Journal of Immunological Methods, 17 (1977) 39--46
39
© Elsevier/North-Holland Biomedical Press
PRODUCTION OF LARGE AMOUNTS OF ANTIBODIES, COMPLEMENT, AND LEUKOCYTES IN ASCITIC FLUIDS OF GUINEA PIGS 1
STEPHEN V. STUX 2, SHYR-TE JU and A L F R E D NISONOFF
Rosenstiel Research Center, Department of Biology, Brandeis University, Waltham, MA 02154, U,S.A. (Received 28 February 1977, accepted 21 March 1977)
A method is described for the production in guinea pigs of large amounts of ascitic fluid containing non-specific IgG, antiprotein antibodies, complement, other serum proteins and leukocytes. The method is an adaptation of a procedure previously applied to mice. A major difference is the extended schedule of inoculations required for the induction of ascites in guinea pigs; a requirement for boosting with antigen intradermally while repeatedly inoculating adjuvant intraperitoneally ; and the much larger quantities obtained. The average yield of ascitic fluid, when antigen was not used, was 113 ml per animal, and the average yield of IgG was 0.87 g. With antigen (keyhole limpet hemocyanin) the average yields were 143 ml and 1.6 g of antibody per guinea pig. Complement titers were 41 to 74% of those in serum. The number of leukocytes per ml of ascites ranged from 7 × 106 to 20 × 106. The method should be useful for the production of large amounts of leukocytes, antibodies and other serum proteins from a small colony of laboratory animals. In addition, cells can be obtained without the need to sacrifice the animal.
INTRODUCTION
A n u m b e r of methods have been described for the production of ascitic fluids containing antibodies (e.g., Munoz, 1957; Lieberman et al., 1959; Lieberman et al., 1961; Lieberman et al., 1962; Sartorelli et al., 1966; Brandt et al., 1967; Sommerville, 1967; Wager and R~is~inen, 1967). The yields of antibodies obtained by these methods are described and summarized by Tung et al. (1976), who cite additional references. Recently (Tung and Nisonoff, 1975; Tung et al., 1976), a procedure was described for producing large volumes of ascitic fluids containing relatively high titers of antibodies in mice. An essential feature of the m e t h o d is the use of a high volume-ratio of adjuvant to antigen solution; the mixture is repeatedly injected intraperitoneally. This m e t h o d has now been adapted, with certain necessary modifications, to guinea pigs. It should provide a convenient and economical source of antibodies, complement and other serum proteins, as well as leukocytes. The m u c h larger size of the guinea pig, as compared to the mouse, per1 This work was supported by Grant AI-12907 from the National Institutes of Health. 2 Postdoctoral Fellow of the National Institutes of Health (Grant AI-05089-01).
40
mits the recovery of correspondingly larger quantities of biologically active materials. M A T E R I A L S AND METHODS
Female guinea pigs of the Hartley strain were obtained from the Camm Research Institute, Wayne, NJ. The animals weighed 350--400 g. Complete Freund's adjuvant (CFA) was obtained from the Difco Co., Detroit, MI; keyhole limpet hemocyanin (KLH) from Calbiochem, San Diego, CA; and lyophilized guinea pig complement (whole serum) from Microbiological Associates, Bethesda, MD. Sheep red blood cells (SRBC) were obtained from the Baltimore Biological Laboratories, Cockeysville, MD, and stored at 4°C for a maximum of two weeks. Rabbit Fc fragments were prepared from IgG by the method of Porter (1959). The crystals were dissolved at pH 4 and filtered through Sephadex G-100 for further purification. The pH was adjusted to 4.7 prior to immunization. KLH was radioiodinated with 12sI by using chloramine T, according to the method of Hunter (1970). Anti-KLH antibodies in ascitic fluids were quantitared by carrying out