Immunochemistry, Vol. 0 Pergamon Press Ltd.

IS,

pp. 371-378

1978

Printed in Great Britain

PURIFICATION OF THE i-ANTIGEN 51 A FROM PARAMECIUM TETRA URELIA BY IMMUNOAFFINITY CHROMATOGRAPHY RICHARD H. DAVIS, JR. and EDWARD STEERS, JR.* National Institutes of Health, Laboratory of Chemical Biology, NIAMDD, 9000 Rockville Pike, Bethesda, MD 20014, U.S.A. (First received I5 August

1977; in revised

form10 October

1977)

Abstract-Purification of the surface antigen of Paramecium tetraurelia (termed i-antigen) by previously published procedures has been shown to result in preparations which frequently contain contaminating proteins including a thiol-activated protease. Alternate procedures for the purification of i-antigen were developed using ion-exchange chromatography. While certain of these procedures result in a homogeneous preparation of i-antigen, the poor yields obtained make these procedures impractical. An alternate method for the purification of i-antigen was developed using immunoaffinity chromatography. Purification by this technique proved to be superior to the various standard procedures described previously. The immunoaffinity column is rapid and results in a recovery, on the average, of over 95% of the applied material. The columns are readily regenerated and may be used numerous times without loss of capacity or purification ability. The i-antigen purified by this method appears homogeneous immunologically and electrophoretically on polyacrylamide gel electrophoresis and isoelectric focusing.

INTRODUCTION

The immobilization antigen (i-antigen) is a protein located on the surface of the protozoan P. tetraurelia. The term i-antigen refers to the fact that paramecia exposed to homologous antisera made against this protein, become immobilized and eventually die. Even though the exact function of this surface protein is unknown, the i-antigen has been of interest to cell biologists and geneticists for several reasons. This type of paramecium is capable of making as many as 12 different i-antigens. Usually, however, only one i-antigen is believed to be expressed by an animal at any given time. This mutual exclusion has been shown to be controlled by the cytoplasmic state of the paramecium which in turn is affected by the environment of the cell. Although the methods for transformation of serotype are well documented, the mechanism(s) controlling this transformation are unknown. This system represents an interesting model for the study of intra- and intercellular differentiation and gene regulation (Preer, 1969; Sommerville, 1970). Researchers agree that the protein responsible for the immobilization reaction in paramecium is a large protein of approximately 300,000 daltons. There is conflicting evidence, however, concerning theexistence and/or number of subunits composing the protein. This controversy has arisen in part because methods previously used to purify the i-antigen have been shown to yield contaminating proteins, some of which contain degradative activity (Hansma, 1975; Steers & Davis, 1976). The study of a homogeneous preparation of i-antigen and its comparison with other serotypic i-antigens should greatly aid in

* To whom correspondence

should be addressed. 371

understanding the mechanism(s) controlling their expression. The subject of this paper is the preparation of homogeneous i-antigen by methods not previously described. MATERIALS

AND METHODS

Culture methods Paramecium tetraurelia, strain 51.7s (isolated at Spencer, Indiana) was used for this study. Cultures were grown from single cell isolations on a Cerophyl infusion (Cerophyl Laboratories, Kansas City, Missouri) in 5 gallon carboys. 12 I. of infusion buffered with Na2HP04.7HZ0 were expanded to 96 I. by procedures previously described (Pollack & Steers, 1973). Culture fluid was inoculated with Klebsiella cloacae (ATCC 27889) 24 hr before use for expansion. The cells were harvested at maximum stationary phase by concentrating in a De Laval modified cream separator (Preer & Preer, 1959). The concentrate (1.4 I.) was further concentrated by centrifuging in an International Model HN centrifuge using 100 ml pear-shaped tubes. The packed cells, averaging 2.5-3.0 ml of packed cells per 12 1. of culture fluid, were extracted for the i-antigen by the procedure of Preer (1959a). SE-Sephadex

column chromatography

SE-Sephadex G-25 (Pharmacia, Uppsala), was swollen and washed with 0.10 M sodium acetate pH 4.2 buffer. After packing, the column (2 x 20 cm) was equilibrated by washing with 0.05 M sodium acetate buffer pH 4.2, overnight at room temperature (25°C). The protein was dialyzed overnight at 4°C and then applied to the column in this same buffer. Elution was performed in 0.05 M sodium acetate buffer with a stepwise gradient of pH 4.2, 4.6 and 5.2. The elution was followed by monitoring the absorbance at 280 nm. Selected fractions were pooled and frozen until needed.

