individual fractions were assayed for reaginic antibody. After the peak of activity was found, all frac tions showing reaginic activity were pooled and concentrated to a final volume of 6 ml by positive pressure dialysis. This material shall be referred to as sample I. Molecular weight determination. The molecular weight of the reaginic antibody was determined using the same column. The column was calibrated by using dextran blue (Pharmacia Fine Chemicals) to measure the void volume, human IgG, la beled with 125I as a marker of 150,000 daltons, ovalbumin (Pharmacia Fine Chemi cals) as a marker of 45,000 daltons, and rabbit IgE as a marker of 215,000 daltons molecular weight. All of the above markers were a generous gift of Dr. Stephen K average is defined as (Ve-Vo)/(Vt-Vo). Ve is the elution volume of the mate
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Stux .
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rial whose molecular weight is to be measured, Vo is the void volume (as measured by dextran blue, and Vt is the total volume of the column. For comparison, serum A was passed on the same column with 12!I-labelled rab bit /-globulin (a generous gift of Dr. M orinobu T akahashi) as marker.
Absorption Chromatography Specific guinea pig (GP) anti-DNP antibodies were obtained by the method of E isen [12]. Briefly, 140 ml of the GP anti-DNP-BGG pool was precipitated by DNP-BF at the equivalence point. The precipitate was then dissolved using 20 ml of 0.1 m DNP-OH. Then 700 mg of streptomycin (Mann Research Laboratories, New York, N.Y.) was added and the mixture was left overnight at 4 °C. The mixture was then centrifuged and the supernatant was dialyzed extensively against 0.005 m PB pH 8.0, layered on a DEAE (Whatman A-52) column and eluted with 0.005 M PB pH 8.0. It has been shown that at this molarity only IgG2 anti-DNP is eluted [13]. The material eluted from the column was concentrated to 13.6 ml (4 mg protein/ml) by positive pressure dialysis, and contained no detectable IgG, (as checked by PCA). This material was coupled to Sepharose 4B beads (Pharmacia Fine Chemicals) by the cyanogen bromide method [14] with an efficiency of 97°/o. The final column measured 25.6X0.6 cm and contained 39 mg total protein. 12 ml of a rabbit anti-GPIgG, (a generous gift of Dr. V ictor N ussenzweig ) was precipitated at a final concentration of 33°/o ammonium sulfate, redissolved in 7 ml of 0.15 m NaCl and extensively dialyzed against BBS. The antiserum was then pas sed through the Sepharose GPIgG, column. The rabbit anti-GPIgG, serum that came through the Sepharose anti-GPIgG2 column was concentrated by positive pressure dialysis to 5 ml (8.2 mg protein/ml) and coupled to Sepharose 4B beads (Pharmacia Fine Chemicals) by the above meth od with an efficiency of 88%. The final rabbit anti-GPIgG, column had a volume of 16X0.6 cm and a total of 36.35 mg protein. 0.5 ml of the purified GPIgE sample I (the material concentrated after Sephadex G-200 filtration) was passed through the Sepharose rabbit anti-GPIgG, column. The material that came through the column was concentrated to a volume of 2.4 ml by positive pressure dialysis and tested for reaginic antibody activity by PCA. The ma terial that remained on the column was eluted using 5 m guanidine HC1 (Eastman Kodak, Rochester, N.Y.) which had been adjusted to pH 7 with Tris (hydroxyme thyl) Aminomethane (Fischer Scientific, Fairlawn, N.J.). This material was dialyzed against BBS, concentrated to a volume of 2.0 ml, and titrated for PCA activity.
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Isoelectric Focusing Isoelectric focusing was performed on 0.5 ml of sample I or on 0.5 ml of serum A or serum B. Ampholine with a range of pH 5-10 (LKB Produkter, Brommal, Sweden) was used. An LKB 3371E power supply was adjusted to a constant 600 V, and the amperage was allowed to decrease until no further change was seen. After 72 h, 2.5-ml fractions were removed by an LKB peristaltic pump with a flow rate of 17 ml/h, and their OD280 was determined by an LKB Uvicord II Absortiometer. The pH of each fraction was measured using a radiometer titrator type TTT1 (Radiome ter, Copenhagen). Each fraction was individually dialyzed against BSS and titrated by PCA for IgE and IgG,.