precipitin reactions with increasing amounts of the labeled antigen and constant amounts of ascitic fluid in the presence of 0.01 M ethylenediaminetetraacetate (EDTA), pH 8.0. The amount of antibody in the precipitate was estimated by dissolving it in 0.04 N NaOH and reading the optical density at 280 nm; the value was corrected for the content of antigen on the basis of the radioactivity present. Concentrations of IgG in ascites were estimated by radial immunodiffusion according to the method of Mancini et al. (1965). Guinea pig IgG was purified by two precipitations with sodium sulfate at a final concentration of 18% (w/v) followed by passage through DEAE cellulose in 0.005 M phosphate buffer, pH 8. Complement activity was assayed by the method of Kabat and Mayer (1961), using optimally sensitized sheep erythrocytes, and smaller quantities of materials. Leukocytes in ascitic fluids were counted in a hemocytometer after lysing RBC with 0.90% NH4C1 (Shortman et al., 1972}. Giemsa stain was used for differential counting. Ascitic fluids were induced by intraperitoneal inoculations of complete Freund's adjuvant or emulsions of adjuvant with antigen solution, using a 9 : 1 volume ratio of adjuvant to antigen solution. A significant difference, as compared to the mouse, was the requirement for a larger number of injections, as well as larger volumes of adjuvant, to induce ascites. Whereas 4 injections of adjuvant (without antigen) suffice for many strains of mice (Tung and Nisonoff, 1975; Tung et al., 1976), 6 inoculations followed by a one month rest period and further injections were found to be necessary for guinea pigs. A typical schedule of inoculations is given in a footnote of table 1. When antigen was present, 4 inoculations of the emulsion were administered at weekly intervals. Adjuvant was then given weekly, but without anti-
9
10
0 0 8 (14) 20 (13) 0 0 60 (5.7) 0 0 0 0 0
0 0 19 7 0 0 110 0 65 0 10 2
(9.8) (9.2)
(5.9)
(4.8)
(11) (15)
58 (15) 0 D 24 (4.8) 0 0 85 (4.3) 0 107 (11) 0 0 0 20 (16) 15 (9.2) 25 (5.9) D 0 62 (3.8) 12 (13) 25 (13) 10 (5.9)
D ** 5 (8.0)
12
75 (4.2) D 0 5 (13) 40 (5.9)
D 3 (8) D
D
13
0 80 (12) 143 (5.9)
D
0
14
7 (16) 50 (6.1) D
25 (8.1)
15
0 75 (7.5)
17
1
1
3 1 Total yield of IgG 10
0 100 (8.0)
D
16
870 40 321 800 365 148 236 315 797 268 116 157 433
Yield of IgG (mg)
Guinea pigs were injected with 0.5 ml CFA i.p. weekly for 6 weeks. After 4 weeks of rest ascites fluid, if present, was tapped. Each animal then received weekly injections of CFA until the end of the experiment. Ascites, when present, was tapped prior to each injection. The numbers in parentheses are mg/ml of IgG. ** Deceased.
1 2 3 4 5 6 7 8 9 10 11 12
11
Weeks after first injection
ml of ascites (mg/ml of IgG)
Guinea pig no.
Production of IgG in ascites of individual Hartley guinea pigs *
TABLE 1
~a
11
12
13
Weeks after first injection
0 73 (16) 0 0 24 (13) 126 (12) 0 0
0 D ** 0 0 0 0 0 0 5 0 48 197 0 20 (15)
(13) (7)
(13)
7 (17)
(10) (12) (6.3) (17)
(3.8)
3 (11) 5 0 43 122 70 52
14
46 (13) 7 (13) 0 62 (7) D 0
0
15
0
0 0 0 39 (11)
0
16
94 (14)
D 0 33 (15) 24 (11)
0
17
0
19
152 1 168 682 0 0 91 23 (16) 16 (14) 2 453 6 (14) 0 5 566 441 0 0 2 500 Total yield of anti-KLH 13 053
0
18
Yield of anti-KLH Ab (mg)
* Animals were injected i.p. with 0.5 mg portions of KLH emulsified in CF A (volume ratio of C F A to antigen solution, 9 : 1, total volume, 0.5 ml). Injections were given weekly for 4 weeks. The animals were then inoculated with CF A alone (0.5 ml) at weekly intervals until the end of the experiment. At week 8 they were also inoculated intradermalty with 0.5 mg KLH in saline; 0.75 mg was given intradermally at weeks 10 and 14. The animals were examined for ascites beginning 11 weeks after the first injection and tapped weekly if fluid was present. ** Deceased.