312

RICHARD

H. DAVIS,

JR. and EDWARD

The DEAE-cellulose (DE-52) was prepared by washing with ().I N HCI followed by 0.1 N NaOH and then with distilled water until the slurry was neutral. The DEAEcellulose was then equilibrated by extensive washing with 0.05 M sodium phosphate buffer pH 7.0.

Polyacrylamide disc gel electrophoresis was carried out according to the manual supplied by the Canal Industrial Corp. (Rockville, MD). Polyacrylamide gels containing 0.1% Na dodecylsulfate (SDS) were prepared and electro-

Ag-4

Ag-4 +DTT

JR.

phoresed according to the procedures described by Shapiro et ul. (1967). The SDS gels were 5% with respect to acrylamide concentration. Electrophoresis wascarried out at room temperature with a current of 6 ma per tube for 4 hr. Gels were stained for protein with 0.1% AmidoSchwartz in 755% acetic acid. Preparation

Isoelectric focusing was performed by the method of Doerr and Chrambach (1971) with the following modifications. The gels were made 4% in acrylamide. Temed was not included. The upper reservoir contained0.01 MH,PO, and the lower reservoir, 0.02 M NaOH. The protein to be electrofocused was either included in the polymerized gel or layered on the top of the polymerized gel under a protective layer of sucrose and ampholine. The gels were electrofocused at a constant 200 V for 14 hr. The electrofocused gels were stained by the method of Reisner et (II. (1975).

STEERS,

of untisrrtr

Antisera utilized in these experiments were prepared by injecting rabbits subcutaneously with approx 0.5 mg of the appropriate protein together with an equal volume of Freund‘s complete adjuvant. The injections were once a week for a period of three weeks after which blood was collected hy ear-bleedings at one week intervals.

Two-dimensional double diffusion tests (Ouchterlony, 1948) were carried out in Hyland agar plates (Tavenol Labs. Inc. Costa Mesa, CA). Incubations proceeded for 18-24 hr after which the plates were observed and photo graphically recorded. Double diffusion in 0.9% agar layered in I .7 mm (i.d.) Pyrex tubes was used to determine the relative efficiency of antigen binding of the immunoaffinity column (Preer, 1956).

BSA

Ag-4 + BSA

Ag-4 + BSA + DTT

Fig. 1. Polyacrylamide gel electrophoresis (5% polyacrylamide disc gels containing 0. I% Na dodecyl sulfate) of i-antigen purified by the procedure of Preer (1959u), and showing the presence of sulfhydrylactivated protease. Ag l-4, i-antigen preparation; Ag 14 + DTT, i-antigen preincubated with 0.01 M DTT: BSA. bovine serum albumin: Ag l-4 + BSA. mixture of i-antigen and bovine serum albumin; Ag l-4 + BSA + DTT, mixture of i-antigen and bovine serum albumin preincubated with 0.01 M DTT. Gels were stained with 0.1% Amido-Schwartz. 100 fig of each protein sample was applied to each gel.

lmmunoaffinity

Prepurution of anfi-i-antigen

Purification

of i-antigen

ujinity column

Gel. 15 g of Sepharose

4B was washed with 250 ml of distilled H,O on a sintered glass funnel under suction. The gel was then washed with 4-25 ml volumes of H,O. centrifuging between washes: decanting the supernatant and . line particles ‘_ IgG. IgG was isolated from whole sera by the method of Livingston (1974). The separated IgG was concentrated to S-IO mg/ml by adjusting to SO% saturation with (NH,)$O, and then dialyzed against 1 I. of 0.1 M NaHCOiI. pH 8. I at 4°C for 24 hr. The dialyzed IgG was adjusted to pH 9.6 with 0.2 M Na,CO. 0.5 M NaCl and stored in an ice bucket.

