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Comparative studies of monkey and human immunoglobulins
Immunochemistry. Pergamon Press 1970. Vol.7, pp. 839-849. Printed in Great Britain
C O M P A R A T I V E STUDIES OF MONKEY AND H U M A N IMMUNOGLOBULINS* VICTORIA MONTE-WICHERt, KONRAD WICHER and CARL E. ARBESMAN Departments of Medicine and Microbiology, State University of New York at Buffalo, School of Medicine and the Allergy Research Laboratory at Buffalo General Hospital, Buffalo, New York, U.S.A.
(First received 1June 1970; in revisedform 19June 1970) Abstract- Immunoglobulins G, A and M of monkey Macaca mulatta were separated by methods similar t o t h o s e used for the separation of human serum immunoglobulins. Monospecific antisera to the monkey and human IgG, IgA and IgM were prepared and used for examination of the cross reactivity between the monkey and human immunoglobulins. When antisera to human immunoglobulins were applied, definite differences were seen between the IgG and IgA but not between the IgM. However, when antisera to monkey immunoglobulins were used, definite differences were seen in all three immunoglobulins. In the quantitative precipitation reaction, the IgG of the monkey cross reacted with the antibodies to human IgG in a range of 56-60 per cent and the IgG of human cross reacted with antibodies to monkey IgG in a range of 57-68 per cent. The single radial diffusion test was utilized for the studies of cross reactivity. Larger diameters of the precipitation rings with the heterologous than with the same concentration of the homologous antigen were obtained. The concentration of the serum immunoglobulins of Macaca mulatta monkeys were determined using monospecific antisera. The mean value of the IgG was 12.43 mg/ml, the IgA 3.5 mg/ml and the IgM 0.87 mg/ml.
INTRODUCTION Newly d e v e l o p e d or i m p r o v e d techniques such as electrophoresis, i m m u n o diffusion, gel filtration, c h r o m a t o g r a p h y , enzymatic digestion o f proteins, etc., have c o n t r i b u t e d significantly to the studies o f s e r u m p r o t e n s in m a n a n d animals. While the i m m u n o g l o b u l i n s G, A, M, D a n d E o f h u m a n s e r u m are r a t h e r well characterized, the m o n k e y i m m u n o g l o b u l i n s have b e e n less extensively studied. T h e first a t t e m p t s to characterize rhesus m o n k e y i m m u n o g l o b u l i n s were m a d e by Kabat[1] a n d Tiselius a n d Kabat[2]. In o t h e r s u b s e q u e n t studies p e r f o r m e d by o t h e r a u t h o r s [3-14] m o n k e y s e r u m fractions were e x a m i n e d by chemical a n d electrophoretic m e t h o d s a n d c o m p a r e d with h u m a n fractions. I m m u n o g l o b u l i n G o f m o n k e y was isolated by several authors [15-21, 24, 26]. S e r u m fractions rich in I g M [24] a n d i m m u n o c h e m i c a l l y p u r e IgM [28], as well as an I g A rich fraction[26] a n d a fraction c o n t a i n i n g I g E have b e e n p r e p a r e d [26, 27]. *This study was presented in part at the 53rd Annual Meeting of the Federation of American Societies for Experimental Biology in Atlantic City, New Jersey, April, 1969 and was supported in part by Unites States Public Health Service, NIAID Research Grant No. 5-R01-AI-01303. ?Dr. Victoria Monte-Wicher's present address: McMaster University, Department of Pediatrics, Hamilton, Ontario, Canada. 839
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VICTORIA MONTE-WICHER, K. WICHER and C. E. ARBESMAN
Enzymatic cleavage of monkey IgG has been pertormed and the Fab and Fc fragments examined in immunoelectrophoresis[20]. Also, the L and H chains of monkey IgG have been produced and compared with those of other animals [21, 28] In most cases, the comparison of immunoglobulins of monkey and human, applying serological methods, was performed by using only antisera to human immunoglobulins. No reports could be found as to the preparation and use of monospecific antisera to rhesus monkey IgM and IgA. Utilizing such antisera, one might expect to obtain more specific characteristics of monkey immunoglobulins and more information about the cross reactivity between human and monkey sera. In the present studies, antisera to IgG, IgA and IgM of monkey Macaca mulatta were produced in rabbits. Utilizing monospecific antisera to monkey and human, the immunoglobulins of both species were examined and the concentration of immunoglobulins in monkey sera were determined by the single radial diffusion method. MATERIALS AND METHODS
Monkey serum. Sera were obtained from Macaca mulatta monkeys. These animals were purchased from Shamrock Farms, Inc., Middletown, New York. Antisera to human immunoglobulins. Monospecific antisera to human IgG, IgA, IgM, IgD, to Fab and Fc fragments of IgG, and antisera to whole serum were produced in rabbits in our laboratory. Immunoelectrophoresis ( I E P ) - w a s carried out according to the method of Scheidegger[29], using LKB equipment and barbital buffer, pH 8-6, ionic strength - 0-05. Starch block electrophoresis (SEP)-was performed as described by Kunkel and Trautman [30]. The double diffusion gel precipitation test ( D D G P ) - w a s carried ont according to the method of Ouchterlony[31], using 1.5 per cent Nobel Agar* in small plastic Petri dishes. A b s o r p t i o n - Gluteraldehyde? was used to polymerize antigens or antibodies for absorption. This was done according to the method described by Avrameas and Ternynck [32]. Determination of proteins. The amount of immunoglobulins in fractions obtained from gel filtration or ion exchange chromatography was determined with a Beckman Spectrophotometer at 280 mk~ assuming 1 mg/ml = 1.40.D..,s0. The concentrations of proteins of the eluates obtained from starch block electrophoresis were determined by the Folin-Ciocalteau method of Lowry[33], standardized with HGG. Isolation of monkey immunoglobulins. The serum immunoglobulins of monkeys were isolated by methods similar to those used for human immunoglobulins. The IgG and IgA were prepared by DE-525 cellulose chromatography according *Special Agar-Noble, Difco Laboratories, Detroit, Michigan. tGlutaraldehyde,J. T. Baker Chem. Co., Philipsburg, New York. $DE52 Pre-swoilen diethylaminoethyl cellulose, Whatman, W. and Balston, R. Ltd., England.
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841
to the method of Ishizaka and Ishizaka[34]. The Igm was prepared by the method of Fahey, and McLaughlin [35], using starch* block electrophoresis and Sephadex G-200t gel filtration. The Fab and the Fc fragments of monkey IgG were prepared in a way similar to that used for the fragments of human IgG [36]. Production of antisera. Antisera to monkey immunoglobulins or their fragments were produced in rabbits. The immunization schedule was the same in all cases. Rabbits received two to three foot pad injections of antigens incorporated in complete Freund's adjuvantS. The injections were given every 10 days. The final bleedings were taken 2-3 weeks after the last injection of antigen. The antiserum to whole monkey serum was produced by injecting a total of 2 ml of whole monkey serum in complete Freund's adjuvant. The antisera to immunoglobulins G, A and M were produced by using 1.25 mg protein of each antigen. The antiserum to IgG was absorbed with Fab fragment of the monkey IgG. The antisera to IgA and IgM were rendered monospecific by absorption with monkey IgG. The antiserum to IgA required further absorption with cord blood. The antisera to Fab and Fc fragments of the IgG were obtained by injecting a total of 0.937 mg protein. All antisera were examined by IEP and DDGP.
