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Electrophoretic isolation of circumoval precipitins in the serum of individuals infected with Schistosoma mansoni
EXPERIMENTAL PARASITOLOGY 8, 549-556
(1959)
Electrophoretic Isolation of Circumoval Precipitins in the Serum of Individuals Infected with
Schistosoma mansoni Marta Cancio, 1 Amina R. de Sala, 2 and Rafael Rodriguez-Molina ~ Veterans Administration Center, General Medical Research Laboratory, San Juan, Puerto Rico
(Submitted for publication, 26 September 1958) The circumoval precipitin reaction seems to be the result of the interaction of specific substances in the eggs of Schistosoma mansoni with an antibody present in the serum of individuals infected with this parasite. (Oliver-Gonzhlez et al., 1955). Since most antibodies appear to be specific proteins, it was our purpose to separate the protein fractions of the blood serum of patients infected with Schistosoma mansoni in order to determine experimentally which of the fractions contains the precipitin. The relationships of such factors as electrolyte concentration, p H , and osmotic pressure, to the precipitin reaction were also considered. MATERIALS AND METHODS
The serum proteins of individuals passing viable eggs of Schistosoma mansoni and of non-infected controls were separated using the Spinco CP-Continuous Flow Paper Electrophoresis Cell enclosed in a special refrigerator kept at 5°C. 4 Pre-cut upper and lower curtains and wicks Supervisory Biochemist, General Medical Research Laboratory, San Patricio Hospital, U. S. A. Veterans Administration, San Juan, Puerto Rico, and Associate in Biochemistry, School of Medicine, School of Tropical Medicine, San Juan, Puerto Rico. Medical Biology Technician, General Medical Research Laboratory, San Patrieio Hospital, U. S. A. Veterans Administration, San Juan, Puerto Rico. 3 Assistant Director, Professional Services for Research, San Patricio Hospital, U. S. A. Veterans Administration, San Juan, Puerto Rico, and Clinical Professor of Medicine, School of Medicine, School of Tropical Medicine, University of Puerto Rico. 4 Spinco Division of Beckman Instruments, Inc., Belmont, California. Operating Instructions: Spinco Model CP, Continuous Flow Paper Electrophoresis Cell. 549
550
CANCIO, DE SALA AND RODRIGUEZ-MOLINA
from Schleicher and Schuel No. 470 filter paper were used; barbital buffer of p H 8.6 and ionic strength 0.02 was used as the electrolyte. A continuous and steady flow of buffer was maintained with the wick siphons and the overflow tube in the electrolyte reservoir set at a level of 10 cm and 5.5 cm, respectively. The current from a Spinco Constat power supply was adjusted to 30 milliamps which required 600V. The curtain was allowed to come to equilibrium by applying the current for 30 minutes prior to sample fractionation. Before sample separation the serum was dialyzed overnight in the refrigerator against the electrolyte solution. The serum sample was applied at a rate of 1.5 ml per hour to the top of the lower curtain using a tab 4 inches from the negative side of the cell. The sample was then carried downward by the flow of the buffer. A 0.5 ml. aliquot of each tube was qualitatively analyzed for protein with 10 % trichloroacetic acid. The protein fractions were then concentrated by dialysis at 5°C against 30% polyvinyl pyrrolidine solution (PVP) and the concentrates were then identified by electrophoresis employing filter paper strips (3 X 30.6 cm Whatman 3 ram) as stabilizer and barbital buffer (pH 8.6 and ionic strength, 0.05) as electrolyte. A Spinco Duostat power supply was used to apply 240 volts for 4 hours. The strips were dried at 120-130°C for 30 minutes and stained 30 minutes using bromphenol blue in methanol. 5 Aliquots of the whole and dialyzed sera were run simultaneously so as to identify each fraction by its mobility. Once the fractions were identified, they were dialyzed in the refrigerator against sera from individuals known to be free of schistosomiasis. Total protein determinations were performed on the unfractionated sera as well as on M1 the fractions by the standard biuret technique as used b y Reinhold, 1953. Circumoval tests were performed with each of the fractions as well as with an aliquot of the unfractionated serum by the technique of OliverGonz£1ez (1954), and also as described by Rodrlguez-Molina et al. (1956). Eggs of S. mansoni obtained from livers of infected mice were incubated in human serum overnight at 37°C. In the case of sera from human beings infected with Schistosoma mansoni, a precipitate was formed around the egg membrane, which was interpreted as resulting from the reaction be5 Spinco Division of Beckman Instruments, 1957. Paper electrophoresis system. Operating instructions. Revised serum protein analysis. Technical serum protein analysis. Technical Bulletin No. 6027A.