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Table I. IgE and IgEi titers of English Short Hair guinea pigs 7 days after boosters Guinea pig No.1
1 2 3 4 5 6 7 8 9 10
Boost second2 IgE
IgGi
fifth2 IgE
IgGi
0 100 100 0 100 100 0 0 0 100
20 20 20 0 20 20 0 0 0 20
0 8,000 4,000 1,000 8,000 8,000 0 0 0 0
100 500 2,000 500 500 500 0 100 0 0
1 Guinea pigs immunized according to schedule I. 2 Figures in table refer to reciprocal of last dilution giving reaction (6 mm blue spot) in 3 of 4 guinea pigs.
0.5 ml of serum A and serum B were also passed through the column before and after the reaginic antibody to test the effectiveness of the column.
Of all the strains tested, the best results were obtained with the English Short Hair strain. Generally, about half of these animals produced IgE antibody. In table I, results from one experiment are summarized. The worst response was obtained with the Hartley strain. Only 1 of 10 animals responded, and the PCA titer of IgE antibody was never higher than ten. In the other two strains (Camm Responder and Non-responder), 2-4 of 10 animals produced IgE antibody, but the titers were lower than in the English Short Hair strain. In every case, heated and unheated sera from individual animals were titrated using a SP of 4 h, 2 and 7 days. Though generally IgE and IgG, were detected in the same sera, there was no correlation between titers of IgE and IgG,. It must be emphasized that in some cases a high titer of IgE was associated with a low IgG, antibody titer (table I).
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Results
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Table II. Effect of different SP on heated and unheated serum from guinea pig No. 25, scored as reaction size (mm diameter) performed in four guinea pigs Serum, days after immuni zation
Dilution
Heating at 56°C 1h
Sensitization period 4h 2 days
7 days
1:100 1:400 1:800 1:1,600
-
18 20 20 18 13 14 13 14 10 13 12 10 0 12 8 8
20 17 15 10
1:10 1:100 1:400 1:800 1:1,600
+ + + + +
11 10 12 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
9 0 0 0 0
9 0 0 0
1:100 1:400 1:800 1:1,600
-
20 20 20 18 14 15 14 14 12 10 12 12 8 8 10 10
30 22 17 15
30 25 19 18
ND 12 6 0 0
ND 12 0 0 0
0 0 0 0
1:10 1:100 1:400 1:800 1:1,600
-
+ + + + +
10 12 0 6 0 0 0 0
6 0 0 0
22 23 20 22 18 22 13 17 13 18 12 8 8 12 0 6
0
0 0 0 0 0
8 0 0 0 0
25 20 14 11
18 18 16 14
0 14 0 0 0 0 0 0
18 18 16 12 13 12 11 6 12 10 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
30 20 20 20 22 13 13 13 20 12 13 10 12 8 8 6 ND 0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
For each SP, all dilutions of heated and unheated samples of both sera were injected into the same guinea pig; every vertical column represents one guinea pig.
Persistence in the Skin A typical experiment of PCA reaction is shown in table II, using indi vidual sera from an English Short Hair guinea pig (after the second and
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For those experiments measuring the persistance in the skin, individu al sera were used. As stated above, for DEAE chromatography, molecu lar weight determination, electrofocusing, and absorption experiments, the serum pooled from English Short Hair guinea pigs was used. The IgE produced by these animals was very unstable, and its activity was de creased even when stored at -70 °C. After 3 months, 50-75% of the IgE activity was lost as shown by PCA [K ojima et al., to be published].
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Fraction number
Fig. 1. Sephadex G-200 gel filtration of GPIgE. 2.0-ml fractions were individu ally checked for reaginic antibody activity by PCA (vertical bars).
third boosters). Very similar results were obtained with individual sam ples of sera containing IgE even from other strains taken 7 days after any of the boosters. The only variable was the titer of the sera. Heating for 1 h at 56 °C consistently reduced the titer. As can be seen from table II, the heated serum with a 7-day SP had a lower PCA titer than with a 4-hour SP (91 day bleeding). But even with 4 h SP, the titer was reduced by 75% or more. End dilution titers of heated sera A and B were the same as that of the unheated sera. The persistence in the skin was assayed using individual high titer sera (titer of 1,600). Even with a 21-day SP, the unheated sample gave a PCA reaction at a dilution of 1,600. Identical results were obtained with a 14day SP (not shown).