Activation

of gel (ut 25°C). 5 g of washed Sepharose

48

in I5 ml of H,O was adjusted to pH II with a solution of IO9 NaOH dropwise with constant stirring. At zero time, 2 ml of CNBr/CH,CN (I g/ml) was added maintaining the pH at I I for I2 min by the dropwise addition of 10% NaOH (March et al.. 1974). The temperature was maintained at 25” by the addition of ice. The gel solution is constantly stirred during this time. After I2 min. the gel was transferred to a pre-cooled (0°C. in ice) sintered glass lilter and washed with 100 ml H,O (cold) followed by 20 ml of cold 0.2 M NazC0,.0.5 M NaCl at pH 10.4. The washing was done as quickly as possible due to the instability of the activated gel. _

Couplirfg The activated gel (5 g) was mixed with cold IgG (75 mg in I I ml) and agitated end over end in a plastic centrifuge tube for I7 hr at 4°C (mechanical stirring may damage the gel beads). The gel was centrifuged and the supernatant removed. The difference in 280 nm absorbance of the supernatant before and after coupling was taken to indicate the amount of protein bound to the gel. X2-96% of the IgG was coupled to the gel. The gel was then washed with 100 ml Na,CO,,, pH 10.4, 0.05 M NaCI. The gel was incubated in 1.0 M ethanolamine (pH 8.0 with NaOH) at room temperature for 2.5 hr. The gel was washed on a sintered glass funnel with 100 ml each of: (a) 0. I M Na acetate, 0.5 M NaCl; (b) 2.0 M urea, 0.5 M NaCl; (c) 0.1 M NaHCO,. 0.5 M NaCI. The gel was rinsed with distilled H,O between each wash. Following the final wash, the gel was suspended in 0.05 A4 phosphate buffer. pH 7.3 and stored at 4°C until used.

The immobilization antigen as purified by previously published methods, is not pure. In light of this it is possible that the characterization previously done on this protein is not correct. Analysis of this protein preparation (Ag-4) on standard polyacrylamide gel electrophoresis frequently shows multiple components. Furthermore, in the presence of reducing agents such as dithiothreitol, degradative activity can be demonstrated by SDS acrylamide gel electrophoresis (Fig. I) (Hansma, 1975; Steers & Davis, 1976). Ion exchange chromatography on DEAEcellulose has been used previously as a step in the purification of i-antigen (Ma&doe & Reisner, 1967). In an effort to further purify the i-antigen, DEAE-Sephadex was used as an additional step to the previously described procedure (Steers&Davis, 1976). With DEAE-Sephadex two fractions could be separated (designated D-I and D-II). Although the degradative activity (D-II) could be separated from the immunological activity (D-I), polyacrylamide electrophoresis still showed multiple components in

I

I

SIh .

-I

,IH

pH46

I

02MLA juu t

0

20

40

fi0

RO

100

120

140

FRACTION NUMRER

Fig. 2. Elution pattern obtained from chromatographing i-antigen purified by procedure of Preer (19S9a) on SESephadex A-50column 2 x 15 cm in 0.05 M sodium acetate buffer, pH 4.2. The elution buffers consisted of 0.05 M sodium acetate run as a stepwise gradient of pH 4.2. 4.6 and 5.2. Fractions containing 0.8 ml were collected and read in a spectrophotometer at 280 nm.

the D-I fraction. The heterogeneity clearly discernible on standard 6.7% polyacrylamide gels was further emphasized by chromatography of th_e D-l fraction on SE-Sephadex (Fig. 2). Four separate fractions (designated S-I, S-II, S-III and S-IV) were evident in this elution pattern. Analysis on polyacrylamide disc gels of the separate fractions showed S-I to be very similar to the applied protein fraction. S-I was therefore considered to result from saturating the column. S-II and S-III gave single bands on disc electrophoresis whereas S-IV appeared to consist of multiple components. To determine which fraction was the immobilization antigen, the separate peaks S-II, S-III and S-IV were used to immunize rabbits. These immunization experiments showed that only antisera to fraction S-II was specific for serotype 51 A. Similarly, antiS-II sera which had been absorbed with live cells of serotype 51 A did not give a precipitation band on Ouchterlony plates nor did it cause immobilization when incubated with homologous cells. The technique of Ouchterlony (1948) also showed that components in S-III and S-IV do not cross react with S-II. While the peak fraction of S-II appeared pure on disc gels, the quantitative purification of i-antigen by this technique proved to be impractical for several reasons. First, on an average, as much as 50% of the protein applied to this ion exchange column was not recovered by the elution procedure. This was true whether crude extract or the pooled S-II was applied to the column. Second, pooled fractions of S-II were found to be cross-contaminated by a small fraction of S-III. Also, extracts of certain non-51 A paramecium (those not immobilized by anti-S-II sera) give the same elution pattern on SE-Sephadex as extracts from 51 A-type paramecium. This presents a problem when working with cultures which

