Immunochemical study of the antigens of Trichinella spiralis larvae. II. Some physicochemical properties of these antigens

EXPERIMENTAL

PARASITOLOGY

14, 337-345 (1963)

Immunochemical Study of the Antigens of Trichilzella spiralis Larvae. II. Some Physicochemical Properties of These Antigens1 Charles

E. Tanner

Institute of Parasitology, McGill University, Macdonald College P.O., Quebec, Canada (Submitted

for publication,

11 February

1963)

A study has been made of some physicochemical properties of the precipitating antigens of Trichinella spiralis larvae using immunoelectrophoresis in agar as the principal method of analysis. Evidence is presented which strongly suggests that the isoelectric point of the major antigen of these parasite larvae is similar to that of normal human serum gamma-globulin. Fractionation of pH 7.0 buffered saline extracts of theseparasites with common laboratory reagents (5% trichloroacetic acid, acetone and ammonium sulfate at 50% saturation) has indicated that ten of the eleven antigens of this parasite are proteins. Evidence is also presented which suggests that one of the ten protein antigens. may also contain some polysaccharide associated with the protein molecule. These fractionation studies have been unable to clarify the chemical nature of the major precipitating antigen of these larvae.

The chemical nature of the antigens of Trichinella spiralis has been studied since Strobe1 ( 1911) demonstrated complementfixing activity in alkaline or antiformin extracts of larvae from pepsin-digested rat muscle. The literature on this subject has been reviewed by Gould ( 1945) and Kagan (1960). Witebsky et al. (1942) prepared an antigen for complement fixation by heating an alkaline extract of larvae in a boiling water bath for 1.5 minutes; they claimed that this antigen was superior to crude extracts for serological diagnosis. Melcher (1943) extracted 1 Presented in part at the 37th Annual Meeting of the .4merican Society of Parasitologists in Washington, D.C., June, 1962 [Tanner, C.E. 1962. Journal of Parasitology 48, (suppl.) 181. Contribution from the Institute of Parasitology, McGill University, Macdonald College P.O., Quebec, Canada, with financial assistance from the National Research Council of Canada and with funds provided by Canadian National Health Grant NO. 6047-188.

from larvae a polysaccharide and acid-soluble and acid-insoluble proteins which gave good results in the serological diagnosis of trichinosis. Labzoffsky et al. (1959) prepared eight complement-fixing fractions from pyridineextracted Trichinella larvae, and Sleeman and Muschel (1961) have recently described a method for the extraction of these parasites into serologically active ethanol-soluble and ethanol-insoluble fractions. In a previous communication (Tanner and Gregory, 1961) a report was made of immunoelectrophoretic analyses of various antigenie preparations of Trichinella spiralis larvae. It was reported that these larvae contain at least eleven electrophoretically distinct precipitating antigens. It was shown that. various antigen preparations of T. spiralis larvae were immunologically complex and that all the antigens in those preparations could be identified in crude phosphate-buffered saline extracts.

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Immunoelectrophoresis is an extremely sensitive and accurate technique for the study of biological products (Grabar, 1957). This technique has the advantage over the majority of physicochemical analytical methods in that it is a mild procedure; consequently, denaturation during analysis is minimal. This immunochemical method was used in the present study for a chemical and physicochemical analysis of the precipitating antigens of T. spiralis larvae. MATERIALS AND METHODS

Trichinella

Saline Extract

Antigen

The antigen was prepared as previously described (Tanner and Gregory, 1961). Lyophilized larvae were homogenized in pH 7.0 phosphate-buffered saline, centrifuged, and passed through a sterilizing filter membrane. The antigen was stored at 5°C until used, l:lO,OOO merthiolate being added as a preservative. Electrophoresis

and Immunoelectrophoresis

The method employed in this study was that of Grabar and Williams (1953). The majority of the determinations were done on microscope slides as previously described (Tanner and Gregory, 1961), but a few were done on 5 X 7 inches glass plates. The routine immunoelectrophoretic analyses were done for 2 hours in 15% Difco Noble Agar containing barbiturate buffer at pH 8.6, p = 0.0375 (Block et al., 1955). The current strength used in all determinations was 1.7 ma per centimeter at a voltage of 2 v per centimeter. Normal human serum proteins and the arcs of specific precipitation were stained with Amidoschwarz BlO (Uriel and Grabar, 1956). Antisera

Antisera used to identify the Trichinella antigens after immunoelectrophoresis were pooled sera from experimentally infected rabbits.