1 2 3 4 5 6 7 8
ml of ascites (mg/ml of anti-KLH, in parentheses)
Guinea pig no.
Production of anti-KLH antibodies in ascitic fluids of individual Hartley guinea pigs *
TABLE 2
b0
43
gen. The animals were boosted by an occasional intradermal inoculation of antigen in saline into multiple sites, as described in a f o o t n o t e of table 2. Ascitic fluids were tapped from the peritioneal cavity b y using a 16-gauge, 1.5 inch hypodermic needle w i t h o u t a syringe. The needle was inserted a minimal distance into the peritoneal cavity at the position where fluid had accumulated. When large amounts of fluid were present, it emerged rapidly in a continuous stream. Generally, animals were n o t tapped unless substantial amounts of fluid were obviously present. When tapping was followed by an intraperitoneal injection, the needle was inserted into the same location. During tapping, manual pressure was exerted on the a b d o m e n of the guinea pig. The animals must be treated gently to avoid agitation which sometimes leads to coma and death. The ascites in the guinea pig formed much smaller clots than those seen in mice; when present, clots were removed b y centrifugation. RESULTS
The data in table 1 indicate yields of IgG obtained by repeated administration of complete Freund's adjuvant. The latter was given weekly for 6 weeks; weekly inoculations were continued after a 4-week rest period. When ascites were present, it was tapped prior to each inoculation. The yields in terms of volume and concentration of IgG (in parentheses) are presented in the table. In the 12 individual guinea pigs used, the average volume of ascitic fluid obtained was 113 ml and the average total yield of IgG was 0.87 g. The maximum volume obtained in a single tapping was 143 ml. Table 2 presents results obtained when guinea pigs were immunized with KLH as described under Methods and in f o o t n o t e (*) of the table. The animals were given 4 inoculations of the adjuvant-antigen emulsion (9 : 1 volume ratio); this was followed by weekly injections of adjuvant alone. Animals were boosted occasionally b y an intradermal injection of 0.5--0.75 mg of KLH in saline. Tappings were initiated when ascitic fluid appeared, at the times indicated in table 2. In the 8 individual guinea pigs tested, the average total of ascitic fluid obtained was 143 ml and the average total yield of antiKLH antibodies was 1.6 g per animal. The data in table 3 indicate the yields of complement and leukocytes in ascitic fluids of guinea pigs inoculated with CFA alone or with an emulsion containing Fc fragments of rabbit IgG. The schedules of inoculations were similar to those used to obtain the data of table 1 (CFA) and table 2 (CFA and antigen). The fluid examined was that obtained in the first tapping of each guinea pig. In the 5 guinea pigs tested the titers of complement in ascites were 41--74% as large as the titers in serum taken at the same time. The c o m p l e m e n t titer in guinea pig serum obtained from a commercial source was slightly less than the average values of the sera tested (footnote **, table 3). Since an average of 113 ml of fluid was obtained from individual guinea pigs inoculated with adjuvant alone (table 1) it is evident that ascitic fluids
12 37 48 14 100
1 *** 2 3 4 5
20 13 7 not done 13
No. of leukocytes per ml (× 10 - 6 )
2.4 4.8 3.3 -13
Total yield of leukocytes (× 10 - 8 )
1600 1200 1320 1610 1240
ascites
2860 2280 3230 3450 1670
serum
0.56 0.53 0.41 0.47 0.74
CHs0/ml serum
Complement activity (CHs0 units per ml) ** CHs0/ml ascites
19 44 63 23 124
000 000 000 000 000
Total CHs0 units in the tapping (ascites)
* Induced as described under Materials and Methods. The number of CHso units was determined by the method o f Kabat and Mayer, 1961, on a reduced scale. One CHs0 unit produces 50% hemolysis of 0.05 ml of 1% maximally sensitized SRBC. The data are averages of the results of two separate experiments. ** 1 ml of reconstituted commercial guinea pig complement (Microbiological Associates) contained 2100 CHs0 units. *** Guinea pigs 1 and 2 received CFA alone; the remainder were inoculated with an emulsion of rabbit Fc in CF A by the procedure described in a footnote of table 2.