Examination of cross reactivity between monkey and human immunoglobulins. Three different methods were applied for the determination of the cross reactivity. (1) Double diffusion gel precipitation (DDGP). Whole monkey and human sera were examined using monospecific antisera to monkey and human IgG, IgA and IgM. (2) Quantitative precipitation test. This test was performed in tubes with monkey and human IgG. Antiserum to monkey IgG, containing 320t~g Ab N/ml was distributed into two rows of tubes, 0.5 ml in each. In the first and second rows respectively, the homologous and heterologous antigens were added in increasing concentrations, in a volume of 0"5 ml. Similar experiments were performed with antiserum to human IgG containing 246/~g Ab N/ml. The tubes were left at 37°C for thirty minutes and then were shaken overnight at 4°C. The tubes were centrifuged and the supernatants were examined for the presence of excess antibodies or antigens by DDGP. The precipitates were washed twice with cold saline; each precipitate was dissolved in 1 ml of 0-05 N NaOH and the protein concentration was determined spectrophotometrically at 280 m/~. (3) Single radial diffusion test (SRD). In this test also the cross reactivity of the IgG only was examined. Duplicate samples of 15 t~g and 7"5 p,g of monkey and human IgG were placed into the wells of the agar plate containing antiserum to monkey or human IgG respectively. The plates were kept in cold and 24 hr later the diameter of the precipitation rings was measured. Quantitative Determination of lgG, IgA and IgM in monkey sera. The concentration of immunoglobulins G, A, and M were determined in sera of 20 Macaca mulatta monkeys, 2-3 years old, weighing 6-10 lb. These monkeys, originating *Aroostocrat Potato Starch, Morningstar Paisley, New York, New York. tSephadex G-200. Pharrnacia, Uppsala, Sweden. :~Freund's adjuvant, C. F. A. Difco Laboratories, Detroit, Michigan.
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VICTORIA MONTE-WICHER, K. WICHER and C. E. ARBESMAN
f r o m the West Indies, were kept fbr approximately 4-6 weeks at the cohmy of Shamrock Farms, Inc., Middletown, New York, b e f o r e being sent to o u r animal unit. In both locations, they were fed Purina Monkey Chow. T h e animals a p p e a r e d healthy and had negative tuberculin tests. T h e y were not exposed experimentally to any immunological stimulants but were routinely treated with specific antihelminthic drugs to eliminate the presence o f parasites in the stool. T h e single radial diffusion m e t h o d o f Mancini et a1.[37] was used. Briefly, for the d e t e r m i n a t i o n o f each immunoglobulin, the monospecific antisera, in p r o p e r dilutions, were i n c o r p o r a t e d into the agar. 'Fen milliliters o f this m i x t u r e were p o u r e d on an 8 × 10 cm gloss plate in a plastic frame. F'wentvfive wells, 2 m m dia., were cut. F e n microliters o f five known concentrations for each standard (immunochemically p u r e IgG, IgA* and IgM) and 10 bd o f the samples tinder investigation were placed into the wells. T h e plates were kept at 4°C in a moist c h a m b e r until the diffusion was completed and the results read. T h e a m o u n t o f the immunoglobulins in the serum samples was calculated on the basis o f the standard curve obtained with the five concentrations o f standard antigen. RESULTS
Immunoglobulins and antisera T h e p r e p a r a t i o n o f m o n k e y IgG and IgA is diagramatically p r e s e n t e d in Fig. I. Each chromatographic fraction eluted f r o m the DEAE c o l u m n was exa m i n e d by IEP and DDGP against antiserum to whole m o n k e y serum and antisera to h u m a n IgG, IgA and IgM. Pooled monkey sera precipitated with (NH4)2SO4--50 per cent saturation Precipitate dissolved, dialyzed
DEAE-52 Stepwise elution, phosphate buffer pH 8.0
0"005 M /
I~I'G
0"010M
0.025 M
0.035 M
0-050 M
0'080 M
IgA, Rich fraction Sephadex (2,-200
4.
Refihration
Fig. 1. Fractionation of monkey IgG and IgA. *Purified IgA was prepared by absorption of pooled normal monkey sera or fraction rich in IgA with immunoadsorbent containing monospecific rabbit antisera to monkey IgA. The IgA was then eluted in pH 2"0 and adjusted to pH 7"2.
Monkey Immunoglobulins
843
Immunoglobulin G was detected in all six fractions, from 0.005M to 0.080M; the fractions eluted with 0.005M and 0-010M phosphate buffer contained detectable only IgG. The fractions from 0.025 M to 0.080 M contained IgA and other serum components. The 0"025 M fraction was chosen for further purification of the IgA because it contained lesser amounts of other serum proteins and enough IgA for further processing. The chromatographic fractions of 0.050 M and 0.080 M also contained macroimmunoglobulin which was not purified further. The preparation of the IgM is presented in Fig. 2. Immunochemically pure IgM was obtained by combining starch block electrophoresis with gel filtration in Sephadex G-200. Immunochemically pure IgG (0.005 M), the purified IgA (minute amounts of IgG were still present) and pure IgM were used to prepare the various antisera. The antisera to monkey immunoglobulins G, A, and M, after being rendered monospecific, were examined in IEP using whole monkey and human sera. The results of these experiments are presented in Fig. 3. All three antisera gave only one precipitation line with whole monkey and human sera, demonstrating very similar electrophoretic patterns. The only significant difference was found in the strength of the reaction with the antisera to IgA which reacted more strongly with the homologous immunoglobulins. The DEAE fractions of monkey sera obtained by stepwise elution with phosphate buffer of increasing motarities and of constant pH (8.0) were examined in IEP against antisera to monkey IgG, IgA and IgM (Fig. 4). For the purpose of this experiment, two additional concentrations, 0.10 M and 0.15 M of the phosphate buffer were used for elution of the IgM. All these fractions, prior to the test, were dialyzed against buffered salin, pH 7"2 and the concentrations of the fractions adjusted to 2 mg/ml for IgG and 1.5 mg/ml for IgA and IgM. Differences in the rate of electromigration of these fractions were observed. The rate of electromigration of the IgG present in the fractions of 0.005 M - 0 . 0 8 0 M, when compared with the electromigration of the IgG of the whole serum, increased with the increasing molarities of the phosphate buffer. A similar phenomenon was noted in the IgA containing fractions (0"025M-0"080 M) but not with those containing IgM (0"050 M -- 0.15 M). Pooled monkey sera precipitated with (NH4)2SO4-50 per cent saturation Precipitate dissolved, dialyzed Starch block-veronal buffer pH 8-6 IgM rich fraction Sephadex G-200 borate buffered saline pH 8"0 IgM Fig. 2. Fractionation of monkey IgM.