RESULTS AND DISCUSSION

In previous experiments (Tanner and Gregory, 1961) eleven different precipitating antigens were detected in extracts of Trichinella larvae. These antigens were labelled from A to K from the cathodic end of the electropherogram for purposes of identification. Of these antigens, antigen D is the major precipitating component and migrates electrophoretically in the region of normal human serum gamma-globulins. Antibodies to this antigen were found in all sera from experimentally infected animals; antibodies to the other ten antigens were not always found in every serum. The segregation of these precipitating antigens in immunoelectrophoretic analysis is specific and predictable. In view of this fact, it was decided that it might be possible to determine isoelectric points in agar in much the same way as they are determined for other macromolecules with zone and freeboundary electrophoresis (Kunkel and Tiselius, 1951; Abramson et al., 1942). Simultaneous analyses were therefore made of a buffered saline extract of the larvae and normal human serum proteins. The protein constituents of normal human serum stain well after electrophoresis in agar, but it has been impossible to obtain good and reproducible stains after electrophoresis in agar of the Trichinella saline extract. In view of this, whereas electrophoresis was perfectly adequate for serum, immunoelectrophoresis was used to determine the isoelectric point of the Trichinella antigens. The analyses were made in the pH range from 3.0 to 9.0 with Michaelis’ Verona1 buffer (Bull, 1943) at a molar concentration of 0.05 and in the pH range from 9.0 to 9.6 with glycine-NaOH buffer as the electrolyte at the same molar concentration (Dawson et al., 1959). Electrophoretic mobilities were corrected for conductivity, electroosmosis, voltage fluctuations, and the length and cross sectional area of each agar preparation used,

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as recommended by Kunkel and Tiselius ( 195 1). The conductivity of the different agar media was measured with a Sullivan and Griffiths bridge tester (H. W. Sullivan Ltd., London). Electroosmosis in the agar gels was measured using dextran stained with urea in acidified ethanol (Uriel, 1958). The electrophoretical mobilities of the albumin, beta- and gamma-globulin components in normal human serum in agar at various pH values are illustrated in Fig. 1. The mobilities of three precipitating components of Trichinella extract in the same media are shown in Fig. 2. 40

0

i

0

j g?-----T’15.

A

o Antigen

0

i

ZO-

FIG. 2. Electrophoretic mobility of three precipitating antigens in a buffered (pH 7.0) extract of Trichinella larvae in agar buffered at different pH values. Electrophoreses were done for 2 hours at 1.7 ma/cm and 2 v/cm. Verona1 buffer (Bull, 1943) was used from pH 3.2 to 9.0 ; glycine-NaOH buffer (Dawson et al., 1959) was used from pH 9.0 to 9.6. Antigens were identified after electrophoresis with a pool of sera from experimentally infected rabbits.

o 2 qLDbulin . a globulin q albumin

40 1 0 FIG. 1 Electrophoretic mobility of three components of normal human serum in agar buffered at different pH values. Electrophoreses were done for 2 hours at 1.7 ma/cm and 2 v/cm. Verona1 buffer (Bull, 1943) was used from pH 3.2 to 9.0; glycineNaOH buffer (Dawson et al., 1959) was used from pH 9.0 to 9.6. Proteins were stained with amidoschwarz BlO (Uriel and Grabar, 1956). Three loci on the abscissa indicate the isoelectric points of albumin, beta- and gamma-globulin as determined by paper and free-boundary electrophoresis by other investigators.

Theoretically, “zero” electrical mobility occurs at the isoelectric point of macromolecules, that is, where the line plot of mobility values against pH crosses the “X” (or pH) axis. This type of plot was obtained for all the substances examined, but the “isoelectric points” obtained for the three major components of normal human serum (Fig. 1) do not correspond to the values obtained by freeboundary and zone electrophoresis. The values obtained by the more conventional means are pH 4.75 for albumin (Kunkel and Tiselius, 1951), pH 5.3 for beta-globulin (Abramson et al., 1942), and pH 6.5 for gammaglobulin (Abramson et al., 1942). Using agar as a supporting medium, the result obtained