ml of ascites (single tapping)
Guinea pig number
Leukocytes and complement in guinea pig ascitic fluids *
TABLE 3
45
are potentially a source o f large quantities of complement. In the 4 guinea pigs tested for the content of leukocytes in ascites, the average concentration was approximately 1.3 × 107 cells per ml. Viability exceeded 95% in each case. In each guinea pig more than 33% of the cells were l y m p h o c y t e s ; the remainder were polymorphonuclear cells and macrophages. It should be noted that the leukocytes are obtained without sacrificing the guinea pig. Immunoelectrophoresis indicated the presence in ascitic fluids of numerous serum proteins. DISCUSSION
The method presented here is an adaptation of that previously described for the mouse (Tung and Nisonoff, 1975; Tung et al., 1976). An important modification is the requirement for a considerably larger number of inoculations of CFA for the induction of ascites, and the need to b o o s t intradermally for optimal production of antiprotein antibodies; repeated intraperitoneal inoculations o f antigen results in hardening of the peritoneum. The technique has proven useful in the mouse for the production of substantial amounts of antibodies from individual animals, and has permitted studies of amino acid sequences on H and L chains of antibodies from pools of ascites and N-terminal sequences o f chains from individual mice (Capra et al., 1975a, b; Friendenson et al., 1975). The amounts of IgG or of specific antibodies that can be obtained from guinea pigs are many times larger and should facilitate studies on the antibodies of this species, as well as providing a convenient source of a variety of antibodies from a small laboratory animal. Although the complement activity, as measured b y lysis of sheep red blood cells, averages a b o u t half that of serum, the volumes of ascites obtainable from an individual guinea pig are so large that the total amount of complement in the ascites is much greater than can be obtained from serum. Immunoelectrophoresis indicated the presence of numerous other serum proteins, including IgM, in ascitic fluids. Since the ascites contains, on the average, a b o u t 1 × 107 leukocytes per ml, over 33% of which are lymphocytes, it is also a potentially useful source of large numbers of cells. L e u k o c y t e s derived from the ascites of mice have proven to be useful in adoptive transfers of suppressor T cells (Ju and Nisonoff, unpublished data) and of B cells producing antibodies of defined idiot y p e (Ju et al., 1977). REFERENCES Brandt, W.D., E.L. Buescher and F.M. Hetrick, 1967, Am. J. Trop. Med. 16,339. Capra, J.D., A.S. Tung and A. Nisonoff, 1975a, J. Immunol. 115, 1548. Capra, J.D., A.S. Tung and A. Nisonoff, 1975b, J. Immunol. 115,414. Friedenson, B., A.S. Tung and A. Nisonoff, 1975, Proc. Nat. Acad. Sci. 72, 3676. Hunter, R.. 1970, Proc. Soc. Exp. Biol. Med. 133,989.
46 Ju, S-T., F. Owen and A. Nisonoff, 1976, Cold Spring Harbor Symp. Quant. Biol. 6, 699. Kabat, E.A. and M.M. Mayer, 1961, Experimental Immunochemistry (Charles C. Thomas, Springfield, IL.) p. 135. Lieberman, R.J., O.A. Douglas and N. Mantel, 1959, J. Immunol. 84,514. Lieberman, R., N. Mantel and W. Humphrey, Jr., 1961, Proc. Soc. Exp. Biol. Med. 107, 163. Lieberman, R., N. Mantel, W. Humphrey, Jr and J.G. Blakeley, 1962, Proc. Soc. Exp. Biol. Med. 110,897. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistry 2,235. Munoz, J., 1957, Proc. Soc. Exp. Biol. Med. 95,757. Porter, R.R., 1959, Biochem. J. 73, 119. Sartorelli, A.C., D.S. Fisher and W.G. Downs, 1966, J. Immunol. 96,676. Shortman, K., N. Williams and P. Adams, 1972, J. Immunol. Methods 1,273. Sommerville, R.G., 1967, Arch. Virusforsch. 20,445. Tung, A.S. and A. Nisonoff, 1975, J. Exp. Med. 141,112. Tung, A.S., S-T. Ju, S. Sato and A. Nisonoff, 1976, J. Immunol. 116', 676. Wager, O. and J.A. Rasanen, 1967, Ann. Med. Exp. Biol. Fenn. 45,170.