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VICTORIA MONTE-WICHER, K. W1CHER and C. E. ARBESMAN
Cross reactivity (1) Monkey and human sera were compared in DDGP, using monospecific antisera to monkey and human immunoglobulins (Fig. 5). The antisera to IgG reacted with both the homologous and heterologous proteins, demonstrating cross reactivity. Definite spurs were seen on the side of the homologous antigens. The antisera to monkey and human immunoglobulins gave practically the same strength of reactions. The reactions seen with the antisera to IgA differ from those seen in the IgG system. The differences are more distinctive in the strength of the reaction and also in the size of the spurs that formed over the well with the homologous antigens. In the IgM system, the anti-monkey IgM reacted with the monkey and human sera by formation of a definite spur that extended over the well with the homologous antigen. Anti-human IgM did not detect differences between the IgM of either specie. (2) The results of the quantitative precipitation experiments performed in tubes with monospecific antisera to monkey and human IgG are given in Figs. 6 and 7. Figure 6 presents the precipitation reaction of monkey and human IgG with the antiserum to monkey IgG. The optimal precipitation reaction of the monkey IgG was obtained with 163/xg Ab/N, whereas of human IgG with 93/xg Ab/N. The difference would indicate that the heterologous antigen (human IgG) cross reacted with the antibodies to monkey IgG in 57 per cent. Figure 7 presents the precipitation reaction of human and monkey IgG with the antiserum to human IgG. In this experiment, the optimal precipitation reaction of the human IgG was obtained with 123/xg Ab/N, whereas of monkey antigen with 69/xg Ab/N. The cross reactivity of the monkey IgG in this experiment was calculated to be 56 per cent. (3) The results of the cross reactivity, as examined in single radial diffusion, are summarized in Table 1. Readings of the diameters of the diffusion rings were taken after 24 hr incubation at 4°C. The diameters were read by four reZg G ~92
MONKEY [6c
128
@
96
~5
/ ~
64
32
l
Y I
i
#g Antigen N odded
Fig. 6. Precipitation reaction of monkey and human IgG with antiserum to monkey IgG. The optimal precipitation reaction of the monkey IgG was obtained with 16s/xg Ab N and of human IgG with 93/xg Ab N.
Fig. 3. Immunoelectrophoresis of monkey (M) and human (H) sera. Monospecific antisera to monkey (a-M) immunoglobulins or to human (a-H) immunoglobulins were placed in the troughs. Similarities in the electrophoretic pattern were observed.
(Facing page 844)
T-000 M
:*oso M
_
i.
F. o*l!ioM
F.O*lOOM -
’
‘-
I
‘--
2.
Fiq:. 1Irllnunoelect~ophol-esis of‘ the 1)E:Al-T cellulose fractions (F) of rnonke)- serum elured with phosl’hkite buffer of’increasing molarities and normal monkey serum (NMS). Monospecific antisera (a-14) to monkey I$, IgA 01 Igyt were placed in the troughs. The I<$; and [#A. but not tlle IgM fractions demonstrated differences in the etectrophorctic mobititv.
?*OIOM
0
OM
OH
oti
Fig. 5. Double diffusion gel precipitation of monkey (M) and human (H) whole sera with monospecific antisera to monkey (a-M) and human (a-H) immunoglobulins. Differences in the cross reactivity could be observed.
Fig. 8. Examination of cross reactivity of monkey and human IgG in single radial diffusion. Appearance of precipitation rings of two concentrations 15 and 7.5 pg of monkey (M-IgG) and human (H-IgG) immunoglobulin G in an agar plate containing antibodies to monkey IgG (a-M IgG), and in an agar plate COIItaining antibodies to human IgG (a-H IgG). The diameters of the precipitation rings obtained with homologous antigens are smaller than those obtained with heterologous antigen.
Monkey Immunoglobulins
845
Ig G 144 HUMAN 120 ~b
96
72
48 /
//
24
i
I
,u.g Antigen N added
Fig. 7. Precipitation reaction of human and monkey IgG with antiserum to human IgG. The optimal precipitation reaction of the human IgG was obtained with 123/~g Ab N and of monkey IgG with 69/~g Ab N. searchers and, in no one case did the reading differences exceed 0.3 mm. The numerical values in the table express the diameters in millimeters. The values in the parentheses are of duplicate samples. The diameters of the precipitation rings obtained with the homologous antigens are smaller than those obtained with the heterologous antigen (Fig. 8). These differences are statistically significant (P < 0.01).
Quantitative determinationof immunoglobulins The concentration of the immunoglobulins G, A and M were determined in the sera of 20 monkeys. The individual results are presented in Table 2. The concentration of the IgG varied in the individual monkeys from 8.3 to 17.5 mg/ml. The mean value was 12.43 mg/ml with one standard deviation +--2.3. J
Table 1. Single radial diffusion test. Monkey and human IgG examined against the homologous and heterologous antisera to IgG Concentration of antigen in/zG. Human
Antisera to Human IgG Monkey IgG
15"0
7.6* (7"5)
9"5 (9.4)
7"5
6"3 (6"4)
8-4 (8"5)
15.0
8"6 (8-8)
8.9
7"5
7-9 (7-7)
7"4 (7-3)
igG Monkey
(8.8)
igO *The numerical values indicate diameters in millimeters.
846
VICTORIA MONTE-WICHER, K. WICHER and C. E. ARBESMAN Table 2. Concentration of immunoglobulins in sera of monkey Macaca mulatta Monkey No.
IgG
I mmunoglobulins IgA
IgM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
14.0" 10"0 11"3 15"0 11"3 14"4 11"2 11'0 11"0 16"5 11-4 11 '5 9"5 12"5 8"3 11 '5 17"5 14.7 13.2 12-9
4"5 3"8 3"8 4'5 1"3 2"8 4'0 5'4 4"5 2"3 1"4 3"3 6"0 3"0 2"0 3-9 4"0 3'0 2'6 4'0
0"72 0"42 0"71 1'35 1"15 0"97 0"72 0'76 1"00 1'27 0"90 0"82 0"44 0"62 0"70 0"44 0"94 1'43 1"33 0"85
3"50
0-87
8_+1"50
8-_+0"29
Mean value 12.43 Standard deviation (~i) 8+2.30 *Mg/ml.