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here for albumin (pH 4.3) is about 0.4 pH different from the standard value. The difference between the values reported here for beta- and gamma-globulins is greater by about three whole pH’s from the accepted ones. This difference was obtained even though the mobility measurements were all corrected, as recommended by Kunkel and Tiselius (1951), for fluctuations in voltage, electroosmotic Aow of the buffer, the conductivity of the agar at each pH value, and the length and the cross sectional area of each of the supporting media. One factor which did not enter the corrections was the physical state of the agar at the various pH’s. On the acid side of pH 8.0 the agar medium becomes less firm with decrease in alkalinity and increase in acidity, whereas above this, the medium becomes more firm with an increase in alkalinity. The semisolidity of the agar medium in the acid range probably resembles the physical conditions found in paper electrophoresis and may, perhaps, account for the relative closeness of the values reported here for albumin and those obtained by Kunkel and Tiselius ( 1951) The increase in solidity of the agar medium at the pH’s above 8 which were investigated did not, however, interfere with the mobilities of the protein constituents of normal serum. Even though the values reported here for normal human serum are different from the accepted ones, it is possible to compare these “isoelectric points” with those obtained for three precipitating antigens in extracts of Trichinella larvae (Fig. 2), since the determinations on the serum served as controls. Of the three mobilities plotted, that of the major antigenic component (antigen D) is of the greatest interest. This constituent showed an “isoelectric point” at pH 9.8, which is precisely the same value which was obtained in these investigations for serum gamma-globulin. Further stsudies on the physicochemical definition of the antigens of T. spiralis larvae

concerned an attempt to determine the chemical nature of these substances by fractionation with various common and standard chemical reagents. Precipitation with 5% trichloroacetic acid is one of the more common procedures for the isolation of proteins from biological extracts. The immunoelectrophoretic analysis of the results of such a fractionation of a saline extract of Trichinella larvae is illustrated in Fig. 3. The analysis was made after the trichloroacetic acid sediment had been eluted in alkali, the pH adjusted to neutrality, and NaCl added to a final concentration of 0.15 J4. The results show that most of the antigens of Trichinella larvae are precipitable by 5% trichloroacetic acid, indicating that these antigens are proteins. However, the major antigenie component of the larvae (antigen D) is not sedimented by this fractionation procedure. This substance was also not found in the supernatant of this fractionation, suggesting that the acid treatment hydrolyzed either the whole antigenic molecule or the loci on the molecule responsible for antigenic specificity. As illustrated in Fig. 4, acetone (1, 2, or 3 volumes) and 50% ammonium sulfate precipitation of the eluted trichloroacetic acid sediment did not greatly add to the knowledge of these antigens. Reprecipitation at 50% ammonium sulfate, as shown in the upper half of Fig. 4, yields a sediment which contains all the important protein constituents of the saline extract of Trichinella larvae. The supernatant of this sedimentation shows the presence of two constitutents (antigens G and K) which are only partially precipitable by the salt from the acid precipitation, since both of these antigens are also found in the precipitate. The lower half of Fig. 4 shows the results of reprecipitation of the eluted trichloroacetic acid precipitate with 1, 2, and 3 volumes of acetone. This precipitation also sediments all the protein antigens of the saline extract. The

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FIG. 3. Immunoelectrophoretic analysis of a 5% trichIoroacetic acid precipitate of a buffered (pH 7.0) saline extract of Tvichinella larvae. Electrophoresis was done for 2 hours at 1.7 ma/cm and 2 v/cm in 1% Veronal-buffered agar, pH 8.6. Antigens were identified after electrophoresis with a pool of sera from experimentally infected rabbits.

difference in the acetone sediments seems to be entirely quantitative: the higher acetone concentrations bring down more antigens than the lower acetone concentrations. Qualitatively the antigens precipitated appear to be the same. In order to determine whether other simple fractionation procedures would clarify the nature of the precipitating antigens of Trichinella larvae, the crude saline extract was salted out at 50% ammonium sulfate saturation at various pH levels. The eluted ammonium sulfate precipitates and supernatants were then dialyzed against 0.15 M NaCl until no residual sulfate could be detected with BaCl. The concentration of the eluted precipitates and the supernatants was brought back, where necessary, to the original volume of saline extract fractionated by pervaporation and dialysis. The antigens were identified