39
© Elsevier/North-Holland Biomedical Press
PRODUCTION OF LARGE AMOUNTS OF ANTIBODIES, COMPLEMENT, AND LEUKOCYTES IN ASCITIC FLUIDS OF GUINEA PIGS 1
STEPHEN V. STUX 2, SHYR-TE JU and A L F R E D NISONOFF
Rosenstiel Research Center, Department of Biology, Brandeis University, Waltham, MA 02154, U,S.A. (Received 28 February 1977, accepted 21 March 1977)
A method is described for the production in guinea pigs of large amounts of ascitic fluid containing non-specific IgG, antiprotein antibodies, complement, other serum proteins and leukocytes. The method is an adaptation of a procedure previously applied to mice. A major difference is the extended schedule of inoculations required for the induction of ascites in guinea pigs; a requirement for boosting with antigen intradermally while repeatedly inoculating adjuvant intraperitoneally ; and the much larger quantities obtained. The average yield of ascitic fluid, when antigen was not used, was 113 ml per animal, and the average yield of IgG was 0.87 g. With antigen (keyhole limpet hemocyanin) the average yields were 143 ml and 1.6 g of antibody per guinea pig. Complement titers were 41 to 74% of those in serum. The number of leukocytes per ml of ascites ranged from 7 × 106 to 20 × 106. The method should be useful for the production of large amounts of leukocytes, antibodies and other serum proteins from a small colony of laboratory animals. In addition, cells can be obtained without the need to sacrifice the animal.
INTRODUCTION
A n u m b e r of methods have been described for the production of ascitic fluids containing antibodies (e.g., Munoz, 1957; Lieberman et al., 1959; Lieberman et al., 1961; Lieberman et al., 1962; Sartorelli et al., 1966; Brandt et al., 1967; Sommerville, 1967; Wager and R~is~inen, 1967). The yields of antibodies obtained by these methods are described and summarized by Tung et al. (1976), who cite additional references. Recently (Tung and Nisonoff, 1975; Tung et al., 1976), a procedure was described for producing large volumes of ascitic fluids containing relatively high titers of antibodies in mice. An essential feature of the m e t h o d is the use of a high volume-ratio of adjuvant to antigen solution; the mixture is repeatedly injected intraperitoneally. This m e t h o d has now been adapted, with certain necessary modifications, to guinea pigs. It should provide a convenient and economical source of antibodies, complement and other serum proteins, as well as leukocytes. The m u c h larger size of the guinea pig, as compared to the mouse, per1 This work was supported by Grant AI-12907 from the National Institutes of Health. 2 Postdoctoral Fellow of the National Institutes of Health (Grant AI-05089-01).
40
mits the recovery of correspondingly larger quantities of biologically active materials. M A T E R I A L S AND METHODS
Female guinea pigs of the Hartley strain were obtained from the Camm Research Institute, Wayne, NJ. The animals weighed 350--400 g. Complete Freund's adjuvant (CFA) was obtained from the Difco Co., Detroit, MI; keyhole limpet hemocyanin (KLH) from Calbiochem, San Diego, CA; and lyophilized guinea pig complement (whole serum) from Microbiological Associates, Bethesda, MD. Sheep red blood cells (SRBC) were obtained from the Baltimore Biological Laboratories, Cockeysville, MD, and stored at 4°C for a maximum of two weeks. Rabbit Fc fragments were prepared from IgG by the method of Porter (1959). The crystals were dissolved at pH 4 and filtered through Sephadex G-100 for further purification. The pH was adjusted to 4.7 prior to immunization. KLH was radioiodinated with 12sI by using chloramine T, according to the method of Hunter (1970). Anti-KLH antibodies in ascitic fluids were quantitared by carrying out precipitin reactions with