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excretory antigens produced by tetraphyridia of mesocestoides corti defined by SDS—PAGE and HPLC
Veterinary Parasitology, 10 (1982) 221--228 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
221
S E C R E T O R Y / E X C R E T O R Y A N T I G E N S P R O D U C E D BY T E T R A T H Y R I D I A O F M E S O C E S T O I D E S C O R T I D E F I N E D BY SDS--PAGE AND HPLC
F. SOGANDARES-BERNAL and M.V. DENNIS
Department of Pathology, Baylor University Medical Center, Dallas, TX 75246 (U.S.A.) S. GRAHAM and E. BIEHL Departments of Biology and Chemistry, Southern Methodist University, Dallas, TX 75275 (U.S.A.) M. VOGE
Department of Microbiology and Immunology, University of California, Los Angeles, CA 90024 (U.S.A.)
ABSTRACT Sogandares-Bernal, F., Dennis, M.V., Graham, S., Biehl, E. and Voge, M., 1982. Secretory/ excretory antigens produced by tetrathyridia of Mesocestoides corti defined by SDS--PAGE and HPLC. Vet. Parasitol., 10: 221--228. Antigens derived from culture medium in which metacestodes of Mesocestoides corti Hoeppli, 1925, had been maintained were studied by sodium-dodecyl-sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and high performance liquid chromatography (HPLC). By SDS--PAGE a minimum of 18 Coomassie-staining bands were discerned, of which 4 major bands and 4 major peaks by HPLC of similar molecular weights were observed. The HPLC eluate peaks were analyzed for antigenic activity in vitro by double diffusion in two dimensions, and in vivo in a rabbit. The rabbit had been artificially sensitized with a dialyzable leukocyte extract, showing transfer-factor-like activity, from peritoneal exudate cells removed from mice infected with tetrathyridia. All of the HPLC fractions reacted with rabbit antibody prepared against secretory/excretory antigens, but the sensitized rabbit responded only to two fractions. It is now possible by HPLC to fractionate complex antigens without denaturing them and to elucidate further the role they play during infection.
INTRODUCTION K o w a l s k i a n d T h o r s o n ( 1 9 7 2 ) first d e m o n s t r a t e d a n t i g e n s f r o m a m e d i u m c o n t a i n i n g t e t r a t h y r i d i a o f M e s o c e s t o i d e s corti H o e p p l i , 1 9 2 5 . S o g a n d a r e s B e r n a l et al. ( 1 9 8 1 ) l a t e r r e p o r t e d t h e p r e s e n c e o f c i r c u l a t i n g a n t i g e n s in m i c e i n f e c t e d w i t h t e t r a t h y r i d i a o f M. corti. T h e p r e s e n t s t u d y was i n i t i a t e d t o i d e n t i f y f u r t h e r a n d assess t h e a n t i g e n s c o m p o s i n g t h e s e c r e t o r y / e x c r e t o r y p r o d u c t s o f t e t r a t h y r i d i a m a i n t a i n e d in v i t r o . *Author to whom correspondence should be addressed.
0304-4017/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company
222 MATERIALS AND METHODS
Preparation of secretory/excretory antigens Tetrathyridia were collected in large numbers from the peritoneal cavities of infected ICR/Timco mice (Texas Inbred Mouse Co., Houston, Texas) and Rockland mice (Rockland Co., Gilbertsville, Pennsylvania). The metacestodes removed from mice were washed repeatedly in physiological saline (0.85% NaC1) in order to rid them of peritoneal exudate cells attached to their surfaces (Voge et al., 1979). The tetrathyridia were maintained at 3°C in 500-ml medicine bottles containing about 500 ml lX Joklik-modified, minimum essential medium (JMEM) (Cat. No. 410-1300, Grand Island Biological Co., Grand Island, New York) with 294 mg 1'-~ L-glutamine, 75U ml -~ Penicillin G, 50 pg ml -~ streptomycin, and w i t h o u t sodium bicarbonate, or calcium chloride. When the phenol-red indicator had become straw-colored the medium was replaced. The spent culture medium was collected at about weekly intervals and dialyzed against large volumes of 1% (w/v) a m m o n i u m carbonate at 4°C. When no phenol red indicator could be detected visually in the dialysis sac, the sample was shell-frozen and lyophilized. This antigenic material was designated secretory/excretory antigens (S/E Ags).