SEPARATION OF SERUM CIRCUMOVAL PRECIPITINS
551
tween antigens from the egg and immune bodies or precipitins present in the serum. No such precipitate was formed when eggs were incubated in normal sera. Several experiments were carried out to determine whether factors such as electrolyte concentration, pH and osmotic pressure affect the eggantigen antibody reaction responsible for the eireumoval precipitin test. R E S U L T S AND DISCUSSION
Six fractions were identified by their mobilities in terms of em/sec/volt/era (Fig. 1). This represents distance moved by the components as a function of time at a constant potential gradient. The fastest moving component can be identified as albumin. In order of decreasing mobility the other fractions are alpha-l-globulin, alpha-2-globulin, beta globulin, gamma-l-globulin and gamma-2-globulin. Gamma-l-globulin and gamma-2-globulin migrate in opposite directions from the point, of sample application on the filter paper strip. Gamma-l- migrates towards the positive electrode, and gamma-2- migrates towards the negative electrode. Total protein determinations were l~erformed on the sera before fractionation and on the fractions recovered. The protein recovered was 83 % of that present in the unfraetionated sera. The gamma-l-fraction obtained from the infected sera is the only fraction which gives a positive circumoval test (Fig. 2). The reaction given by this fraction is comparable to that of the unfractionated sera (Fig. 3). The gamma-l-fraction is more marked in the sera from infected Fatients although it is also evident in the control sera. The results of this study indicate that the circumoval preeipitins present in the serum of patients infected with Schistosoma mansord are localized in the gamma-l-globulin fraction. A negative eireumoval reaction was obtained on all other fractions. The isolation of the gamma-l-globulin fraction was made possible by the continuous flow paper curtain electrophoresis method of protein fractionation in which a voltage of 600 was used. The same serum when separated by paper strip electrophoresis at 240 volts showed only five definite fractions. The subfraetionation of gamma globulin into two different fractions proved to be of great importance in the localization of the preeipitins responsible for the circumoval reaction. It was found that dialysis of a circumoval positive ( 3 + ) serum against
FRACTIONS SEPARATED |N CONTINUOUS FLOW CURTAIN ELECTROPHOR|$1SAT $ C, 3~6-,58 I Gamma
Q
B
A2
A I A|b.
¢¢:
Schtslosema
mansoni
0
0
• g
0
•
.
Q
.
.
.
,
0
0
•
0
Q
FIG. 1. F r a c t i o n s s e p a r a t e d in continuous flow p a p e r c u r t a i n eleetrophoresis from S. mansoni-infected h u m a n serum. 552
S E P A R A T I O N OF S E R U M CIRCUMOVAL P R E C I P I T I N S
553
FIG. 2. Egg of S. mansoni showing positive circumoval reaction of gamma-Iglobulin fraction. (800X) the electrolyte had no deleterious effect upon the cireumoval reaction. When the serum was dialyzed against barbital buffer of p H 8.6 and ionic strength of 0.02 the serum was equilibrated with the buffer. This avoided a drastic change in the ionic concentration, osmotic pressure, etc. of the serum when it came in contact with the buffer during sample fractionation. When the circumoval positive serum was diluted 1 : 1 with the electro-
554
CANCIO~ DE SALA AND RODRIGUEZ-MOLINA
]PIG. 3. Egg of S. mansoni showing positive circumoval reaction of unfraetionated human serum. (400X)
lyte solution, a negative circumoval was obtained. This may be the effect of dilution of the serum with the buffer, or the effect of the direct addition of the electrolyte solution to the serum which may cause drastic changes in the ionic and osmotic composition of the serum. It was found that the gamma-l-fraction, as obtained after fraetionation diluted in about 15 ml of electrolyte solution, gave a 1 + reaction. When this fraction was concentrated against PVP, it gave a negative eireumoval test. However, when this concentrated fraction was dialyzed against a normal serum, a 3 + eireumoval reaction was obtained. This indicated that in the process of concentration by dialysis aganist PVP indispensable ions are removed. Dialysis against serum replenishes the lost electrolytes and insures the same osmotic conditions present in the unfraetionated sample.