T h e c o n c e n t r a t i o n o f the I g A varied f r o m 1.3 to 6.0 mg/ml; the m e a n value was 3.5 m g / m l with o n e s t a n d a r d deviation _+1-5. T h e concentration o f the IgM varied f r o m 0"42 to 1"43 mg/ml; the m e a n value was 0.87 m g / m l with o n e stand a r d deviation +0.29. T h e variations o f the concentrations o f the i m m u n o g l o bulins are not too m u c h h i g h e r t h a n those o b s e r v e d by o t h e r a u t h o r s [38-40] in n o r m a l h u m a n populations. DISCUSSION W h o l e m o n k e y s e r u m , w h e n c h r o m a t o g r a p h e d o n DE-52 cellulose a p p l y i n g stepwise elution o f increasing molarities (0.005-0.15 M) o f the p h o s p h a t e buffer at constant p H (8-0) d e m o n s t r a t e d an elution p a t t e r n similar to that o b s e r v e d with h u m a n serum. T h e I g G eluted f r o m the DE-52 cellulose with buffers o f increasing molarities d e m o n s t r a t e d an increasing e l e c t r o m i g r a t i o n in the IEP. T h i s would indicate that the I g G o f whole s e r u m consists o f a h e t e r o g e n e o u s p o p u l a t i o n o f molecules with different rates o f e l e c t r o m i g r a t i o n but with similar antigenic characteristics. A similar p h e n o m e n o n was o b s e r v e d earlier[41, 42] with h u m a n IgG. T h a t the m o n k e y I g G vary not only in physicochemical properties but also in a n t i b o d y activity was observed[43] in sera o f two m o n k e y s
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847
immunized with ragweed pollen. In the experiment, antibody activity as measured by radioimmunoelectrophoresis, was found in the portion of the fast moving IgG (in the /3 region) but not in the portion of the slower moving IgG (y region). Differences in the rate of electromigration were also observed in the monkey IgA of the chromatographic fractions. This immunoglobulin probably also consists of a population of various molecules. However, further studies would be necessary to fully explain this phenomenon. The IgM that was eluted with the different phosphate buffers in the DE-52 cellulose column did not show differences in the IEP. Many authors[6-28] using antiserum to human proteins compared the immunoglobulins of human and monkey. All of them observed cross reactivity between the monkey and human immunoglobulins. According to Achyara, Poulik, and Goodman [14] no differences were visible between human and nonhuman primates IgM when examined by antiserum to human IgM. In our DDGP experiments, we confirmed this observation. However, definite differences were observed when antiserum to monkey IgM was used. This indicates that the IgM of monkey and human are closely related but not identical as others have reported. The use of homologous antiserum to monkey IgM revealed the existing antigenic differences. When the IgG of human and monkey were examined against antihuman or anti-monkey IgG, there were similarities in the strength of the reaction and in the size of the spurs. It has to be stressed that both antisera were prepared with IgA that had been isolated from pooled monkey or human sera. In the IgA system, the antiserum to human IgA reacted with a certain number of monkey sera very weakly and with others not at all in contrast to the anti-monkey IgA which, in all cases, reacted with the human IgA. The differences in the reactivity of the antisera could be due to the source of the IgA used for preparation of the antisera. The monkey IgA was isolated from the pooled monkey sera whereas the IgA of human was separated from an IgA myeloma serum. From the results of the cross reactivity studies by DDGP, the following conclusions can be drawn: when using antisera to human immunoglobulins, there is a complete identity in the IgM system, less cross reactivity in the IgG and much less in the IgA system. When using antisera to monkey immunoglobulins, partial identity was observed in all three systems. However, the most striking differences were observed also in the IgA system. The cross reactivity between the monkey and human IgG was confirmed in precipitation test in tubes. In repeated experiments, the human IgG cross reacted with the antibodies to the monkey IgG in a range of 57-68 per cent, whereas the monkey IgG reacted with the antibodies to human IgG in a range of 56-60 per cent. Ishizaka and Ishizaka[26] using antiserum to human IgG for precipitation of monkey IgG found similar results. The ranges of the cross reactivity are very close between the two species; however, slightly higher cross reactivity was observed when antiserum to monkey IgG was used. From the work of Mancini et al. [37], it is known that in the SRD test, diffusion ring of any antigen applied in the antibody containing plate is directly proportional to the concentration of the antigen and inversely proportional to the con-
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VICTORIA MONTE-WICHER, K. WICHER and C. E. ARBESMAN
centration of the antibodies. The IgG of monkey, when examined in SRD in agar plate containing antibody to human IgG, demonstrated a larger precipitation ring than the same concentration of the human IgG. A similar phenomenon was observed when human IgG was examined in a plate containing antibodies to monkey IgG. The larger diffusion rings observed with the heterologous antigens can be explained on the basis of differences in the number of the antigenic determinants between both antigens reacting with the agar incorporated antibodies. The antiserum in the agar plates has fewer antibodies to the heterologous antigen; therefore, the edge of the precipitation ring for the heterologous antigen would be farther from the wells than the edge of the precipitation ring for the homologous antigen. From our results, one may conclude that when related antigens are examined in single radial diffusion method, the diffusion rate seems to be inversely proportional to the cross reactivity. The results of the cross reactivity obtained by SRD (Fig. 8 and Table 1) support the previously discussed observation of the precipitation test in liquid media; that human IgG cross reacts more with antiserum to monkey IgG than does the monkey lgG with the antiserum to human IgG. In analysis of the results in Table 1, one sees that when both antigens were examined with antiserum to monkey IgG the difference in the diameter of the precipitation rings was smaller (8.9:9.5, dif. = 0.6; or 7-4:8.4, dif. = l'0) than when examined with antiserum to human IgG (7.6 : 8.6, dif. = 1"0 or 6-3 : 7-9, dif. = 1"6). The single radial diffusion method used by us for the determination of heterologous immunoglobulins seems to be a useful tool in studies of the heterogeneity of proteins. However, the patterns of heterologous antigens with unknown grade of polymerization are not the best subjects to be examined by this method. The quantitation of serum immunoglobulins in monkey was determined by SRD. The mean values, when compared with those of human[38-40] showed to be very close in regard to the IgG and IgM whereas the mean value of the IgA was found to be higher in monkeys. It remains to be proven, however, whether we are dealing with the real values or with differences in the physicochemical properties of IgA molecules in monkey sera. Several authors [20, 23] determined the relative values of monkey immunoglobulins using antiserum to human immunoglobulins. However, only Poulik [20] mentioned that such an estimation may not be reliable. In the light of our experiments with the cross reactivity obtained in the SRD with the heterologous antigens, each estimation performed with non-homologous antiserum would be incorrect. We proved that the assumption made by Poulik was very much correct. Among the factors which may influence the protein concentration are: (1) the age of the animal, (2) the time spent in captivity, (3) the diet, and (4) the degree of an active parasitosis. We considered our monkeys as young animals, having been in captivity at least 3 months with a proper diet and without active parasitosis. Our monkeys were routinely treated with specific drugs and did not show parasites in the stool. However, the routine examination did not include any procedures to diagnose lung mites or blood parasites. Therefore, we cannot state that the monkeys were entirely free of parasites. It is known that parasites in monkeys can influence the level of IgE. Whether the remaining serum immunoglobulins are elevated in such monkeys is not well documented.
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REFERENCES Kabat E. A.,J. exp. Med. 69, 103 (1939). Tiselius A. and Kabat E. A.,J. exp. Med. 69, 119 (1939). Deutsch H. F. and Goodloe M. B.,J. biol. Chem. 161, 1 (1945). Moore D. H.,J. biol. Chem. 161, 21 (1945). Greenberg L. D., Hoessly Urs J. P., Brooks R. and Rinehart J. F., Proc. Soc. exp. Biol. Med. 79,425 (1952). 6. Picard J., HeremansJ. and Vanderbroek G., Vox Sang. 7,190 (1962). 7. AUenJ. R. and Carstens L. A., Lab. Animal Care 15, 103 (1965). 8. Boyden A., Serological Approaches to Studies of Protein Structure and Metabolism (Edited by Cole W. H.). Rutgers Univ. Press, New Brunswick, New Jersey (1954). 9. Gemeroy D., Boyden A. and DeFalco R., Comparative Serology of the Primates. Proc. XIV. Intern. Congr. Zool., pp. 327, Copenhagen (1953). 10. Boyden A., Comparative serology: amins, methods and results, In Serological and Biochemical Comparison of Proteins (Edited by Cole W. H,). Rutgers Univ. Press. New Brunswick, New Jersey (1958). 11. Williams C. A.,Jr. and Wemyss C. T., Jr., N.Y. Acad. Sci. 94, 77 (1961). 12. Goodman M., HumanBiol. 34, 104 (1962). 13. Goodman M., Human Biol. 35,377 (1963). 14. Achyara U. S. V., Poulik M. D. and Goodman M., Fedn Proc. Abstract 27,490 (1968). 15. Kabat E. A., Wolf A., Bezer A. E. and Murray J. P.,j. exp. Med. 93,615 (1951). 16. Bangham D. R., Proc. R. Soc. Med. 54,993 (1961). 17. Sgouris J. T., Storey R. W., Wolfe R. W. and Anderson G. R., Transfusion 6, 146 (1966). 18. Quinlivan W. L., Am.J. Physiol. 212,324 (1967). 19. Allen J., Nature, Lond. 209, 324 (1966). 20. Poulik M. D., Primates in Medicine, Vol. 1, p. 68. Karger, Basel/New York (1968). 21. Hood L., Grey W. R., Sanders B. G. and Dreyer W. J., Syrup. quant. Biol. 32, 1331 (1967). 22. LakinJ. D., Patterson R. and PruzanskyJ.J.,FednProc. Abstract 1393 (1966), 23. Lakin J. D., Patterson R. and Pruzansky J. J.,J. Immun. 98, 745 (1967). 24. Lakin J. D., Patterson R. and Pruzansky J. J.,J. Immun. 102,253 (1969). 25. Lakin J. D., Patterson R. and PruzanskyJ. J.,J. lmmun. 102,975 (1969). 26. Ishizaka K. and Ishizaka T.,J. Immun. 100,554 (1968). 27. Patterson R., Roberts M. and PruzanskyJ. J.,J. Immun. 102,466 (1969). 28. Metha P. D. and Tomasi T. B., Jr., Fedn Proc. Abstract 28, 820 (1969). 29. Scheidegger J. J., Int Archs Allergy appl. Immun. 7,103 (1955). 30. Kunkel H. G. and Trautman R., Zone Electrophoresis in various types of supporting media, In Electrophoresis, Theory, Methods and Applications (Edited by Bier M.). Acad. Press., Inc. Publications (1959). 31. Ouchterlony O., Prog. Allergy 5, 1 (1958). 32. Avrameas S. and Ternynck T., lmmunochemistry 6, 53 (1969). 33. Lowery O. H., Rosebrough N. J., Farr A. L. and Randall R. J.,J. biol. Chem. 193, 265 1. 2. 3. 4. 5.