Preparation of antisera The S/E Ags was administered to female New Zealand White rabbits (3 kg) in quantities of 2 mg in 1 ml physiological saline via the ear vein every three days until a precipitation titer of 1:8 was obtained in reaction between the serum and S/E Ags (10 mg m1-1 0.85 NaC1). The titer was established by the Ouchterlony Technique using 1% Agarose in borate-buffered saline (BBS) at pH 8.3 and the plates were examined at 24, 48, and 72 h. The wells were 3 mm in diameter by 2 mm deep and were set at 7 or 8 mm between centers. Normal rabbit serum (NRS) was also reacted against S/E Ag by the same technique as a control. The antisera were designated RAS/E.
Fractionation of S/E Ags SDS--PA GE analysis Lyophilized S/E Ags (30 mg m1-1 buffer) were dissolved in 1.5 M Tris--HC1 buffer (pH 8.8) followed by brief sonication to aid dissolution. The solubilized antigen was then separated by sodium-dodecyl-sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) following the m e t h o d of Laemmli (1970) in 1.54 mm-thick, 9% separating gels measuring 100 (length) X 140 (width) mm in 0.37 M Tris--HCl, pH 8.8, and 3% stacking gel in 0.125 M Tris--HC1, pH 6.8.
223 Prior to electrophoresis, the samples were boiled for 5 min in a sample buffer solution (ratio 1:2) consisting of 4.7 ml glass distilled water, 1 ml 0.5 M Tris--HC1, 1 ml glycerol, 1 ml 10% (w/v) sodium-dodecyl-sulfate (SDS), 0.1 ml 2-mercaptoethanol, and 0.2 ml 0.05% (w/v) bromphenol blue (tracking dye). The electrode buffer was composed of 24 g Tris (hydroxymethyl) aminomethane, 115.2 g glycine, 40 ml 10% (w/v) SDS brought to 4 1 with glass distilled water. A ten-well teflon comb was used to produce wells in the stacking gel. Wells were approximately 20 mm deep × 8 mm wide with an interwell space of approximately 4.8 mm. A Bio-Rad (Richmond, CA) Model 220 electrophoresis chamber connected to a circulating water-chiller maintaining a constant temperature of 20°C was used for all SDS--PAGE analyses. A constant 20 MA current was used once the S/E Ags had entered the separating gel. The gels were tinctured with Coomassie brilliant blue R-250 (Bio-Rad, Richmond, CA), (500 mg Coomassie brilliant blue R-250, 115 ml absolute methanol, 20 ml glacial acetic acid, 115 ml glass distilled water) for 5 min prior to destaining overnight in a 4.5:4.5:1 solution of methanol, water, and acetic acid.
High-performance liquid chromatography of S/E Ags High-performance liquid chromatography (HPLC) of the S/E Ags was performed in a Waters Associates (Milford, MA) Model 6000 A solvent delivery system using a Model 440 UV detector (254 nM), and a Bio-Rad (Richmond, CA) Bio-Sil TSK-250 column (300× 7.5 mm). The HPLC buffer used was 0.1 M Na2SO4 and 0.02 M NaH2PO4 (pH 6.8) to which sodium azide (0.05%) had been added to retard bacterial growth. The buffer was passed through Gelman (Ann Arbor, MI), Metricel membranes (GA-6, 0.45 uM pore size) and degassed prior to use. The S/E Ags (10 mg m1-1 buffer) was dissolved in the buffer, sonicated briefly to aid in dissolution, then centrifuged (4200 g, for 2 min) to remove particulate matter. Samples (60 td) were injected into the system and passed through the column at 350 psi. The samples being eluted were monitored with a chart recorder and peaks were collected in a fraction collector. After each sample was eluted, the column was flushed with a volume of buffer prior to injection of new samples to be fractionated. The eluates were pooled if their peaks matched positions when the chart strips were overlaid on a light-box. The four pooled peaks of 14 separate HPLC runs were dialyzed overnight in 4 1 of 1% ammonium carbonate, followed by lyophilization until powdered. Prior to use, the four pooled HPLC peaks were each dissolved in 200 pl 0.85% NaC1. Antigenic activity of the pooled peaks was tested in Ouchterlony plates as indicated above under preparation of antisera. A rabbit was sensitized to tetrathyridial antigens. Delayed-type hypersensitivity (DTH) was established by use of soluble peritoneal l y m p h o c y t e extracts (250 ~g TFD) (M.V. Dennis, 1980, personal communication) from infected mice. This sensitized rabbit