RICHARD H. DAVIS. JR. and EDWARD STEERS, JR

374 2.4,

“7 AS-1

2.0

E = 1.6

i

w 5 2 1.2 B $ $ 0.8

0.4

0

. 0 Fraction

Number

Fig. 3. Elution pattern obtained from chromatographing i-antigen purified by procedure of Preer (1959~) on an immunoaffinity column 0.6 x 10 cm. Sample, containing 15 mg of protein in 1 ml, applied in 0.05 M phosphate buffer, pH 7.3, and eluted with 0.05 M glycine-NaOH buffer, pH 10.4. 0.8 ml fractions collected and read spectrophotometrically at 280 nm. Arrow indicates start of elution buffer.

AS-1

AS-2

may contain ceils of other serotypes or with cultures which consist of antigenically unstable cell lines (contain more than one antigenic type) (Seed et al., 1964; Finger et al., 1969). The di~culties encountered in the purification of the i-antigen with Sephadex ion-exchange chromatography, prompted us to employ the specific binding of antibody with antigen in the form of an immunoaffinity column. Aliquots from the leading edge of the S-II peak (the second peak to elute from SE-Sephadex, Fig. 2), shown to be pure for serotype 5 1 A by immunoanalysis and by polyacrylamide gel electrophoresis, were used to immunize rabbits. The antisera obtained against this select fraction was found by immunodiffusion and immobilization tests to be specific for the 51 A antigen only. The immunoaffinity column was made by reacting 75 mg of purified anti-S-II IgG to 5 g of CNBractivated Sepharose 4B. The coupling was 82% efficient with 12.3 mg IgG per g of Sepharose in the final product. The elution profile of acolumn packed with this gel is shown in Fig. 3. Using the standard Preer preparation as the applied protein sample, two fractions, designated AS-1 and AS-2, were eluted from the column. In 0.05 M PO, buffer, pH 7.3, AS-I appeared in

BSA

AS-I, BSA, OTT

AS-2, BSA, OTT

Fig. 4. Polyacrylamide gel electrophoresis of i-antigen fractions etuted from immunoaffinity column. 5% polyacrylamide disc gels containing 0.1% Na dodecyl sulfate. AS-I, fall-through peak which does not bind to column in 0.05 M Na phosphate buffer, pH 7.3. AS-2. peak which binds to column and is eluted with 0.05 glycine-NaOH buffer, pH 10.4; BSA, bovine serum albumin standard: AS-I, BSA, DTT, mixture of peak AS-1 and bovine serum albumin preincubated with 0.01 M DTT showing degradation; AS-2 BSA, DTT, mixture of peak AS-2, bovine serum atbumin preincubated with 0.01 M DTT. Gels stained with 0.1% Amido-Schwartz. 100 /lg of each protein sample applied to each gel.

Immunoal?inity

Purification

375

of i-antigen

Anti-S-IILSepharose’

S-II

S-l 3.0

B

1.0

p

2.0 IL_&_ 0

0.6

-

0.4

-

0.2

-

I -I

10

20

30

rESA

2.0

0.8

1.0

L Fraction

Number

L 0 Fraction

Number

1 10 Fraction

20

30

Number

Fig. 5. Elution patterns on immunoafinity column of fractions separated on SE-Sephadex and control fractions: non-51 A i-antigen and bovine serum albumin samples applied and washed with 0.05 M sodium phosphate buffer, pH 7.3. Eluted with 0.05 Mglycine-NaOH buffer, pH 10.4, arrow indicates start of elution buffer. 0.8 ml fractions collected and read spectrophotometrically at 280 nm.

the void volume of the column. This fraction contained some immunological activity resulting from applying an excess of protein to the column. The excess immunological activity could be removed quantitatively by recycling the fall-through fraction a second time. Polyacrylamide electrophoresis showed the AS-1 fraction to contain multiple components and SDS gel electrophoresis indicated that this fraction still contained all the degradative activity of the starting preparation (Fig. 4). AS-2 could be eluted with 6 M urea or 0.05 M glycine-NaOH buffer, pH 10.4. On polyacrylamide electrophoresis, only one component was noted in the AS-2 fraction and on SDS gels, no degradative activity was evident. AS-2 reacted with antisera prepared against the starting antigen preparation (Ag-4) as well as antisera prepared against whole cell homogenates to give a single precipitin band in agar double diffusion tests over an antigen concentration range of 4.0 mg per ml to 0.25 per ml. The efficiency of anti-S-II IgG coupled to Sepharose in binding AS-2 was compared by the quantitative method of Preer (1956) to anti-S-II IgG which had not been coupled to Sepharose. The bound anti-S-II IgG was found to be 57% efficient when compared to the unbound anti-S-II IgG (100%). One of the difficulties encountered using ionexchange chromatography for the isolation of the i-antigen is the cross-contamination of pooled fractions of S-I, S-III and S-IV with S-II and the contamination of S-II by S-III. To examine the specificity of the anti-S-II Sepharose and to compare its efficiency of separation to ion-exchange chromatography, pooled fractions of S-I, S-II, S-III and