SEPARATION OF SERUM CIRCUMOVAL PRECIPITINS
555
Sera from non-infected h u m a n s which were circumoval negative were used as controls. Control sera were fractionated and all experiments were performed on the fractions obtained as in the infected sera. Negative results were obtained with all fractions from the control sera. Our results with h u m a n sera are in agreement with those of E v a n s and Stirewalt (1957) who reported an increase in the g a m m a - b e t a globulin region of the sera of mice with progressive S. mansoni disease using the paper electrophoretic technique. Fiorillo (1954), from similar studies on ten h u m a n patients with S. mansoni infection associated with splenomegaly, also found a marked increase in relative b e t a - g a m m a components with a fusion of the two peaks so t h a t they could not be distinguished one from the other. In a previously reported study, E v a n s et al. (1955) indicated t h a t certain serological phenomena could be identifled with specific schistosome-antiserum fractions. When fractions obrained b y the classical Tiselius electrophoresis method were tested in vitro against living cercariae of S. mansoni, cercaricidal and pericercarial envelope forming activity were ascribed to the gamma-globulin fraction. Electrophoretic techniques have been rarely used as a tool for the study of parasitic diseases. Stauber (1954) stated t h a t the studies of Stirewalt and E v a n s illustrate the usefulness of the electrophoretic method in solving problems of interest to the parasitologist. More work along these lines should help in the elucidation of the biochemical aspects of immunoparasitology. SUMMARY
Sera from h u m a n s infected with Schistosoma mansoni and from noninfected controls were fractionated b y continuous flow paper curtain electrophoresis. The circumoval precipitin test was performed on all fractions obtained. I t was observed t h a t the circumoval precipitin antibody present in the blood sera of individuals infected with Schistosoma mansoni is located in the gamma-l-globulin fraction. The other fractions, i.e., albumin, alpha-l, alpha-2, beta and g a m m a - 2 were negative for circumoval precipitins. ADDENDUM
Since the present paper was accepted for publication, the work of Evans and Stirewalt (1959) has appeared describing the localization of CHR and cercarial agglutinating activity in a fast-moving portion of the gamnm-l-globulin (Tglobulin) of electrophoretically isolated serum from untreated human cases of schistosomiasis mansoni.
556
CANCIO~ DE SALA AND RODRIGUEZ-MOLINA I~EFERENCES
EVANS, A. S., AND STIREWALT, M. A. 1957. Serologic reactions in Schistosoma mansion infections. III. Ionographic f r a c t i o n a t i o n of sera of mice with progressive disease. Exptl. Parasitol. 6, 8-17. EVANS, A. S., STIREWALT, M. A., AND MACKENZIE, M. 1955. Serologic reactions in Schistosoma mansoni infections. II. Cercarial b e h a v i o r in electrophoretically separated fractions of sera of infected and uninfected mice. Exptl. Parasitol. 4, 419-426. EVANS, A. S., AND STIREWALT, M. A. 1959. Serologic reactions in Schistosoma mansoni infections. V. Localization of C H R a n d cercarial a g g l u t i n a t i n g factors in electrochromatographieally fraction.~ted hum.m sera. Exptl. Parasitol. 8: 1-9. FIOmLLO, A. M. 1954. E s t u d o electrofordtico de soro de pacientes portadores de esquistossomose mansoni hepato~esplenica. Hospital 0 Rio de Janeiro 45, 647 651. OLIVER-GoNZXLEZ, J. 1954. Anti egg precipitins in the serum from h u m a n s infected with Sehistosoma mansoni. J. Infectious Diseases 95, 86-91. OLIVER-GONZALEZ, J., BAUMAN, PRESTON, M., AND BENENSON, A. S. 1955. Species specificity of the anti-egg precipitin in schistosome serums. J. Infectious Diseases 96, 95-100. REINHOLD, J. G. 1953. Total P r o t e i n , Albumin, and Globulin. S t a n d a r d M e t h o d s of Clinical Chemistry. Vol. 1. Academic Press, Inc., New York, pp. 88-97. RODRIGUEZ-MOLINA, R., OLIVER-GoNZXLEZ, J., AND SERRANO, DIANA G. 1956. Studies oi1 i m m u n i t y to Schistosomiasis mansoni. I: E v a l u a t i o n of t h e circumoval prccipitin test as a diagnostic procedure in clinical Schistosomiasis mansoni. R e p o r t of 46 cases. Bol. asoc. m~d. Puerto Rico 48, 389-392. STAUBER, L. A. 1954. Application of clectrophoretic techniques in the field of parasitic diseases. Exptl. Parasitol. 3, 558-561.