(1951). 34. 35. 36. 37. 38. 39. 40. 41.
Ishizaka K. and Ishizaka T.,J. Allergy37, 169 (1966). FaheyJ. L. and McLaughlin C.,J. Immun. 91,484 (1963). Porter R. R., Biochem.J. 73,119 (1949). Mancini G., Carbonara A. O. and Heremans J. F., lmmunochemistry 2, 235 (1965). FaheyJ. L. and McKelvey E. M.,J. Immun. 94, 84 (1965). Chodirker W. B. and Tomasi T. B.,Jr. Science 142, 1080 (1963). Stiehm E. R. and Fudenberg H. H., ~lm.J. Med. 40, 805 (1966). Sorber H. A., Gutter F. J., Wyckoff M. M. and Peterson E. A., J. Am. chem. Soc. 78, 756 (1956). 42. FaheyJ. L. and Hobert A. P.,J. biol. Chem. 234, 2645 (1959). 43. Wicher K., Unpublished observation.
C O M P A R A T I V E STUDIES OF MONKEY AND H U M A N IMMUNOGLOBULINS* VICTORIA MONTE-WICHERt, KONRAD WICHER and CARL E. ARBESMAN Departments of Medicine and Microbiology, State University of New York at Buffalo, School of Medicine and the Allergy Research Laboratory at Buffalo General Hospital, Buffalo, New York, U.S.A.
(First received 1June 1970; in revisedform 19June 1970) Abstract- Immunoglobulins G, A and M of monkey Macaca mulatta were separated by methods similar t o t h o s e used for the separation of human serum immunoglobulins. Monospecific antisera to the monkey and human IgG, IgA and IgM were prepared and used for examination of the cross reactivity between the monkey and human immunoglobulins. When antisera to human immunoglobulins were applied, definite differences were seen between the IgG and IgA but not between the IgM. However, when antisera to monkey immunoglobulins were used, definite differences were seen in all three immunoglobulins. In the quantitative precipitation reaction, the IgG of the monkey cross reacted with the antibodies to human IgG in a range of 56-60 per cent and the IgG of human cross reacted with antibodies to monkey IgG in a range of 57-68 per cent. The single radial diffusion test was utilized for the studies of cross reactivity. Larger diameters of the precipitation rings with the heterologous than with the same concentration of the homologous antigen were obtained. The concentration of the serum immunoglobulins of Macaca mulatta monkeys were determined using monospecific antisera. The mean value of the IgG was 12.43 mg/ml, the IgA 3.5 mg/ml and the IgM 0.87 mg/ml.
INTRODUCTION Newly d e v e l o p e d or i m p r o v e d techniques such as electrophoresis, i m m u n o diffusion, gel filtration, c h r o m a t o g r a p h y , enzymatic digestion o f proteins, etc., have c o n t r i b u t e d significantly to the studies o f s e r u m p r o t e n s in m a n a n d animals. While the i m m u n o g l o b u l i n s G, A, M, D a n d E o f h u m a n s e r u m are r a t h e r well characterized, the m o n k e y i m m u n o g l o b u l i n s have b e e n less extensively studied. T h e first a t t e m p t s to characterize rhesus m o n k e y i m m u n o g l o b u l i n s were m a d e by Kabat[1] a n d Tiselius a n d Kabat[2]. In o t h e r s u b s e q u e n t studies p e r f o r m e d by o t h e r a u t h o r s [3-14] m o n k e y s e r u m fractions were e x a m i n e d by chemical a n d electrophoretic m e t h o d s a n d c o m p a r e d with h u m a n fractions. I m m u n o g l o b u l i n G o f m o n k e y was isolated by several authors [15-21, 24, 26]. S e r u m fractions rich in I g M [24] a n d i m m u n o c h e m i c a l l y p u r e IgM [28], as well as an I g A rich fraction[26] a n d a fraction c o n t a i n i n g I g E have b e e n p r e p a r e d [26, 27]. *This study was presented in part at the 53rd Annual Meeting of the Federation of American Societies for Experimental Biology in Atlantic City, New Jersey, April, 1969 and was supported in part by Unites States Public Health Service, NIAID Research Grant No. 5-R01-AI-01303. ?Dr. Victoria Monte-Wicher's present address: McMaster University, Department of Pediatrics, Hamilton, Ontario, Canada. 839
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VICTORIA MONTE-WICHER, K. WICHER and C. E. ARBESMAN
Enzymatic cleavage of monkey IgG has been pertormed and the Fab and Fc fragments examined in immunoelectrophoresis[20]. Also, the L and H chains of monkey IgG have been produced and compared with those of other animals [21, 28] In most cases, the comparison of immunoglobulins of monkey and human, applying serological methods, was performed by using only antisera to human immunoglobulins. No reports could be found as to the preparation and use of monospecific antisera to rhesus monkey IgM and IgA. Utilizing such antisera, one might expect to obtain more specific characteristics of monkey immunoglobulins and more information about the cross reactivity between human and monkey sera. In the present studies, antisera to IgG, IgA and IgM of monkey Macaca mulatta were produced in rabbits. Utilizing monospecific antisera to monkey and human, the immunoglobulins of both species were examined and the concentration of immunoglobulins in monkey sera were determined by the single radial diffusion method. MATERIALS AND METHODS
Monkey serum. Sera were obtained from Macaca mulatta monkeys. These animals were purchased from Shamrock Farms, Inc., Middletown, New York. Antisera to human immunoglobulins. Monospecific antisera to human IgG, IgA, IgM, IgD, to Fab and Fc fragments of IgG, and antisera to whole serum were produced in rabbits in our laboratory. Immunoelectrophoresis ( I E P ) - w a s carried out according to the method of Scheidegger[29], using LKB equipment and barbital buffer, pH 8-6, ionic strength - 0-05. Starch block electrophoresis (SEP)-was performed as described by Kunkel and Trautman [30]. The double diffusion gel precipitation test ( D D G P ) - w a s carried ont according to the method of Ouchterlony[31], using 1.5 per cent Nobel Agar* in small plastic Petri dishes. A b s o r p t i o n - Gluteraldehyde? was used to polymerize antigens or antibodies for absorption. This was done according to the method described by Avrameas and Ternynck [32]. Determination of proteins. The amount of immunoglobulins in fractions obtained from gel filtration or ion exchange chromatography was determined with a Beckman Spectrophotometer at 280 mk~ assuming 1 mg/ml = 1.40.D..,s0. The concentrations of proteins of the eluates obtained from starch block electrophoresis were determined by the Folin-Ciocalteau method of Lowry[33], standardized with HGG. Isolation of monkey immunoglobulins. The serum immunoglobulins of monkeys were isolated by methods similar to those used for human immunoglobulins. The IgG and IgA were prepared by DE-525 cellulose chromatography according *Special Agar-Noble, Difco Laboratories, Detroit, Michigan. tGlutaraldehyde,J. T. Baker Chem. Co., Philipsburg, New York. $DE52 Pre-swoilen diethylaminoethyl cellulose, Whatman, W. and Balston, R. Ltd., England.