increasing amounts of the labeled antigen and constant amounts of ascitic fluid in the presence of 0.01 M ethylenediaminetetraacetate (EDTA), pH 8.0. The amount of antibody in the precipitate was estimated by dissolving it in 0.04 N NaOH and reading the optical density at 280 nm; the value was corrected for the content of antigen on the basis of the radioactivity present. Concentrations of IgG in ascites were estimated by radial immunodiffusion according to the method of Mancini et al. (1965). Guinea pig IgG was purified by two precipitations with sodium sulfate at a final concentration of 18% (w/v) followed by passage through DEAE cellulose in 0.005 M phosphate buffer, pH 8. Complement activity was assayed by the method of Kabat and Mayer (1961), using optimally sensitized sheep erythrocytes, and smaller quantities of materials. Leukocytes in ascitic fluids were counted in a hemocytometer after lysing RBC with 0.90% NH4C1 (Shortman et al., 1972}. Giemsa stain was used for differential counting. Ascitic fluids were induced by intraperitoneal inoculations of complete Freund's adjuvant or emulsions of adjuvant with antigen solution, using a 9 : 1 volume ratio of adjuvant to antigen solution. A significant difference, as compared to the mouse, was the requirement for a larger number of injections, as well as larger volumes of adjuvant, to induce ascites. Whereas 4 injections of adjuvant (without antigen) suffice for many strains of mice (Tung and Nisonoff, 1975; Tung et al., 1976), 6 inoculations followed by a one month rest period and further injections were found to be necessary for guinea pigs. A typical schedule of inoculations is given in a footnote of table 1. When antigen was present, 4 inoculations of the emulsion were administered at weekly intervals. Adjuvant was then given weekly, but without anti-
9
10
0 0 8 (14) 20 (13) 0 0 60 (5.7) 0 0 0 0 0
0 0 19 7 0 0 110 0 65 0 10 2
(9.8) (9.2)
(5.9)
(4.8)
(11) (15)
58 (15) 0 D 24 (4.8) 0 0 85 (4.3) 0 107 (11) 0 0 0 20 (16) 15 (9.2) 25 (5.9) D 0 62 (3.8) 12 (13) 25 (13) 10 (5.9)
D ** 5 (8.0)
12
75 (4.2) D 0 5 (13) 40 (5.9)
D 3 (8) D
D
13
0 80 (12) 143 (5.9)
D
0
14
7 (16) 50 (6.1) D
25 (8.1)
15
0 75 (7.5)
17
1
1
3 1 Total yield of IgG 10
0 100 (8.0)
D
16
870 40 321 800 365 148 236 315 797 268 116 157 433
Yield of IgG (mg)
Guinea pigs were injected with 0.5 ml CFA i.p. weekly for 6 weeks. After 4 weeks of rest ascites fluid, if present, was tapped. Each animal then received weekly injections of CFA until the end of the experiment. Ascites, when present, was tapped prior to each injection. The numbers in parentheses are mg/ml of IgG. ** Deceased.
1 2 3 4 5 6 7 8 9 10 11 12
11
Weeks after first injection
ml of ascites (mg/ml of IgG)
Guinea pig no.
Production of IgG in ascites of individual Hartley guinea pigs *
TABLE 1
~a
11
12
13
Weeks after first injection
0 73 (16) 0 0 24 (13) 126 (12) 0 0
0 D ** 0 0 0 0 0 0 5 0 48 197 0 20 (15)
(13) (7)
(13)
7 (17)
(10) (12) (6.3) (17)
(3.8)
3 (11) 5 0 43 122 70 52
14
46 (13) 7 (13) 0 62 (7) D 0
0
15
0
0 0 0 39 (11)
0
16
94 (14)
D 0 33 (15) 24 (11)
0
17
0
19
152 1 168 682 0 0 91 23 (16) 16 (14) 2 453 6 (14) 0 5 566 441 0 0 2 500 Total yield of anti-KLH 13 053
0
18
Yield of anti-KLH Ab (mg)
* Animals were injected i.p. with 0.5 mg portions of KLH emulsified in CF A (volume ratio of C F A to antigen solution, 9 : 1, total volume, 0.5 ml). Injections were given weekly for 4 weeks. The animals were then inoculated with CF A alone (0.5 ml) at weekly intervals until the end of the experiment. At week 8 they were also inoculated intradermalty with 0.5 mg KLH in saline; 0.75 mg was given intradermally at weeks 10 and 14. The animals were examined for ascites beginning 11 weeks after the first injection and tapped weekly if fluid was present. ** Deceased.