(1959)
Electrophoretic Isolation of Circumoval Precipitins in the Serum of Individuals Infected with
Schistosoma mansoni Marta Cancio, 1 Amina R. de Sala, 2 and Rafael Rodriguez-Molina ~ Veterans Administration Center, General Medical Research Laboratory, San Juan, Puerto Rico
(Submitted for publication, 26 September 1958) The circumoval precipitin reaction seems to be the result of the interaction of specific substances in the eggs of Schistosoma mansoni with an antibody present in the serum of individuals infected with this parasite. (Oliver-Gonzhlez et al., 1955). Since most antibodies appear to be specific proteins, it was our purpose to separate the protein fractions of the blood serum of patients infected with Schistosoma mansoni in order to determine experimentally which of the fractions contains the precipitin. The relationships of such factors as electrolyte concentration, p H , and osmotic pressure, to the precipitin reaction were also considered. MATERIALS AND METHODS
The serum proteins of individuals passing viable eggs of Schistosoma mansoni and of non-infected controls were separated using the Spinco CP-Continuous Flow Paper Electrophoresis Cell enclosed in a special refrigerator kept at 5°C. 4 Pre-cut upper and lower curtains and wicks Supervisory Biochemist, General Medical Research Laboratory, San Patricio Hospital, U. S. A. Veterans Administration, San Juan, Puerto Rico, and Associate in Biochemistry, School of Medicine, School of Tropical Medicine, San Juan, Puerto Rico. Medical Biology Technician, General Medical Research Laboratory, San Patrieio Hospital, U. S. A. Veterans Administration, San Juan, Puerto Rico. 3 Assistant Director, Professional Services for Research, San Patricio Hospital, U. S. A. Veterans Administration, San Juan, Puerto Rico, and Clinical Professor of Medicine, School of Medicine, School of Tropical Medicine, University of Puerto Rico. 4 Spinco Division of Beckman Instruments, Inc., Belmont, California. Operating Instructions: Spinco Model CP, Continuous Flow Paper Electrophoresis Cell. 549
550
CANCIO, DE SALA AND RODRIGUEZ-MOLINA
from Schleicher and Schuel No. 470 filter paper were used; barbital buffer of p H 8.6 and ionic strength 0.02 was used as the electrolyte. A continuous and steady flow of buffer was maintained with the wick siphons and the overflow tube in the electrolyte reservoir set at a level of 10 cm and 5.5 cm, respectively. The current from a Spinco Constat power supply was adjusted to 30 milliamps which required 600V. The curtain was allowed to come to equilibrium by applying the current for 30 minutes prior to sample fractionation. Before sample separation the serum was dialyzed overnight in the refrigerator against the electrolyte solution. The serum sample was applied at a rate of 1.5 ml per hour to the top of the lower curtain using a tab 4 inches from the negative side of the cell. The sample was then carried downward by the flow of the buffer. A 0.5 ml. aliquot of each tube was qualitatively analyzed for protein with 10 % trichloroacetic acid. The protein fractions were then concentrated by dialysis at 5°C against 30% polyvinyl pyrrolidine solution (PVP) and the concentrates were then identified by electrophoresis employing filter paper strips (3 X 30.6 cm Whatman 3 ram) as stabilizer and barbital buffer (pH 8.6 and ionic strength, 0.05) as electrolyte. A Spinco Duostat power supply was used to apply 240 volts for 4 hours. The strips were dried at 120-130°C for 30 minutes and stained 30 minutes using bromphenol blue in methanol. 5 Aliquots of the whole and dialyzed sera were run simultaneously so as to identify each fraction by its mobility. Once the fractions were identified, they were dialyzed in the refrigerator against sera from individuals known to be free of schistosomiasis. Total protein determinations were performed on the unfractionated sera as well as on M1 the fractions by the standard biuret technique as used b y Reinhold, 1953. Circumoval tests were performed with each of the fractions as well as with an aliquot of the unfractionated serum by the technique of OliverGonz£1ez (1954), and also as described by Rodrlguez-Molina et al. (1956). Eggs of S. mansoni obtained from livers of infected mice were incubated in human serum overnight at 37°C. In the case of sera from human beings infected with Schistosoma mansoni, a precipitate was formed around the egg membrane, which was interpreted as resulting from the reaction be5 Spinco Division of Beckman Instruments, 1957. Paper electrophoresis system. Operating instructions. Revised serum protein analysis. Technical serum protein analysis. Technical Bulletin No. 6027A.