224
was then skin-tested with 25 pl of each of the HPLC-derived samples. Equal amounts of Keyhole-Limpet Hemocyanin (KLH) (Cal-Biochem, Behring Co., La Jolla, CA), (1 mg m1-1 0.85% NaC1) and S/E Ags (2 mg m1-1 0.85% NaC1) served, respectively, as negative and positive controls. Bio-Rad (Richmond, CA) standards used for estimation of the higher molecular weights were (in kilodaltons) a mixture of myosin (200), ~-galactosidase (116.5) phosphorylase B (94), bovine serum albumin (68}, and ovalbumin (43), carbonic anhydrase (30), soybean trypsin inhibitor (21) and lysozyme (14). In the lower molecular standards used, carbonic anhydrase and soybean trypsin inhibitor must have become denatured as judged by their absence in their usual position in the gels. The higher molecular weight standards were used both in HPLC and SDS--PAGE analyses, and the lower molecular weight standards were used only in SDS--PAGE analyses. RESULTS
SDS--PAGE analyses of S/E Ags revealed the presence of at least 18 Coomassie-staining bands ranging in molecular weights from 140 to 12 kilodaltons (Fig. 1). The relative abundance of the different substances in S/E Ags can be seen in Fig. 1. HPLC (Fig. 2) revealed four major peaks of absorbance at 254 nm. The approximate molecular weights of the four peaks were estimated from peaks produced by the separately eluted molecular weight standards passed through the same column under identical conditions. The
3 Fig. 1. S D S - - P A G E o f S / E Ags. A and G represent the molecular weight standards × 1000 in daltons. B - - F represent different loadings of the antigen in 300 7 increments per well. See t e x t for details.
225
S/E
ANTIGENS
OF
M. CORTI. HPLC
0.35
1~
PROFILE
SAMPLE
STANDARD I
0.30 0.25 >I-
.J
0.20 0.15
I
o oJo
6x5
000 0.00
;; '\
I,
',
i , 1. . . . . , ,
I
I ,
43
%
,-
3
-,
5
7
9
II
15
13
CENTIMETERS
Fig. 2. S/E Ags of M. corti fractionated by HPLC. The molecular weight of the standards (dashed-line) are indicated, in kilodaltons, above the eluate peaks. See text for details. TABLE I Estimated molecular weight of the major components of S/E Ags by SDS--PAGE and HPLC Coomassie-staining band number from origin
SDS--PAGE estimated molecular weight in kilodaltons
HPLC elution peaks measured at 254 nm
Estimated molecular weight in kilodaltons
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
140 96 82 72 70 65 58 54 52 45 41 35 31 28 26 20 15 12
1
140
2 3
82 73
4
65
226
estimated molecular weights, in kilodaltons, of the four peaks eluted by HPLC are 140, 82, 73, and 66. In approximately the same sample amounts (Fig. 1C) as used in HPLC (~6007) SDS--PAGE indicated the presence of eight Coomassie-staining bands with approximate molecular weights, in kilodaltons, of 140, 72, 65, 45, 35, 31, and 20. Table I shows the approximate molecular weights of all substances found in optimum concentrations of S/E Ags for both SDS--PAGE and HPLC. Antibodies prepared in rabbits by the administration of S/E Ags produced (Fig. 3) a maximum of four precipitin bands in reaction with S/E Ags by the Ouchterlony technique. The four HPLC peaks (Fig. 3) each produced single bands in reaction with RAS/E, but enough sample was not available to determine the complementarity or non-complementarity of the pooled peaks with each other. The rabbit, sensitized to DTH by soluble peritoneal lymphocyte products of mice infected with tetrathyridia, gave a positive skin test. It produced an obvious wheal when injected with S/E Ag (positive control) and with HPLC fractions 1 and 3, but not fractions 2 or 4, or with KLH (negative control).