Monkey Immunoglobulins
841
to the method of Ishizaka and Ishizaka[34]. The Igm was prepared by the method of Fahey, and McLaughlin [35], using starch* block electrophoresis and Sephadex G-200t gel filtration. The Fab and the Fc fragments of monkey IgG were prepared in a way similar to that used for the fragments of human IgG [36]. Production of antisera. Antisera to monkey immunoglobulins or their fragments were produced in rabbits. The immunization schedule was the same in all cases. Rabbits received two to three foot pad injections of antigens incorporated in complete Freund's adjuvantS. The injections were given every 10 days. The final bleedings were taken 2-3 weeks after the last injection of antigen. The antiserum to whole monkey serum was produced by injecting a total of 2 ml of whole monkey serum in complete Freund's adjuvant. The antisera to immunoglobulins G, A and M were produced by using 1.25 mg protein of each antigen. The antiserum to IgG was absorbed with Fab fragment of the monkey IgG. The antisera to IgA and IgM were rendered monospecific by absorption with monkey IgG. The antiserum to IgA required further absorption with cord blood. The antisera to Fab and Fc fragments of the IgG were obtained by injecting a total of 0.937 mg protein. All antisera were examined by IEP and DDGP.
Examination of cross reactivity between monkey and human immunoglobulins. Three different methods were applied for the determination of the cross reactivity. (1) Double diffusion gel precipitation (DDGP). Whole monkey and human sera were examined using monospecific antisera to monkey and human IgG, IgA and IgM. (2) Quantitative precipitation test. This test was performed in tubes with monkey and human IgG. Antiserum to monkey IgG, containing 320t~g Ab N/ml was distributed into two rows of tubes, 0.5 ml in each. In the first and second rows respectively, the homologous and heterologous antigens were added in increasing concentrations, in a volume of 0"5 ml. Similar experiments were performed with antiserum to human IgG containing 246/~g Ab N/ml. The tubes were left at 37°C for thirty minutes and then were shaken overnight at 4°C. The tubes were centrifuged and the supernatants were examined for the presence of excess antibodies or antigens by DDGP. The precipitates were washed twice with cold saline; each precipitate was dissolved in 1 ml of 0-05 N NaOH and the protein concentration was determined spectrophotometrically at 280 m/~. (3) Single radial diffusion test (SRD). In this test also the cross reactivity of the IgG only was examined. Duplicate samples of 15 t~g and 7"5 p,g of monkey and human IgG were placed into the wells of the agar plate containing antiserum to monkey or human IgG respectively. The plates were kept in cold and 24 hr later the diameter of the precipitation rings was measured. Quantitative Determination of lgG, IgA and IgM in monkey sera. The concentration of immunoglobulins G, A, and M were determined in sera of 20 Macaca mulatta monkeys, 2-3 years old, weighing 6-10 lb. These monkeys, originating *Aroostocrat Potato Starch, Morningstar Paisley, New York, New York. tSephadex G-200. Pharrnacia, Uppsala, Sweden. :~Freund's adjuvant, C. F. A. Difco Laboratories, Detroit, Michigan.
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VICTORIA MONTE-WICHER, K. WICHER and C. E. ARBESMAN
f r o m the West Indies, were kept fbr approximately 4-6 weeks at the cohmy of Shamrock Farms, Inc., Middletown, New York, b e f o r e being sent to o u r animal unit. In both locations, they were fed Purina Monkey Chow. T h e animals a p p e a r e d healthy and had negative tuberculin tests. T h e y were not exposed experimentally to any immunological stimulants but were routinely treated with specific antihelminthic drugs to eliminate the presence o f parasites in the stool. T h e single radial diffusion m e t h o d o f Mancini et a1.[37] was used. Briefly, for the d e t e r m i n a t i o n o f each immunoglobulin, the monospecific antisera, in p r o p e r dilutions, were i n c o r p o r a t e d into the agar. 'Fen milliliters o f this m i x t u r e were p o u r e d on an 8 × 10 cm gloss plate in a plastic frame. F'wentvfive wells, 2 m m dia., were cut. F e n microliters o f five known concentrations for each standard (immunochemically p u r e IgG, IgA* and IgM) and 10 bd o f the samples tinder investigation were placed into the wells. T h e plates were kept at 4°C in a moist c h a m b e r until the diffusion was completed and the results read. T h e a m o u n t o f the immunoglobulins in the serum samples was calculated on the basis o f the standard curve obtained with the five concentrations o f standard antigen. RESULTS
Immunoglobulins and antisera T h e p r e p a r a t i o n o f m o n k e y IgG and IgA is diagramatically p r e s e n t e d in Fig. I. Each chromatographic fraction eluted f r o m the DEAE c o l u m n was exa m i n e d by IEP and DDGP against antiserum to whole m o n k e y serum and antisera to h u m a n IgG, IgA and IgM. Pooled monkey sera precipitated with (NH4)2SO4--50 per cent saturation Precipitate dissolved, dialyzed
DEAE-52 Stepwise elution, phosphate buffer pH 8.0
0"005 M /
I~I'G
0"010M
0.025 M
0.035 M
0-050 M
0'080 M
IgA, Rich fraction Sephadex (2,-200
4.
Refihration
Fig. 1. Fractionation of monkey IgG and IgA. *Purified IgA was prepared by absorption of pooled normal monkey sera or fraction rich in IgA with immunoadsorbent containing monospecific rabbit antisera to monkey IgA. The IgA was then eluted in pH 2"0 and adjusted to pH 7"2.