after fractionation and electrophoretic segregation, as usual, by their reaction with antibodies in a pool of sera from experimentally infected rabbits. The results of this experiment are illustrated in Fig. 5. Fractionation of crude buffered saline extracts of the larvae at various pH values with ammonium sulfate at SO% saturation reveals the reluctance, as it were, of the major antigen (antigen D) to segregate entirely into either the precipitate or the supernatant of these fractionations. There seems to be, however, a quantitative difference in the segregation of this antigen. Apparently more antigen D is found in the precipitate than in the supernatant of the fractionations done at pH’s 5.5 and 6.0; at pH 7.0 most of the antigen is found in the supernatant; at pH 8.0 most of the antigen is again found in the precipitate,

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FIG. 4. Immunoelectrophoretic analysis roacetic acid sediment of a buffered (pH sulfate saturation (upper) or with acetone 2 v/cm in 1% Veronal-buffered agar, pH sera from experimentally infected rabbits.

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of fractions obtained by reprecipitating the eluted 5% trichlo7.0) saline extract of Trichinella larvae with 50% ammonium (lower). Electrophoresis was done for 2 hours at 1.7 ma/cm and 8.6. Antigens were identified after electrophoresis with a pool of Arrows indicate precipitates (4 ) and supernatants ( +).

and at pH 9.0 the great majority of this antigen is in the sediment. These results seem to suggest that it would be possible to isolate the major antigenic component of antigenic extracts of Trichinella

larvae by simple salt fractionation at different pH’s. There is one difficulty, however, which is illustrated here: antigen D, where it segregates preferentially into either the precipitate or the supernatnat, is always accompanied by ___-

FIG. 5. Immunoelectrophoretic analysis of fractions obtained by precipitating a buffered (pH 7.0) saline extract of Trichinella larvae with 50% ammonium sulfate saturation at different pH values. Electroagar, pH 8.6. .4ntigens phoresis was done for 2 hours at 1.7 ma/cm and 2 v/cm in 1% Veronal-buffered were identified after electrophoresis with a pool of scra from experimentally infected rabbits. Arrows indicate precipitates ( 4 ) and supernatants ( 1‘ ).

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at least one other antigen. The most tenacious of these companions is antigen B, which segregates immunoelectrophoretically immediately to the cathodic end of antigen D. A detailed observation of the arc of specific immune preciptation given by antigen D in both the precipitate and the sediment of the ammonium sulfate fractionations reveals the presence of other minor arcs within the major one. The seems to indicate that this antigen is not one substance, but a constellation of substances having the same, or very similar, electrophoretical mobilities and dissimilar rates of diffusion through the agar gel. Kaminski (1955) has observed, however, the formation of multiple arcs in systems containing, presumably, only one antigen and one antibody in an excess of one or the other reagent. She concluded that the most likely explanation for this phenomenon was the heterogeneity of the antibodies reacting at different sites of the same antigenic locus. This may very well be the explanation of the multiple arcs appearing with antigen D, since they appear only after salt fractionation has partially precipitated the antigen and the antiserum is in excess with respect to the concentration of the antigen. In a previous communication (Tanner and Gregory, 1961), it was demonstrated that acid or alkali fractionation of extracts of Trichinella larvae did not isolate the antigens of this parasite. These present results indicate that such is also the case with trichloroacetic acid and ammonium sulfate at 507% saturation. The inability of ammonium sulfate at 50% saturation to segregate antigen D into either the precipitate or the supernatant and its solubility in 5% trichloroacetic acid suggests that this antigen is not a protein. In 1942, Witebsky et al, prepared an antigen for complement fixation by subjecting an alkaline extract of the larvae to boiling water for 15 minutes. Since relative heat stability is one of the characterististics of

polysaccharides, a saline extract of larvae was placed in a boiling water bath for 15 minutes and then analyzed immunoelectrophoretically. Whereas Witebsky et al. ( 1942) obtained a sediment after similar treatment of the alkaline extract, there was no gross change in the saline extract after heating. The results of the immunoelectrophoretic analysis of this heated saline extract are illustrated in Fig. 6.

Immunoelectrophoreticanalysis of a buf7.0) saline extract of Trichinella larvae after the extract was heated for 15 minutes in a boiling water bath. Electrophoresis was done for 2 FIG. 6. fered (pH

hours at 1.7 ma/cm and 2 v/cm in 1% Veronalbuffered agar, pH 8.6. Antigens were identified after electrophoresis with a pool of sera from experimentally infected rabbits.