1 2 3 4 5 6 7 8
ml of ascites (mg/ml of anti-KLH, in parentheses)
Guinea pig no.
Production of anti-KLH antibodies in ascitic fluids of individual Hartley guinea pigs *
TABLE 2
b0
43
gen. The animals were boosted by an occasional intradermal inoculation of antigen in saline into multiple sites, as described in a f o o t n o t e of table 2. Ascitic fluids were tapped from the peritioneal cavity b y using a 16-gauge, 1.5 inch hypodermic needle w i t h o u t a syringe. The needle was inserted a minimal distance into the peritoneal cavity at the position where fluid had accumulated. When large amounts of fluid were present, it emerged rapidly in a continuous stream. Generally, animals were n o t tapped unless substantial amounts of fluid were obviously present. When tapping was followed by an intraperitoneal injection, the needle was inserted into the same location. During tapping, manual pressure was exerted on the a b d o m e n of the guinea pig. The animals must be treated gently to avoid agitation which sometimes leads to coma and death. The ascites in the guinea pig formed much smaller clots than those seen in mice; when present, clots were removed b y centrifugation. RESULTS
The data in table 1 indicate yields of IgG obtained by repeated administration of complete Freund's adjuvant. The latter was given weekly for 6 weeks; weekly inoculations were continued after a 4-week rest period. When ascites were present, it was tapped prior to each inoculation. The yields in terms of volume and concentration of IgG (in parentheses) are presented in the table. In the 12 individual guinea pigs used, the average volume of ascitic fluid obtained was 113 ml and the average total yield of IgG was 0.87 g. The maximum volume obtained in a single tapping was 143 ml. Table 2 presents results obtained when guinea pigs were immunized with KLH as described under Methods and in f o o t n o t e (*) of the table. The animals were given 4 inoculations of the adjuvant-antigen emulsion (9 : 1 volume ratio); this was followed by weekly injections of adjuvant alone. Animals were boosted occasionally b y an intradermal injection of 0.5--0.75 mg of KLH in saline. Tappings were initiated when ascitic fluid appeared, at the times indicated in table 2. In the 8 individual guinea pigs tested, the average total of ascitic fluid obtained was 143 ml and the average total yield of antiKLH antibodies was 1.6 g per animal. The data in table 3 indicate the yields of complement and leukocytes in ascitic fluids of guinea pigs inoculated with CFA alone or with an emulsion containing Fc fragments of rabbit IgG. The schedules of inoculations were similar to those used to obtain the data of table 1 (CFA) and table 2 (CFA and antigen). The fluid examined was that obtained in the first tapping of each guinea pig. In the 5 guinea pigs tested the titers of complement in ascites were 41--74% as large as the titers in serum taken at the same time. The c o m p l e m e n t titer in guinea pig serum obtained from a commercial source was slightly less than the average values of the sera tested (footnote **, table 3). Since an average of 113 ml of fluid was obtained from individual guinea pigs inoculated with adjuvant alone (table 1) it is evident that ascitic fluids
12 37 48 14 100
1 *** 2 3 4 5
20 13 7 not done 13
No. of leukocytes per ml (× 10 - 6 )
2.4 4.8 3.3 -13
Total yield of leukocytes (× 10 - 8 )
1600 1200 1320 1610 1240
ascites
2860 2280 3230 3450 1670
serum
0.56 0.53 0.41 0.47 0.74
CHs0/ml serum
Complement activity (CHs0 units per ml) ** CHs0/ml ascites
19 44 63 23 124
000 000 000 000 000
Total CHs0 units in the tapping (ascites)
* Induced as described under Materials and Methods. The number of CHso units was determined by the method o f Kabat and Mayer, 1961, on a reduced scale. One CHs0 unit produces 50% hemolysis of 0.05 ml of 1% maximally sensitized SRBC. The data are averages of the results of two separate experiments. ** 1 ml of reconstituted commercial guinea pig complement (Microbiological Associates) contained 2100 CHs0 units. *** Guinea pigs 1 and 2 received CFA alone; the remainder were inoculated with an emulsion of rabbit Fc in CF A by the procedure described in a footnote of table 2.