SEPARATION OF SERUM CIRCUMOVAL PRECIPITINS
551
tween antigens from the egg and immune bodies or precipitins present in the serum. No such precipitate was formed when eggs were incubated in normal sera. Several experiments were carried out to determine whether factors such as electrolyte concentration, pH and osmotic pressure affect the eggantigen antibody reaction responsible for the eireumoval precipitin test. R E S U L T S AND DISCUSSION
Six fractions were identified by their mobilities in terms of em/sec/volt/era (Fig. 1). This represents distance moved by the components as a function of time at a constant potential gradient. The fastest moving component can be identified as albumin. In order of decreasing mobility the other fractions are alpha-l-globulin, alpha-2-globulin, beta globulin, gamma-l-globulin and gamma-2-globulin. Gamma-l-globulin and gamma-2-globulin migrate in opposite directions from the point, of sample application on the filter paper strip. Gamma-l- migrates towards the positive electrode, and gamma-2- migrates towards the negative electrode. Total protein determinations were l~erformed on the sera before fractionation and on the fractions recovered. The protein recovered was 83 % of that present in the unfraetionated sera. The gamma-l-fraction obtained from the infected sera is the only fraction which gives a positive circumoval test (Fig. 2). The reaction given by this fraction is comparable to that of the unfractionated sera (Fig. 3). The gamma-l-fraction is more marked in the sera from infected Fatients although it is also evident in the control sera. The results of this study indicate that the circumoval preeipitins present in the serum of patients infected with Schistosoma mansord are localized in the gamma-l-globulin fraction. A negative eireumoval reaction was obtained on all other fractions. The isolation of the gamma-l-globulin fraction was made possible by the continuous flow paper curtain electrophoresis method of protein fractionation in which a voltage of 600 was used. The same serum when separated by paper strip electrophoresis at 240 volts showed only five definite fractions. The subfraetionation of gamma globulin into two different fractions proved to be of great importance in the localization of the preeipitins responsible for the circumoval reaction. It was found that dialysis of a circumoval positive ( 3 + ) serum against
FRACTIONS SEPARATED |N CONTINUOUS FLOW CURTAIN ELECTROPHOR|$1SAT $ C, 3~6-,58 I Gamma
Q
B
A2
A I A|b.
¢¢:
Schtslosema
mansoni
0
0
• g
0
•
.
Q
.
.
.
,
0
0
•
0
Q
FIG. 1. F r a c t i o n s s e p a r a t e d in continuous flow p a p e r c u r t a i n eleetrophoresis from S. mansoni-infected h u m a n serum. 552
S E P A R A T I O N OF S E R U M CIRCUMOVAL P R E C I P I T I N S
553
FIG. 2. Egg of S. mansoni showing positive circumoval reaction of gamma-Iglobulin fraction. (800X) the electrolyte had no deleterious effect upon the cireumoval reaction. When the serum was dialyzed against barbital buffer of p H 8.6 and ionic strength of 0.02 the serum was equilibrated with the buffer. This avoided a drastic change in the ionic concentration, osmotic pressure, etc. of the serum when it came in contact with the buffer during sample fractionation. When the circumoval positive serum was diluted 1 : 1 with the electro-
554
CANCIO~ DE SALA AND RODRIGUEZ-MOLINA
]PIG. 3. Egg of S. mansoni showing positive circumoval reaction of unfraetionated human serum. (400X)
lyte solution, a negative circumoval was obtained. This may be the effect of dilution of the serum with the buffer, or the effect of the direct addition of the electrolyte solution to the serum which may cause drastic changes in the ionic and osmotic composition of the serum. It was found that the gamma-l-fraction, as obtained after fraetionation diluted in about 15 ml of electrolyte solution, gave a 1 + reaction. When this fraction was concentrated against PVP, it gave a negative eireumoval test. However, when this concentrated fraction was dialyzed against a normal serum, a 3 + eireumoval reaction was obtained. This indicated that in the process of concentration by dialysis aganist PVP indispensable ions are removed. Dialysis against serum replenishes the lost electrolytes and insures the same osmotic conditions present in the unfraetionated sample.