1
/
/
2 c
4
3
Fig. 3. HPLC fractions of S/E Ags tested for antigenicity by gel diffusion in two dimensions (Ouchterlony). The center well (C) contained rabbit anti-S/E Ags. Wells 1--4 contained the eluate peaks indicated in Fig. 2 from left to right. Well 5 contained S/E Ags prior to being subjected to HPLC. See text for details.
227 DISCUSSION The more prominent Coomassie-staining bands in Fig. 1, sample B, represent the components found in the greatest amounts in S/E Ags and some of these are estimated to have similar molecular weights by HPLC and may be the same substance. Because of technical difficulties related to bed volume, it is possible to overload the small, commercially-available HPLC columns with the compounds being analyzed. This results in either clogging of the column or in faulty resolution of the eluates, a feature which is of lesser importance in SDS--PAGE. The HPLC profile shown in Fig. 2 represents the maximum amount of antigen (60 ul of a 10 mg S/E Ags m1-1 HPLC buffer) which could be fractionated without loss of resolution by our HPLC unit. It is possible that in instances where the Coomassie-staining bands are close together some of the substances might be present on the up or down slopes of the peaks obtained by HPLC. Reduction of the substances in the SDS buffer probably results in additional bands. It is also possible that measurement at another wavelength might, under certain circumstances, provide higher sensitivity for certain of the compounds than the standard UV detector at 254 nm. Although SDS--PAGE possibly provides greater resolution than HPLC, the SDS results in great difficulties when dealing with very small samples. HPLC, on the other hand, provides relatively clean samples of antigen and may lead to one of the best preparative antigen fractionation procedures. The major spectrophotometric peaks obtained by HPLC produce single bands in reaction with RAS/E. This suggests, b u t does not prove, that each peak is composed of a single antigen. The fact that the sensitized rabbit produced a delayed type skin test reaction to S/E Ags and to HPLC peaks 1 and 3, but not to 2 or 4 or the negative control (KLH) suggests the different roles which the S/E Ags play during infection. Observations by Scheinman et al. (1976) and others that human mesangial cells kept in culture have characteristics of smooth muscle cells are perhaps suggestive that these cells may play a hemodynamic role in the function of the glomerulus. The dual role of these mesangial cells in presumably transporting immune complexes to the juxtaglomerular apparatus is also of great interest. In the case of mice infected with tetrathyridia, large immune complexes have been visualized in the mesangium by transmission electron microscopy and identified as containing S/E Ags and 7S71 antibody by immunofluorescence (Sogandares-Bernal et al., 1981). It would seem that fractionation of antigens, if coupled with the preparative procedure of low pressureHPLC as described by Meyers et al. (1979), might produce enough purified antigens to study (i) the trapping of immune complexes, and (ii) the hemodynamic and transport role of the mesangium without the complicating side-effects of concomitant infection.
228 CONCLUSION
HPLC seems to lend itself well to the fractionation of complex antigens, seemingly without denaturing them, and for the collection of samples sufficiently large for study. ACKNOWLEDGEMENT
This investigation was supported in part by a Research Grant, PCM-7718646, from the National Science Foundation.
REFERENCES Kowalski, J.C. and Thorson, R.E., 1972. Immunization of laboratory mice against tetrathyridia of Mesocestoides corti (Cestoda) using secretory and excretory antigen and soluble somatic antigen. J. Parasitol., 58: 7 3 2 - - 7 3 4 . Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature, 22 : 680--685. Meyers, A.I., Slade, J., Smith, R.K. and Mihelich, E.D., 1979. Separation of diastereomers using a low cost preparative medium-pressure liquid chromatograph. J. Org. Chem. 44: 2247--2249. Scheinman, J.I., Fish, A.J., Brown, D.M. and Michael, A.J., 1976. Human glomerular smooth muscle (mesangial) cells in culture. Lab. Invest., 34: 150--158. Sogandares-Bernal, F., Race, M.C., Dennis, M.V. and Voge, M., 1981. Circulating antigens in infections of mice by tetrathyridia of Mesocestoides corti Hoeppli, 1925. Z. Parasitenkd., 64: 157--167. Voge, M., Sogandares-Bernal, F. and Martin, J.H., 1979. Fine structure of the tegument of Mesocestoides tetrathyridia by scanning and transmission electron microscopy. J. Parasitol., 65: 562--567.