Sephadex G-200 Chromatography The fraction from the DEAE chromatography was concentrated from 77.5 to 5 ml and put on a Sephadex G-200 column. The peak of PCA ac tivity ran faster than the bulk of the protein (fig. 1). That this PCA activ-
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DEAE Chromatography The heat-labile PCA activity was eluted by the 0.035 and the 0.05 m PB. The pooled fractions, with a volume of 77.5 ml, showed a PCA titer of 20, but upon heating only the undiluted sample gave a PCA reaction. It is well known that IgG! is also eluted at 0.035-0.05 M [13].
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Fig. 2. Estimation of the molecular weight of GPIgE by Sephadex G-200 gel fil tration markers. Ovalbumin = 45,000 daltons; 125I human IgG, = 150,000 daltons; rabbit IgE = 215,000 daltons.
ity was actually due to IgE, was shown by the fact that the pooled frac tions, which had a PCA titer of 200 unheated with a 7-day SP, had a PCA titer of only 10 upon heating for 1 h at 56 °C with a 4-hour or 7-day SP. The Sephadex G-200 column was also used for determination of the molecular weight. The K average of the markers and the unknown prote in were determined, as seen in figure 2. The molecular weight of the GPIgE was found to be 185,000 daltons. The peak of IgG, PCA activity coincided exactly with the peak of the t25I-labelled rabbit y-globulin which was taken as 140,000 daltons. Isoelectric Focusing Isoelectric focusing was also performed on two sera with high titer IgGi. When serum A was fractionated, the PCA activity was found in fractions ranging from pH 7.01-6.3, with a peak at 6.6. When serum B was used, the PCA activity was found in the range pH 6.87-6.20, with a peak at 6.87. When sample I was electrofocused, the IgE titer was found between pH 6.2 and pH 7.1 with a peak at pH 6.53. Another trial gave a range of pH 6.08-6.5, with a peak at 6.48. These results indicate that the anti-DNP IgE is only slightly more electronegative than the IgG,.
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Affinity Chromatography Samples of both IgG, and IgE containing antisera were passed through the column containing rabbit anti-GPIgG, coupled to Sepharose 4B
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beads. Serum A was first passed through the column. After passage through the column, the serum which had an original PCA titer of 8,000 had a titer less than 5 when normalized to the same volume. After elution with 5 m guanidine HC1, the fraction eluted showed a titer of 650 when normalized. Sample I, at the time used for affinity chromatography, had an IgE ti ter of 200. After passage through the same anti-GPIgG, column, the normalized sample had an IgE titer of 100, a reduction of only 50%. Whereas a line was observed in double diffusion in agar against the rabbit anti-GPIgG! before passage through the column, no line was observed af ter passage. After elution with guanidine HC1, 5 M, no PCA activity was detected. Finally, serum B was passed through the same rabbit anti-GPIgG, col umn to ascertain that the column was still active. After passage through the column, the serum, when normalized, had an IgG, activity of only 8% as compared to the original sample. The fraction eluted from the column by 5 m guanidine HC1 had an IgG, titer by PCA of 500 when normalized.
It is well known that high amounts of antigen and CFA produce IgG, and IgG, antibodies in guinea pigs [1-3]. It is remarkable to note that low amounts of antigen and Al(OH)3 as adjuvant (schedule I) produces high titer IgE antibodies in most of those animals that are producing IgE anti bodies. The influence of low doses of antigen and Al(OH)3 as adjuvant for preferential production of IgE over IgG was already observed in other species such as the rabbit [15] and the mouse [16], The IgG, content us ing schedule I for immunization is generally much less than that observed in animals immunized with large doses of antigen and CFA. Moreover, some animals did not produce antibodies using schedule I for immuniza tion (table I). It is also interesting to note that the IgE antibody content of the sera in those animals that responded, progressively increased after the boosters. The first to observe heat labile (reaginic or IgE) antibodies in guinea pigs was C atty [4] in animals infested with Trichinella spiralis. D obson et al. [17, 18] used A. suum infestation for IgE production in guinea pigs. P arish [5] was the first to obtain IgE antibodies against a natural protein and also against a haptenic determinant. He was also the first to use alum
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Discussion