SEPARATION OF SERUM CIRCUMOVAL PRECIPITINS
555
Sera from non-infected h u m a n s which were circumoval negative were used as controls. Control sera were fractionated and all experiments were performed on the fractions obtained as in the infected sera. Negative results were obtained with all fractions from the control sera. Our results with h u m a n sera are in agreement with those of E v a n s and Stirewalt (1957) who reported an increase in the g a m m a - b e t a globulin region of the sera of mice with progressive S. mansoni disease using the paper electrophoretic technique. Fiorillo (1954), from similar studies on ten h u m a n patients with S. mansoni infection associated with splenomegaly, also found a marked increase in relative b e t a - g a m m a components with a fusion of the two peaks so t h a t they could not be distinguished one from the other. In a previously reported study, E v a n s et al. (1955) indicated t h a t certain serological phenomena could be identifled with specific schistosome-antiserum fractions. When fractions obrained b y the classical Tiselius electrophoresis method were tested in vitro against living cercariae of S. mansoni, cercaricidal and pericercarial envelope forming activity were ascribed to the gamma-globulin fraction. Electrophoretic techniques have been rarely used as a tool for the study of parasitic diseases. Stauber (1954) stated t h a t the studies of Stirewalt and E v a n s illustrate the usefulness of the electrophoretic method in solving problems of interest to the parasitologist. More work along these lines should help in the elucidation of the biochemical aspects of immunoparasitology. SUMMARY
Sera from h u m a n s infected with Schistosoma mansoni and from noninfected controls were fractionated b y continuous flow paper curtain electrophoresis. The circumoval precipitin test was performed on all fractions obtained. I t was observed t h a t the circumoval precipitin antibody present in the blood sera of individuals infected with Schistosoma mansoni is located in the gamma-l-globulin fraction. The other fractions, i.e., albumin, alpha-l, alpha-2, beta and g a m m a - 2 were negative for circumoval precipitins. ADDENDUM
Since the present paper was accepted for publication, the work of Evans and Stirewalt (1959) has appeared describing the localization of CHR and cercarial agglutinating activity in a fast-moving portion of the gamnm-l-globulin (Tglobulin) of electrophoretically isolated serum from untreated human cases of schistosomiasis mansoni.
556
CANCIO~ DE SALA AND RODRIGUEZ-MOLINA I~EFERENCES
EVANS, A. S., AND STIREWALT, M. A. 1957. Serologic reactions in Schistosoma mansion infections. III. Ionographic f r a c t i o n a t i o n of sera of mice with progressive disease. Exptl. Parasitol. 6, 8-17. EVANS, A. S., STIREWALT, M. A., AND MACKENZIE, M. 1955. Serologic reactions in Schistosoma mansoni infections. II. Cercarial b e h a v i o r in electrophoretically separated fractions of sera of infected and uninfected mice. Exptl. Parasitol. 4, 419-426. EVANS, A. S., AND STIREWALT, M. A. 1959. Serologic reactions in Schistosoma mansoni infections. V. Localization of C H R a n d cercarial a g g l u t i n a t i n g factors in electrochromatographieally fraction.~ted hum.m sera. Exptl. Parasitol. 8: 1-9. FIOmLLO, A. M. 1954. E s t u d o electrofordtico de soro de pacientes portadores de esquistossomose mansoni hepato~esplenica. Hospital 0 Rio de Janeiro 45, 647 651. OLIVER-GoNZXLEZ, J. 1954. Anti egg precipitins in the serum from h u m a n s infected with Sehistosoma mansoni. J. Infectious Diseases 95, 86-91. OLIVER-GONZALEZ, J., BAUMAN, PRESTON, M., AND BENENSON, A. S. 1955. Species specificity of the anti-egg precipitin in schistosome serums. J. Infectious Diseases 96, 95-100. REINHOLD, J. G. 1953. Total P r o t e i n , Albumin, and Globulin. S t a n d a r d M e t h o d s of Clinical Chemistry. Vol. 1. Academic Press, Inc., New York, pp. 88-97. RODRIGUEZ-MOLINA, R., OLIVER-GoNZXLEZ, J., AND SERRANO, DIANA G. 1956. Studies oi1 i m m u n i t y to Schistosomiasis mansoni. I: E v a l u a t i o n of t h e circumoval prccipitin test as a diagnostic procedure in clinical Schistosomiasis mansoni. R e p o r t of 46 cases. Bol. asoc. m~d. Puerto Rico 48, 389-392. STAUBER, L. A. 1954. Application of clectrophoretic techniques in the field of parasitic diseases. Exptl. Parasitol. 3, 558-561.