221
S E C R E T O R Y / E X C R E T O R Y A N T I G E N S P R O D U C E D BY T E T R A T H Y R I D I A O F M E S O C E S T O I D E S C O R T I D E F I N E D BY SDS--PAGE AND HPLC
F. SOGANDARES-BERNAL and M.V. DENNIS
Department of Pathology, Baylor University Medical Center, Dallas, TX 75246 (U.S.A.) S. GRAHAM and E. BIEHL Departments of Biology and Chemistry, Southern Methodist University, Dallas, TX 75275 (U.S.A.) M. VOGE
Department of Microbiology and Immunology, University of California, Los Angeles, CA 90024 (U.S.A.)
ABSTRACT Sogandares-Bernal, F., Dennis, M.V., Graham, S., Biehl, E. and Voge, M., 1982. Secretory/ excretory antigens produced by tetrathyridia of Mesocestoides corti defined by SDS--PAGE and HPLC. Vet. Parasitol., 10: 221--228. Antigens derived from culture medium in which metacestodes of Mesocestoides corti Hoeppli, 1925, had been maintained were studied by sodium-dodecyl-sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and high performance liquid chromatography (HPLC). By SDS--PAGE a minimum of 18 Coomassie-staining bands were discerned, of which 4 major bands and 4 major peaks by HPLC of similar molecular weights were observed. The HPLC eluate peaks were analyzed for antigenic activity in vitro by double diffusion in two dimensions, and in vivo in a rabbit. The rabbit had been artificially sensitized with a dialyzable leukocyte extract, showing transfer-factor-like activity, from peritoneal exudate cells removed from mice infected with tetrathyridia. All of the HPLC fractions reacted with rabbit antibody prepared against secretory/excretory antigens, but the sensitized rabbit responded only to two fractions. It is now possible by HPLC to fractionate complex antigens without denaturing them and to elucidate further the role they play during infection.
INTRODUCTION K o w a l s k i a n d T h o r s o n ( 1 9 7 2 ) first d e m o n s t r a t e d a n t i g e n s f r o m a m e d i u m c o n t a i n i n g t e t r a t h y r i d i a o f M e s o c e s t o i d e s corti H o e p p l i , 1 9 2 5 . S o g a n d a r e s B e r n a l et al. ( 1 9 8 1 ) l a t e r r e p o r t e d t h e p r e s e n c e o f c i r c u l a t i n g a n t i g e n s in m i c e i n f e c t e d w i t h t e t r a t h y r i d i a o f M. corti. T h e p r e s e n t s t u d y was i n i t i a t e d t o i d e n t i f y f u r t h e r a n d assess t h e a n t i g e n s c o m p o s i n g t h e s e c r e t o r y / e x c r e t o r y p r o d u c t s o f t e t r a t h y r i d i a m a i n t a i n e d in v i t r o . *Author to whom correspondence should be addressed.
0304-4017/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company
222 MATERIALS AND METHODS
Preparation of secretory/excretory antigens Tetrathyridia were collected in large numbers from the peritoneal cavities of infected ICR/Timco mice (Texas Inbred Mouse Co., Houston, Texas) and Rockland mice (Rockland Co., Gilbertsville, Pennsylvania). The metacestodes removed from mice were washed repeatedly in physiological saline (0.85% NaC1) in order to rid them of peritoneal exudate cells attached to their surfaces (Voge et al., 1979). The tetrathyridia were maintained at 3°C in 500-ml medicine bottles containing about 500 ml lX Joklik-modified, minimum essential medium (JMEM) (Cat. No. 410-1300, Grand Island Biological Co., Grand Island, New York) with 294 mg 1'-~ L-glutamine, 75U ml -~ Penicillin G, 50 pg ml -~ streptomycin, and w i t h o u t sodium bicarbonate, or calcium chloride. When the phenol-red indicator had become straw-colored the medium was replaced. The spent culture medium was collected at about weekly intervals and dialyzed against large volumes of 1% (w/v) a m m o n i u m carbonate at 4°C. When no phenol red indicator could be detected visually in the dialysis sac, the sample was shell-frozen and lyophilized. This antigenic material was designated secretory/excretory antigens (S/E Ags).