S-IV were chromatographed through the immunoaffinity column. As seen in Fig. 5, the elution profiles of these fractions are as might be predicted. The major portion of fractions S-I, S-III and S-IV was not retained by the column, and material which appeared in the void volume was devoid of immunological activity. Bovine serum albumin (BSA), was also not retained by the column. With the pooled S-II fraction, however, the main portion of the applied protein was retained by the column and eluted with 0.05 M glycine-NaOH buffer, pH 10.4. This latter material as compared to the applied material, was devoid of S-III. In a control experiment in which i-antigen was chromatographed on unmodified Sepharose 4B, more than 95% of the applied protein eluted in the void volume suggesting negligible non-specific adsorption. The literature contains many examples of protein preparations which appear to consist of a single component on polyacrylamide disc gels but which give multiple components when subjected to isoelectric focusing. Therefore, as a further test of the purity of our i-antigen preparation, AS-2 was electrofocused on polyacrylamide gels. As shown in Fig. 6, both Ag-4 and AS-I contained multiple components while AS-2 appears as a single, major component. AS-2 could be shown by the method of immunoelectric focusing to contain all of the immunological activity. The isoelectric point of AS-2 (i-antigen) determined in this manner was 4.1 which is in agreement with the value previously reported by Preer (1959b) and Steers (1961) (Fig. 7). The AS-2 (i-antigen) fraction appears as a single molecular species on polyacrylamide disc gels and on isoelectric focusing.

376

RICHARD

H. DAVIS.

JR. and EDWARD

STEERS,

JR

ELECTROFOCUSING

43 4

AS-1

AS-2

Fig. 6. Is oelectric focusing in 4% ac:rylan nide gels using pH 44 ampholytes. Gels contained I .2 mg Ag-4, I.5 mg AS-I and 1.8 Img AS-2. Gels were electrofocused and stained as described in Materials and Methods.

Previous methods used to isolate the i-antigen do not give preparations suitable for characterization of this protein. The answer to the questions of subunits for example, has been hampered by the presence of an apparent degradative activity which co-purifies with the i-antigen. This degradative activity could not be separated from the i-antigen on Sephadex G-200, hydroxylapatite, or CM-cellulose ion-exchange chromatography. As purified by these procedures, the degradative activity appears to be closely associated with the i-antigen preparation. Although the function of the i-antigen in paramecium is unknown, the separation of this degradative activity by DEAE-Sephadex and SE-Sephadex

demonstrates that the two substances are distinct. Whether an in viva relationship exists between the two or not is not evident from this study. The immobilization antigen is functionally defined as the surface protein of P. tetraurelia which reacts with homologous antisera raised against it and which causes immobilization of the cells when incubated with this anti-serum. We have used this definition in principle. The special biological reaction of antigen with its specific antibody, has been utilized to purify the i-antigen. We have been able to produce antisera specific for that one component which in previous purification schemes is responsible for the immobilization reaction. By chemically binding the i-antigen antibodies to Sepharose-4B, an immunoaffinity column specific for the i-antigen was made.

Immunoaffinity

Purification

377

of i-antigen

I a

IO

Slice Fig. 7. Isoelectric

I

I

20

30

number

focusing of Ag-4 (1.2 mg) in 4% acrylamide

gels with pH 4-6 ampholytes. The gel was sliced at 2 mm intervals and the separate slices incubated in 1 ml of distilled H,O. After I2 hr of incubation at 2°C. the pH of the H,O containing the gel slices was measured.