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as an adjuvant. Others could also obtain IgE antibodies in guinea pigs against haptenic determinants [19-21] or against natural proteins [19, 21-23] using as adjuvant alum [20] pertussis vaccine [19], lipopolysaccharides [22, 23], or synthetic double stranded RNA [23]. The original observations of C atty [4] and P arish [5] showed that heating at 56 °C for 1 h destroyed reaginic guinea pig antibodies. Other species also exhibit this general property of reaginic antibodies, including the mouse [24], the rat [25], the rabbit [26, 27] and man [28]. We also observed that heating at 56 °C for 1 h drastically reduced reaginic anti body activity. With a sensitization period of 7 days, the titer of the heated serum was consistently less than that seen at 4 h. The small residual ac tivity after heating might be due to some IgGlb contamination, especially as the titer seems to drop with longer sensitization periods. However, it is not impossible that a heat-stable subpopulation of the IgE exists, similar to what has been suggested in the rabbit [27]. L indquist [29] described a heat-stable rabbit IgE antibody. However, he titrated his sera by the diameter of the reaction, and not by end-dilu tion titer. It has been pointed out that end-dilution titer is more reliable and more sensitive than the measurement of the size of the reaction [27]. It is remarkable that IgE activity can persist in the skin for up to 21 days. This is similar to what is seen in the case of human IgE [30]. Samples of pooled and individual sera gave similar results when heat ed and unheated specimens were titrated for PCA activity. Because of this similarity, and because there was not enough IgE containing serum from a single guinea pig, it was necessary to pool several sera for molecular weight determination, electrofocusing, and absorbtion experiments. The molecular weight of GPIgE in these experiments was shown to be 185,000 daltons. This confirms and extends the observations of D obson et al. [17, 18]. These authors, however, did not give precise figures and they also found that a fraction of IgE had an S rate of 8, whereas another fraction had an S rate of 11 and concluded that the 11 S fraction is probably a polymer or an aggregate of the 8 S fraction. Contrary to rabbit IgE [27], electrofocusing showed very little differ ence between GPIgE and IgGj. The molecular weight of 185,000 daltons is substantially higher than that of IgG,. The higher molecular weight of IgE is consistent with data from man [31] and rabbits [27, 29, 32]. In addition to heat lability and long persistance in the skin, affinity chromatography was used to definitely differentiate between IgG, and IgE.
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The Sepharose 4B specific rabbit anti-GPIgG, column absorbed all the IgG, content of the sera which were passed through the column and after elution with 5 M guanidine a part of the IgG, content was recovered. There was only 50% absorption of IgE by the column. Elution with 5 M guanidine HC1 is a very drastic treatment, and it is not surprising that only a fraction of the IgG, antibody could be re covered. Keeping in mind the fragility of IgE, a reduction in titer from 200 to 100 after passage through this column is not surprising. However, it is also possible that our rabbit antiserum contained traces of anti-IgE. These results show that the rabbit anti-GPIgG, antiserum can distinguish between IgG, and IgE, and does not absorb the latter. Therefore, cross reacting determinants were not demonstrated between these two classes of immunoglobulins. Of course, this does not eliminate the possibility of their existence. However, with carefully selected anti-GPIgG, antisera, it is possible to differentiate between IgG, and IgE. Until sufficient amounts of purified GPIgE antibodies can be isolated for sequencing or until an IgE guinea pig myeloma is obtained for com plete physicochemical and immunological characterization, IgE antibod ies in this species can be defined as those heat-labile antibodies which persist in the injected skin sites for weeks and which are not absorbed by anti-IgG, antibodies. Acknowledgements
de
The authors wish to thank Mr. C saba de Szalay and Mrs. Z suzsanna F ekete N agyivany for their excellent technical help, and Mr. F rank R eilly for his effi
cacious secretarial assistance.
1 Benacerraf, B.; O vary, Z.; Bloch , K. J., and F ranklin, E. C.: Properties of guinea pig 7 S antibodies. I. Electrophoretic separation of two types of guinea pig 7 S antibodies. J. exp. Med. 117: 937-949 (1963). 2 O vary, Z.; Benacerraf, B., and B loch, K. J.: Properties of guinea pig 7 S anti bodies. II. Identification of antibodies involved in passive cutaneous and system ic anaphylaxis. J. exp. Med. 117: 951-964 (1963). 3 B loch , K. J.; K ourilsky, F . M.; O vary, Z., and Benacerraf, B.: Properties of guinea pig 7 S antibodies. III. Identification of antibodies involved in comple ment fixation and hemolysis. J. exp. Med. 117: 965-981 (1963).
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References
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4 C atty, D.: The immunology of nematode infections Trichinosis in guinea pigs as a model. Monogr. Allergy, vol. 5, p. 1 (Karger, Basel 1969).
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Correspondence to: Dr. Z oltán O vary, Department of Pathology, New York Uni versity Medical Center, 550 First Avenue, New York, N Y 10016 (USA)