Preparation of antisera The S/E Ags was administered to female New Zealand White rabbits (3 kg) in quantities of 2 mg in 1 ml physiological saline via the ear vein every three days until a precipitation titer of 1:8 was obtained in reaction between the serum and S/E Ags (10 mg m1-1 0.85 NaC1). The titer was established by the Ouchterlony Technique using 1% Agarose in borate-buffered saline (BBS) at pH 8.3 and the plates were examined at 24, 48, and 72 h. The wells were 3 mm in diameter by 2 mm deep and were set at 7 or 8 mm between centers. Normal rabbit serum (NRS) was also reacted against S/E Ag by the same technique as a control. The antisera were designated RAS/E.
Fractionation of S/E Ags SDS--PA GE analysis Lyophilized S/E Ags (30 mg m1-1 buffer) were dissolved in 1.5 M Tris--HC1 buffer (pH 8.8) followed by brief sonication to aid dissolution. The solubilized antigen was then separated by sodium-dodecyl-sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) following the m e t h o d of Laemmli (1970) in 1.54 mm-thick, 9% separating gels measuring 100 (length) X 140 (width) mm in 0.37 M Tris--HCl, pH 8.8, and 3% stacking gel in 0.125 M Tris--HC1, pH 6.8.
223 Prior to electrophoresis, the samples were boiled for 5 min in a sample buffer solution (ratio 1:2) consisting of 4.7 ml glass distilled water, 1 ml 0.5 M Tris--HC1, 1 ml glycerol, 1 ml 10% (w/v) sodium-dodecyl-sulfate (SDS), 0.1 ml 2-mercaptoethanol, and 0.2 ml 0.05% (w/v) bromphenol blue (tracking dye). The electrode buffer was composed of 24 g Tris (hydroxymethyl) aminomethane, 115.2 g glycine, 40 ml 10% (w/v) SDS brought to 4 1 with glass distilled water. A ten-well teflon comb was used to produce wells in the stacking gel. Wells were approximately 20 mm deep × 8 mm wide with an interwell space of approximately 4.8 mm. A Bio-Rad (Richmond, CA) Model 220 electrophoresis chamber connected to a circulating water-chiller maintaining a constant temperature of 20°C was used for all SDS--PAGE analyses. A constant 20 MA current was used once the S/E Ags had entered the separating gel. The gels were tinctured with Coomassie brilliant blue R-250 (Bio-Rad, Richmond, CA), (500 mg Coomassie brilliant blue R-250, 115 ml absolute methanol, 20 ml glacial acetic acid, 115 ml glass distilled water) for 5 min prior to destaining overnight in a 4.5:4.5:1 solution of methanol, water, and acetic acid.
High-performance liquid chromatography of S/E Ags High-performance liquid chromatography (HPLC) of the S/E Ags was performed in a Waters Associates (Milford, MA) Model 6000 A solvent delivery system using a Model 440 UV detector (254 nM), and a Bio-Rad (Richmond, CA) Bio-Sil TSK-250 column (300× 7.5 mm). The HPLC buffer used was 0.1 M Na2SO4 and 0.02 M NaH2PO4 (pH 6.8) to which sodium azide (0.05%) had been added to retard bacterial growth. The buffer was passed through Gelman (Ann Arbor, MI), Metricel membranes (GA-6, 0.45 uM pore size) and degassed prior to use. The S/E Ags (10 mg m1-1 buffer) was dissolved in the buffer, sonicated briefly to aid in dissolution, then centrifuged (4200 g, for 2 min) to remove particulate matter. Samples (60 td) were injected into the system and passed through the column at 350 psi. The samples being eluted were monitored with a chart recorder and peaks were collected in a fraction collector. After each sample was eluted, the column was flushed with a volume of buffer prior to injection of new samples to be fractionated. The eluates were pooled if their peaks matched positions when the chart strips were overlaid on a light-box. The four pooled peaks of 14 separate HPLC runs were dialyzed overnight in 4 1 of 1% ammonium carbonate, followed by lyophilization until powdered. Prior to use, the four pooled HPLC peaks were each dissolved in 200 pl 0.85% NaC1. Antigenic activity of the pooled peaks was tested in Ouchterlony plates as indicated above under preparation of antisera. A rabbit was sensitized to tetrathyridial antigens. Delayed-type hypersensitivity (DTH) was established by use of soluble peritoneal l y m p h o c y t e extracts (250 ~g TFD) (M.V. Dennis, 1980, personal communication) from infected mice. This sensitized rabbit