Using this material, pure i-antigen can now be obtained for physical and chemical characterization. The bound i-antigen can be removed by relatively mild conditions and after re-equilibration with 0.05 M PO,, pH 7.3, the column material can be used again. In our hands, the binding capacity and specificity of this column has not changed after repeated use under these conditions. When compared to other purification methods, the use of an immunoaffinity column is obviously superior. The classical method of Preer (1959) gives a preparation contaminated not only by other minor components of apparently similar physical and chemical characteristics, but also by a thiolactivated degradative activity. With ion-exchange chromatography, although the degradative activity can be removed, over 50% of the applied material cannot be recovered and pooled fractions were not homogeneous. Furthermore, certain non-51 A serotype preparations chromatographed similarly to 5 1 A serotypes. Considering the small amount of material to work with, these drawbacks are clearly unacceptable. The i-antigen purified by the immunoaffinity column, in contrast to other purification

procedures, can be demonstrated to be a single component by both polyacrylamide gel electrophoresis and isoelectric focusing. Furthermore, antisera raised against the AS-2 fraction obtained from immunoaffinity chromatography reacted with extracts of the starting i-antigen material (Ag-4 preparation) to give a single precipitin band in agar double diffusion tests over an antigen concentration of 4.0 mg per ml to 0.25 mg per ml. This antisera reacted only with 51 A cells in viva immobilization tests further indicating the purity of the AS-2 fraction. The nature of the contaminating substances is not clear from these studies. Because of the close physical and chemical similarity between the i-antigen 51 A and the contaminating substances. the possibility exists that other i-antigen substances may be present in the cultures being harvested. These i-antigens, if present, must exist as secondary antigens (small amounts of i-antigen which are cytoplasmically located or are undetectable by the immobilization test) however, as the various lots which were harvested for these studies were pure for the 51 A serotype by the in rGvo immobilization test. The immunoaffinity column, as described herein,

318

RICHARD H. DAVIS, JR. and EDWARD STEERS, JR.

is relatively quick and far more efficient than previously described procedures. All material applied to the column can be recovered and most important, the i-antigen can be recovered as a homogeneous preparation. The purity of the preparation has been demonstrated immunologically and electrophoretically both on polyacrylamide gels and by isoelectric focusing. Acknowledgements-The authors would like to acknowledge the special technical assistance of Mr. Clitford E. Lee in the isolation, cultivation and harvesting of cell cultures and various other aspects of this work. REFERENCES

Doerr & Chrambach A. (1971) Analyt. Biochem. 42, 96. Finger I., Heller C. & Dilworth L. (1969) J. Protozool. 16, 12. Hansma H. G. (1975) J. Protozool. 22, 257. Livingston D. M. (1974) In Methods in Enzymology (Edited by Jacoby W. & Wilchek M.) Vol. 34, pp. 723-726. Academic Press, New York.

Macindoe H. & Reisner A. H. (1967) Aust. J. /Go/. Sci. 20, 141. March S. C.. Parek I. & Cuatrecasas P. (1974) Anulyt. Biochem. 60, 149. Ouchterlony 0. (1948) Acta path. microhiol. stand. 25, 186. Pollack S. & Steers E. (1973) Exp[ Cell Res. 78, 186. Preer J. R., Jr. (1956) J. fmmun. 77, 52. Preer J. R., Jr. (1959a) J. Immun. 83, 378. Preer J. R., Jr. (1959b) J. Immun. 83, 276. Preer J. R., Jr. In Research in Protozoology (Edited by Chen T. T.) Vol. 3. pp. 130-278. Pergamon Press, New York. Preer J. R., Jr. & Preer L. B. (1959) 1. Protozool. 6, 88. Reisner A. M., Newes P. & Rucholtz C. (1975) Analyt. Biochem. 64, 509. SeedJ.R..Shafer,S.,FingerI.&HellerC.(l964)J.Genef. Res. 5, 137. Shapiro A. L., Vinuela E. & Maize1 J. V. (1967) B&hem. hiophys.

Res. Commun.

28, 815.

Sommerville J. (1970) Adv. Microbial. Physiol. Steers E., Jr. (1961) Science 133, 2010. Steers E., Jr. & Davis R. H.. Jr. (1976) Comp. Physiol.

53B,

195.

4, 131. B&hem.

Purification of the i-antigen 51A from Paramecium tetraurelia by immunoaffinity chromatography.

Immunochemistry, Vol. 0 Pergamon Press Ltd. IS, pp. 371-378 1978 Printed in Great Britain PURIFICATION OF THE i-ANTIGEN 51 A FROM PARAMECIUM TETR...
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