Monkey Immunoglobulins
843
Immunoglobulin G was detected in all six fractions, from 0.005M to 0.080M; the fractions eluted with 0.005M and 0-010M phosphate buffer contained detectable only IgG. The fractions from 0.025 M to 0.080 M contained IgA and other serum components. The 0"025 M fraction was chosen for further purification of the IgA because it contained lesser amounts of other serum proteins and enough IgA for further processing. The chromatographic fractions of 0.050 M and 0.080 M also contained macroimmunoglobulin which was not purified further. The preparation of the IgM is presented in Fig. 2. Immunochemically pure IgM was obtained by combining starch block electrophoresis with gel filtration in Sephadex G-200. Immunochemically pure IgG (0.005 M), the purified IgA (minute amounts of IgG were still present) and pure IgM were used to prepare the various antisera. The antisera to monkey immunoglobulins G, A, and M, after being rendered monospecific, were examined in IEP using whole monkey and human sera. The results of these experiments are presented in Fig. 3. All three antisera gave only one precipitation line with whole monkey and human sera, demonstrating very similar electrophoretic patterns. The only significant difference was found in the strength of the reaction with the antisera to IgA which reacted more strongly with the homologous immunoglobulins. The DEAE fractions of monkey sera obtained by stepwise elution with phosphate buffer of increasing motarities and of constant pH (8.0) were examined in IEP against antisera to monkey IgG, IgA and IgM (Fig. 4). For the purpose of this experiment, two additional concentrations, 0.10 M and 0.15 M of the phosphate buffer were used for elution of the IgM. All these fractions, prior to the test, were dialyzed against buffered salin, pH 7"2 and the concentrations of the fractions adjusted to 2 mg/ml for IgG and 1.5 mg/ml for IgA and IgM. Differences in the rate of electromigration of these fractions were observed. The rate of electromigration of the IgG present in the fractions of 0.005 M - 0 . 0 8 0 M, when compared with the electromigration of the IgG of the whole serum, increased with the increasing molarities of the phosphate buffer. A similar phenomenon was noted in the IgA containing fractions (0"025M-0"080 M) but not with those containing IgM (0"050 M -- 0.15 M). Pooled monkey sera precipitated with (NH4)2SO4-50 per cent saturation Precipitate dissolved, dialyzed Starch block-veronal buffer pH 8-6 IgM rich fraction Sephadex G-200 borate buffered saline pH 8"0 IgM Fig. 2. Fractionation of monkey IgM.
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VICTORIA MONTE-WICHER, K. W1CHER and C. E. ARBESMAN
Cross reactivity (1) Monkey and human sera were compared in DDGP, using monospecific antisera to monkey and human immunoglobulins (Fig. 5). The antisera to IgG reacted with both the homologous and heterologous proteins, demonstrating cross reactivity. Definite spurs were seen on the side of the homologous antigens. The antisera to monkey and human immunoglobulins gave practically the same strength of reactions. The reactions seen with the antisera to IgA differ from those seen in the IgG system. The differences are more distinctive in the strength of the reaction and also in the size of the spurs that formed over the well with the homologous antigens. In the IgM system, the anti-monkey IgM reacted with the monkey and human sera by formation of a definite spur that extended over the well with the homologous antigen. Anti-human IgM did not detect differences between the IgM of either specie. (2) The results of the quantitative precipitation experiments performed in tubes with monospecific antisera to monkey and human IgG are given in Figs. 6 and 7. Figure 6 presents the precipitation reaction of monkey and human IgG with the antiserum to monkey IgG. The optimal precipitation reaction of the monkey IgG was obtained with 163/xg Ab/N, whereas of human IgG with 93/xg Ab/N. The difference would indicate that the heterologous antigen (human IgG) cross reacted with the antibodies to monkey IgG in 57 per cent. Figure 7 presents the precipitation reaction of human and monkey IgG with the antiserum to human IgG. In this experiment, the optimal precipitation reaction of the human IgG was obtained with 123/xg Ab/N, whereas of monkey antigen with 69/xg Ab/N. The cross reactivity of the monkey IgG in this experiment was calculated to be 56 per cent. (3) The results of the cross reactivity, as examined in single radial diffusion, are summarized in Table 1. Readings of the diameters of the diffusion rings were taken after 24 hr incubation at 4°C. The diameters were read by four reZg G ~92
MONKEY [6c
128
@
96
~5
/ ~
64
32
l
Y I
i
#g Antigen N odded
Fig. 6. Precipitation reaction of monkey and human IgG with antiserum to monkey IgG. The optimal precipitation reaction of the monkey IgG was obtained with 16s/xg Ab N and of human IgG with 93/xg Ab N.
Fig. 3. Immunoelectrophoresis of monkey (M) and human (H) sera. Monospecific antisera to monkey (a-M) immunoglobulins or to human (a-H) immunoglobulins were placed in the troughs. Similarities in the electrophoretic pattern were observed.
(Facing page 844)
T-000 M
:*oso M
_
i.
F. o*l!ioM
F.O*lOOM -
’
‘-
I
‘--
2.
Fiq:. 1Irllnunoelect~ophol-esis of‘ the 1)E:Al-T cellulose fractions (F) of rnonke)- serum elured with phosl’hkite buffer of’increasing molarities and normal monkey serum (NMS). Monospecific antisera (a-14) to monkey I$, IgA 01 Igyt were placed in the troughs. The I<$; and [#A. but not tlle IgM fractions demonstrated differences in the etectrophorctic mobititv.
?*OIOM
0
OM
OH
oti
Fig. 5. Double diffusion gel precipitation of monkey (M) and human (H) whole sera with monospecific antisera to monkey (a-M) and human (a-H) immunoglobulins. Differences in the cross reactivity could be observed.
Fig. 8. Examination of cross reactivity of monkey and human IgG in single radial diffusion. Appearance of precipitation rings of two concentrations 15 and 7.5 pg of monkey (M-IgG) and human (H-IgG) immunoglobulin G in an agar plate containing antibodies to monkey IgG (a-M IgG), and in an agar plate COIItaining antibodies to human IgG (a-H IgG). The diameters of the precipitation rings obtained with homologous antigens are smaller than those obtained with heterologous antigen.
Monkey Immunoglobulins
845
Ig G 144 HUMAN 120 ~b
96
72
48 /
//
24
i
I
,u.g Antigen N added
Fig. 7. Precipitation reaction of human and monkey IgG with antiserum to human IgG. The optimal precipitation reaction of the human IgG was obtained with 123/~g Ab N and of monkey IgG with 69/~g Ab N. searchers and, in no one case did the reading differences exceed 0.3 mm. The numerical values in the table express the diameters in millimeters. The values in the parentheses are of duplicate samples. The diameters of the precipitation rings obtained with the homologous antigens are smaller than those obtained with the heterologous antigen (Fig. 8). These differences are statistically significant (P < 0.01).
Quantitative determinationof immunoglobulins The concentration of the immunoglobulins G, A and M were determined in the sera of 20 monkeys. The individual results are presented in Table 2. The concentration of the IgG varied in the individual monkeys from 8.3 to 17.5 mg/ml. The mean value was 12.43 mg/ml with one standard deviation +--2.3. J
Table 1. Single radial diffusion test. Monkey and human IgG examined against the homologous and heterologous antisera to IgG Concentration of antigen in/zG. Human
Antisera to Human IgG Monkey IgG
15"0
7.6* (7"5)
9"5 (9.4)
7"5
6"3 (6"4)
8-4 (8"5)
15.0
8"6 (8-8)
8.9
7"5
7-9 (7-7)
7"4 (7-3)
igG Monkey
(8.8)
igO *The numerical values indicate diameters in millimeters.
846
VICTORIA MONTE-WICHER, K. WICHER and C. E. ARBESMAN Table 2. Concentration of immunoglobulins in sera of monkey Macaca mulatta Monkey No.
IgG
I mmunoglobulins IgA
IgM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
14.0" 10"0 11"3 15"0 11"3 14"4 11"2 11'0 11"0 16"5 11-4 11 '5 9"5 12"5 8"3 11 '5 17"5 14.7 13.2 12-9
4"5 3"8 3"8 4'5 1"3 2"8 4'0 5'4 4"5 2"3 1"4 3"3 6"0 3"0 2"0 3-9 4"0 3'0 2'6 4'0
0"72 0"42 0"71 1'35 1"15 0"97 0"72 0'76 1"00 1'27 0"90 0"82 0"44 0"62 0"70 0"44 0"94 1'43 1"33 0"85
3"50
0-87
8_+1"50
8-_+0"29
Mean value 12.43 Standard deviation (~i) 8+2.30 *Mg/ml.