As illustrated in this figure, only one antigen (antigen H) survived the heat treatment. These results seem to indicate that this one antigen of Trichinella larvae may have some polysaccharide material in the molecule. Antigen H is not, however, entirely polysaccharide in nature since it can be precipitated with both trichloroacetic acid and ammonium sulfate. ACKNOWLEDGMENTS

The author wishes to express his gratitude to Professors W. Rowles and B. P. Warkentin of the Department of Physics, Macdonald College, McGill University, for the use of the Sullivan and Griffiths bridge tester, and to the Canadian Red Cross for generously supplying the blood from which the normal human serum used in these studies was

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obtained. The technical Gyapay is also gratefully

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assistance of Mrs. acknowledged.

OF ANTIGENS

Clara

REFERENCES

ABRAMSON, H. A., MOYER, L. S., AND GORIN, M. H. 1942. “Electrophoresis of Proteins and the Chemistry of Cell Surfaces.” Reinhold, New York. BLOCK, R. J., DURRUM, E. L., AND ZWEIG, G. 1955. “A Manual of Paper Chromatography and Paper Electrophoresis.” Academic Press, New York. BULL, H. B. 1943. “Physical Biochemistry.” Wiley, New York. DAWSON, R. M. C., ELLIOTT, D. C., ELLIOT, W .H., AND JONES, K. M. 1959. “Data for Biochemical Research.” Oxford Univ. Press, London. GOULD, S. E. 1945. “Trichinosis.” Thomas, Springfield, Illinois. GRABAR, P. 1957. Agar-gel diffusion and immunoelectrophoretic analysis. Annals of the New York Academy of Sciences 69, 591-607. GRABAR, P., AND WILLIAMS, C. A. 1953. M&hode permettant 1’Ctude conjugke des propriCtCes 8ectrophorCtiques et immunochimiques d’un melange de protiines. Application au &urn sanguin. Biochimica et Biophysics Acta 10, 193-194. KAGAN, I. G. 1960. Trichinosis: A review of biologic, serologic and immunologic aspects. Journal of Infectious Diseases 10’7, 65-93. KAMINSKI, M. 1955. Formation de lignes multiples par un systtme prkcipitant simple lors de la prCcipitation spCcifique par double diffusion en milieu gCliii& Actes du Colloque sur la Diffusion, Montpellier, France.

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H. G., AND TISELIUS, A. 1951. Electrophoresis of proteins on filter paper. Journal of General Physiology 35, 89-118. LABZOFFSKY, N. A., KUITUNEN, E., MORRISSEY, L. P., AND HAMVAS, J. J. 1959. Studies on the antigenie structure of Trichinella spira!is larvae. Canadian Journal of Microbiology 5, 395-403. MELCHER, L. R. 1943. An antigenic analysis of Trichinella spiral&. Journal of Infectious Diseases 73, 31-39. SLEEMAN, H. K., AND MUSCHEL, L. H. 1961. Studies on complement fixing antigens isolated from Tvichinella spiralis. 1. Isolation, purification and evaluation as diagnostic agents. American Journal of Tropical Medicine and Hygiene 10, 821833. STR~BEL, H. 1911. Die Serodiagnostik der Trichinosis. Miinchen Medizin Wochenschrift 58, 672-674. TANNER, C. E., AND GREGORY, J. 1961. Immunochemical study of the antigens of Trichinella spiralis larvae. I. Identification and enumeration of antigens. Canadian Journal of Microbiology 7, KUNKEL,

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J. 1958. Interprbtation quantitative des rt%ultats apr&s Clectrophor&e en gClose. II. Les prot&nes et les lipoprot&ines du &rum humain normal. Clinica Chimica Acta 3, 384-396. URIEL, J., AND GRABAR, P. 1956. Emploi de colorants dans l’analyse ClectrophorCtique et immuno&lectrophorbtique en milieu g&lit%. Annales de l’lnstitut Pasteur 90, 427-440. WITEBSKY, E., WELS, P., AND HEIDE, A. 1942. Serodiagnosis of trichinosis by means of complement fixation. New York State Medical Journal 42, 431-43s. URIEL,