224
was then skin-tested with 25 pl of each of the HPLC-derived samples. Equal amounts of Keyhole-Limpet Hemocyanin (KLH) (Cal-Biochem, Behring Co., La Jolla, CA), (1 mg m1-1 0.85% NaC1) and S/E Ags (2 mg m1-1 0.85% NaC1) served, respectively, as negative and positive controls. Bio-Rad (Richmond, CA) standards used for estimation of the higher molecular weights were (in kilodaltons) a mixture of myosin (200), ~-galactosidase (116.5) phosphorylase B (94), bovine serum albumin (68}, and ovalbumin (43), carbonic anhydrase (30), soybean trypsin inhibitor (21) and lysozyme (14). In the lower molecular standards used, carbonic anhydrase and soybean trypsin inhibitor must have become denatured as judged by their absence in their usual position in the gels. The higher molecular weight standards were used both in HPLC and SDS--PAGE analyses, and the lower molecular weight standards were used only in SDS--PAGE analyses. RESULTS
SDS--PAGE analyses of S/E Ags revealed the presence of at least 18 Coomassie-staining bands ranging in molecular weights from 140 to 12 kilodaltons (Fig. 1). The relative abundance of the different substances in S/E Ags can be seen in Fig. 1. HPLC (Fig. 2) revealed four major peaks of absorbance at 254 nm. The approximate molecular weights of the four peaks were estimated from peaks produced by the separately eluted molecular weight standards passed through the same column under identical conditions. The
3 Fig. 1. S D S - - P A G E o f S / E Ags. A and G represent the molecular weight standards × 1000 in daltons. B - - F represent different loadings of the antigen in 300 7 increments per well. See t e x t for details.
225
S/E
ANTIGENS
OF
M. CORTI. HPLC
0.35
1~
PROFILE
SAMPLE
STANDARD I
0.30 0.25 >I-
.J
0.20 0.15
I
o oJo
6x5
000 0.00
;; '\
I,
',
i , 1. . . . . , ,
I
I ,
43
%
,-
3
-,
5
7
9
II
15
13
CENTIMETERS
Fig. 2. S/E Ags of M. corti fractionated by HPLC. The molecular weight of the standards (dashed-line) are indicated, in kilodaltons, above the eluate peaks. See text for details. TABLE I Estimated molecular weight of the major components of S/E Ags by SDS--PAGE and HPLC Coomassie-staining band number from origin
SDS--PAGE estimated molecular weight in kilodaltons
HPLC elution peaks measured at 254 nm
Estimated molecular weight in kilodaltons
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
140 96 82 72 70 65 58 54 52 45 41 35 31 28 26 20 15 12
1
140
2 3
82 73
4
65
226
estimated molecular weights, in kilodaltons, of the four peaks eluted by HPLC are 140, 82, 73, and 66. In approximately the same sample amounts (Fig. 1C) as used in HPLC (~6007) SDS--PAGE indicated the presence of eight Coomassie-staining bands with approximate molecular weights, in kilodaltons, of 140, 72, 65, 45, 35, 31, and 20. Table I shows the approximate molecular weights of all substances found in optimum concentrations of S/E Ags for both SDS--PAGE and HPLC. Antibodies prepared in rabbits by the administration of S/E Ags produced (Fig. 3) a maximum of four precipitin bands in reaction with S/E Ags by the Ouchterlony technique. The four HPLC peaks (Fig. 3) each produced single bands in reaction with RAS/E, but enough sample was not available to determine the complementarity or non-complementarity of the pooled peaks with each other. The rabbit, sensitized to DTH by soluble peritoneal lymphocyte products of mice infected with tetrathyridia, gave a positive skin test. It produced an obvious wheal when injected with S/E Ag (positive control) and with HPLC fractions 1 and 3, but not fractions 2 or 4, or with KLH (negative control).