T h e c o n c e n t r a t i o n o f the I g A varied f r o m 1.3 to 6.0 mg/ml; the m e a n value was 3.5 m g / m l with o n e s t a n d a r d deviation _+1-5. T h e concentration o f the IgM varied f r o m 0"42 to 1"43 mg/ml; the m e a n value was 0.87 m g / m l with o n e stand a r d deviation +0.29. T h e variations o f the concentrations o f the i m m u n o g l o bulins are not too m u c h h i g h e r t h a n those o b s e r v e d by o t h e r a u t h o r s [38-40] in n o r m a l h u m a n populations. DISCUSSION W h o l e m o n k e y s e r u m , w h e n c h r o m a t o g r a p h e d o n DE-52 cellulose a p p l y i n g stepwise elution o f increasing molarities (0.005-0.15 M) o f the p h o s p h a t e buffer at constant p H (8-0) d e m o n s t r a t e d an elution p a t t e r n similar to that o b s e r v e d with h u m a n serum. T h e I g G eluted f r o m the DE-52 cellulose with buffers o f increasing molarities d e m o n s t r a t e d an increasing e l e c t r o m i g r a t i o n in the IEP. T h i s would indicate that the I g G o f whole s e r u m consists o f a h e t e r o g e n e o u s p o p u l a t i o n o f molecules with different rates o f e l e c t r o m i g r a t i o n but with similar antigenic characteristics. A similar p h e n o m e n o n was o b s e r v e d earlier[41, 42] with h u m a n IgG. T h a t the m o n k e y I g G vary not only in physicochemical properties but also in a n t i b o d y activity was observed[43] in sera o f two m o n k e y s
Monkey Immunoglobulins
847
immunized with ragweed pollen. In the experiment, antibody activity as measured by radioimmunoelectrophoresis, was found in the portion of the fast moving IgG (in the /3 region) but not in the portion of the slower moving IgG (y region). Differences in the rate of electromigration were also observed in the monkey IgA of the chromatographic fractions. This immunoglobulin probably also consists of a population of various molecules. However, further studies would be necessary to fully explain this phenomenon. The IgM that was eluted with the different phosphate buffers in the DE-52 cellulose column did not show differences in the IEP. Many authors[6-28] using antiserum to human proteins compared the immunoglobulins of human and monkey. All of them observed cross reactivity between the monkey and human immunoglobulins. According to Achyara, Poulik, and Goodman [14] no differences were visible between human and nonhuman primates IgM when examined by antiserum to human IgM. In our DDGP experiments, we confirmed this observation. However, definite differences were observed when antiserum to monkey IgM was used. This indicates that the IgM of monkey and human are closely related but not identical as others have reported. The use of homologous antiserum to monkey IgM revealed the existing antigenic differences. When the IgG of human and monkey were examined against antihuman or anti-monkey IgG, there were similarities in the strength of the reaction and in the size of the spurs. It has to be stressed that both antisera were prepared with IgA that had been isolated from pooled monkey or human sera. In the IgA system, the antiserum to human IgA reacted with a certain number of monkey sera very weakly and with others not at all in contrast to the anti-monkey IgA which, in all cases, reacted with the human IgA. The differences in the reactivity of the antisera could be due to the source of the IgA used for preparation of the antisera. The monkey IgA was isolated from the pooled monkey sera whereas the IgA of human was separated from an IgA myeloma serum. From the results of the cross reactivity studies by DDGP, the following conclusions can be drawn: when using antisera to human immunoglobulins, there is a complete identity in the IgM system, less cross reactivity in the IgG and much less in the IgA system. When using antisera to monkey immunoglobulins, partial identity was observed in all three systems. However, the most striking differences were observed also in the IgA system. The cross reactivity between the monkey and human IgG was confirmed in precipitation test in tubes. In repeated experiments, the human IgG cross reacted with the antibodies to the monkey IgG in a range of 57-68 per cent, whereas the monkey IgG reacted with the antibodies to human IgG in a range of 56-60 per cent. Ishizaka and Ishizaka[26] using antiserum to human IgG for precipitation of monkey IgG found similar results. The ranges of the cross reactivity are very close between the two species; however, slightly higher cross reactivity was observed when antiserum to monkey IgG was used. From the work of Mancini et al. [37], it is known that in the SRD test, diffusion ring of any antigen applied in the antibody containing plate is directly proportional to the concentration of the antigen and inversely proportional to the con-
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VICTORIA MONTE-WICHER, K. WICHER and C. E. ARBESMAN
centration of the antibodies. The IgG of monkey, when examined in SRD in agar plate containing antibody to human IgG, demonstrated a larger precipitation ring than the same concentration of the human IgG. A similar phenomenon was observed when human IgG was examined in a plate containing antibodies to monkey IgG. The larger diffusion rings observed with the heterologous antigens can be explained on the basis of differences in the number of the antigenic determinants between both antigens reacting with the agar incorporated antibodies. The antiserum in the agar plates has fewer antibodies to the heterologous antigen; therefore, the edge of the precipitation ring for the heterologous antigen would be farther from the wells than the edge of the precipitation ring for the homologous antigen. From our results, one may conclude that when related antigens are examined in single radial diffusion method, the diffusion rate seems to be inversely proportional to the cross reactivity. The results of the cross reactivity obtained by SRD (Fig. 8 and Table 1) support the previously discussed observation of the precipitation test in liquid media; that human IgG cross reacts more with antiserum to monkey IgG than does the monkey lgG with the antiserum to human IgG. In analysis of the results in Table 1, one sees that when both antigens were examined with antiserum to monkey IgG the difference in the diameter of the precipitation rings was smaller (8.9:9.5, dif. = 0.6; or 7-4:8.4, dif. = l'0) than when examined with antiserum to human IgG (7.6 : 8.6, dif. = 1"0 or 6-3 : 7-9, dif. = 1"6). The single radial diffusion method used by us for the determination of heterologous immunoglobulins seems to be a useful tool in studies of the heterogeneity of proteins. However, the patterns of heterologous antigens with unknown grade of polymerization are not the best subjects to be examined by this method. The quantitation of serum immunoglobulins in monkey was determined by SRD. The mean values, when compared with those of human[38-40] showed to be very close in regard to the IgG and IgM whereas the mean value of the IgA was found to be higher in monkeys. It remains to be proven, however, whether we are dealing with the real values or with differences in the physicochemical properties of IgA molecules in monkey sera. Several authors [20, 23] determined the relative values of monkey immunoglobulins using antiserum to human immunoglobulins. However, only Poulik [20] mentioned that such an estimation may not be reliable. In the light of our experiments with the cross reactivity obtained in the SRD with the heterologous antigens, each estimation performed with non-homologous antiserum would be incorrect. We proved that the assumption made by Poulik was very much correct. Among the factors which may influence the protein concentration are: (1) the age of the animal, (2) the time spent in captivity, (3) the diet, and (4) the degree of an active parasitosis. We considered our monkeys as young animals, having been in captivity at least 3 months with a proper diet and without active parasitosis. Our monkeys were routinely treated with specific drugs and did not show parasites in the stool. However, the routine examination did not include any procedures to diagnose lung mites or blood parasites. Therefore, we cannot state that the monkeys were entirely free of parasites. It is known that parasites in monkeys can influence the level of IgE. Whether the remaining serum immunoglobulins are elevated in such monkeys is not well documented.
Monkey Immunoglobulins
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