ml of ascites (single tapping)
Guinea pig number
Leukocytes and complement in guinea pig ascitic fluids *
TABLE 3
45
are potentially a source o f large quantities of complement. In the 4 guinea pigs tested for the content of leukocytes in ascites, the average concentration was approximately 1.3 × 107 cells per ml. Viability exceeded 95% in each case. In each guinea pig more than 33% of the cells were l y m p h o c y t e s ; the remainder were polymorphonuclear cells and macrophages. It should be noted that the leukocytes are obtained without sacrificing the guinea pig. Immunoelectrophoresis indicated the presence in ascitic fluids of numerous serum proteins. DISCUSSION
The method presented here is an adaptation of that previously described for the mouse (Tung and Nisonoff, 1975; Tung et al., 1976). An important modification is the requirement for a considerably larger number of inoculations of CFA for the induction of ascites, and the need to b o o s t intradermally for optimal production of antiprotein antibodies; repeated intraperitoneal inoculations o f antigen results in hardening of the peritoneum. The technique has proven useful in the mouse for the production of substantial amounts of antibodies from individual animals, and has permitted studies of amino acid sequences on H and L chains of antibodies from pools of ascites and N-terminal sequences o f chains from individual mice (Capra et al., 1975a, b; Friendenson et al., 1975). The amounts of IgG or of specific antibodies that can be obtained from guinea pigs are many times larger and should facilitate studies on the antibodies of this species, as well as providing a convenient source of a variety of antibodies from a small laboratory animal. Although the complement activity, as measured b y lysis of sheep red blood cells, averages a b o u t half that of serum, the volumes of ascites obtainable from an individual guinea pig are so large that the total amount of complement in the ascites is much greater than can be obtained from serum. Immunoelectrophoresis indicated the presence of numerous other serum proteins, including IgM, in ascitic fluids. Since the ascites contains, on the average, a b o u t 1 × 107 leukocytes per ml, over 33% of which are lymphocytes, it is also a potentially useful source of large numbers of cells. L e u k o c y t e s derived from the ascites of mice have proven to be useful in adoptive transfers of suppressor T cells (Ju and Nisonoff, unpublished data) and of B cells producing antibodies of defined idiot y p e (Ju et al., 1977). REFERENCES Brandt, W.D., E.L. Buescher and F.M. Hetrick, 1967, Am. J. Trop. Med. 16,339. Capra, J.D., A.S. Tung and A. Nisonoff, 1975a, J. Immunol. 115, 1548. Capra, J.D., A.S. Tung and A. Nisonoff, 1975b, J. Immunol. 115,414. Friedenson, B., A.S. Tung and A. Nisonoff, 1975, Proc. Nat. Acad. Sci. 72, 3676. Hunter, R.. 1970, Proc. Soc. Exp. Biol. Med. 133,989.
46 Ju, S-T., F. Owen and A. Nisonoff, 1976, Cold Spring Harbor Symp. Quant. Biol. 6, 699. Kabat, E.A. and M.M. Mayer, 1961, Experimental Immunochemistry (Charles C. Thomas, Springfield, IL.) p. 135. Lieberman, R.J., O.A. Douglas and N. Mantel, 1959, J. Immunol. 84,514. Lieberman, R., N. Mantel and W. Humphrey, Jr., 1961, Proc. Soc. Exp. Biol. Med. 107, 163. Lieberman, R., N. Mantel, W. Humphrey, Jr and J.G. Blakeley, 1962, Proc. Soc. Exp. Biol. Med. 110,897. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistry 2,235. Munoz, J., 1957, Proc. Soc. Exp. Biol. Med. 95,757. Porter, R.R., 1959, Biochem. J. 73, 119. Sartorelli, A.C., D.S. Fisher and W.G. Downs, 1966, J. Immunol. 96,676. Shortman, K., N. Williams and P. Adams, 1972, J. Immunol. Methods 1,273. Sommerville, R.G., 1967, Arch. Virusforsch. 20,445. Tung, A.S. and A. Nisonoff, 1975, J. Exp. Med. 141,112. Tung, A.S., S-T. Ju, S. Sato and A. Nisonoff, 1976, J. Immunol. 116', 676. Wager, O. and J.A. Rasanen, 1967, Ann. Med. Exp. Biol. Fenn. 45,170.