1
/
/
2 c
4
3
Fig. 3. HPLC fractions of S/E Ags tested for antigenicity by gel diffusion in two dimensions (Ouchterlony). The center well (C) contained rabbit anti-S/E Ags. Wells 1--4 contained the eluate peaks indicated in Fig. 2 from left to right. Well 5 contained S/E Ags prior to being subjected to HPLC. See text for details.
227 DISCUSSION The more prominent Coomassie-staining bands in Fig. 1, sample B, represent the components found in the greatest amounts in S/E Ags and some of these are estimated to have similar molecular weights by HPLC and may be the same substance. Because of technical difficulties related to bed volume, it is possible to overload the small, commercially-available HPLC columns with the compounds being analyzed. This results in either clogging of the column or in faulty resolution of the eluates, a feature which is of lesser importance in SDS--PAGE. The HPLC profile shown in Fig. 2 represents the maximum amount of antigen (60 ul of a 10 mg S/E Ags m1-1 HPLC buffer) which could be fractionated without loss of resolution by our HPLC unit. It is possible that in instances where the Coomassie-staining bands are close together some of the substances might be present on the up or down slopes of the peaks obtained by HPLC. Reduction of the substances in the SDS buffer probably results in additional bands. It is also possible that measurement at another wavelength might, under certain circumstances, provide higher sensitivity for certain of the compounds than the standard UV detector at 254 nm. Although SDS--PAGE possibly provides greater resolution than HPLC, the SDS results in great difficulties when dealing with very small samples. HPLC, on the other hand, provides relatively clean samples of antigen and may lead to one of the best preparative antigen fractionation procedures. The major spectrophotometric peaks obtained by HPLC produce single bands in reaction with RAS/E. This suggests, b u t does not prove, that each peak is composed of a single antigen. The fact that the sensitized rabbit produced a delayed type skin test reaction to S/E Ags and to HPLC peaks 1 and 3, but not to 2 or 4 or the negative control (KLH) suggests the different roles which the S/E Ags play during infection. Observations by Scheinman et al. (1976) and others that human mesangial cells kept in culture have characteristics of smooth muscle cells are perhaps suggestive that these cells may play a hemodynamic role in the function of the glomerulus. The dual role of these mesangial cells in presumably transporting immune complexes to the juxtaglomerular apparatus is also of great interest. In the case of mice infected with tetrathyridia, large immune complexes have been visualized in the mesangium by transmission electron microscopy and identified as containing S/E Ags and 7S71 antibody by immunofluorescence (Sogandares-Bernal et al., 1981). It would seem that fractionation of antigens, if coupled with the preparative procedure of low pressureHPLC as described by Meyers et al. (1979), might produce enough purified antigens to study (i) the trapping of immune complexes, and (ii) the hemodynamic and transport role of the mesangium without the complicating side-effects of concomitant infection.
228 CONCLUSION
HPLC seems to lend itself well to the fractionation of complex antigens, seemingly without denaturing them, and for the collection of samples sufficiently large for study. ACKNOWLEDGEMENT
This investigation was supported in part by a Research Grant, PCM-7718646, from the National Science Foundation.
REFERENCES Kowalski, J.C. and Thorson, R.E., 1972. Immunization of laboratory mice against tetrathyridia of Mesocestoides corti (Cestoda) using secretory and excretory antigen and soluble somatic antigen. J. Parasitol., 58: 7 3 2 - - 7 3 4 . Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature, 22 : 680--685. Meyers, A.I., Slade, J., Smith, R.K. and Mihelich, E.D., 1979. Separation of diastereomers using a low cost preparative medium-pressure liquid chromatograph. J. Org. Chem. 44: 2247--2249. Scheinman, J.I., Fish, A.J., Brown, D.M. and Michael, A.J., 1976. Human glomerular smooth muscle (mesangial) cells in culture. Lab. Invest., 34: 150--158. Sogandares-Bernal, F., Race, M.C., Dennis, M.V. and Voge, M., 1981. Circulating antigens in infections of mice by tetrathyridia of Mesocestoides corti Hoeppli, 1925. Z. Parasitenkd., 64: 157--167. Voge, M., Sogandares-Bernal, F. and Martin, J.H., 1979. Fine structure of the tegument of Mesocestoides tetrathyridia by scanning and transmission electron microscopy. J. Parasitol., 65: 562--567.