Immunology, 1968, 15, 789.

Further Studies with Artificial Antigens and Immunity to

Mouse Typhoid

I. USE OF 0 ACETYLATED GALACTANS IN THE PURIFICATIONOF SPECIFIC ANTIBODY AGAINST ANTIGEN 5

G. D. F. JACKSON, D. ROWLEY AND C. R. JENKIN

Department of Microbiology, University of Adelaide, South Australia

(Received 8th May 1968)

Summary. Data presented in this paper indicate that by column chromato-graphy using an 0 acetylated galactan, a considerable degree of purification ofspecific antibody to 0 antigen 5 may be achieved.

INTRODUCTION

Recent investigations concerning the immunity of mice to Salmonella typhimuriuminfections have stressed the importance of specific antibody in determining an animal'sresistance to this infection (Jenkin and Rowley, 1963b; Rowley, Turner andJenkin, 1964).

Certain evidence would also suggest that a particular antibody to a heat labile antigenof the cell wall is of great importance (Auzins and Rowley, 1963). From experimentsinvolving cross protection between strains of bacteria sharing one or more of the antigenicsites of the 0 somatic antigen of S. typhimurium, it was suggested that the heat labileantigen was antigen 5, the immunodominant sugar being an 0 acetyl galactose (Kotelko,Staub and Tinelli, 1961 ; Jenkin and Rowley, 1965). This suggestion has been strengthenedby the finding that an 0 acetylated galactan will immunize mice against this infection,the resulting antibody reacting with antigen 5 (Jenkin, Karnovsky and Rowley, 1967).In a previous study it was shown that Salmonella adelaide which on the Kaufmann-Whiteclassification would appear to be antigenically unrelated to the S. typhimurium, will immu-nize mice against this disease. Cross-absorption studies indicated that serum from micevaccinated with S. adelaide contained antibody against S. typhimurium (Jenkin and Rowley,1965). It is interesting with regard to the above that Auzins (1967) has recently reportedthat the polysaccharide of the 0 somatic antigen of S. adelaide contains 0 acetylatedsugar(s).The present paper is an expansion of a previous study on the immunity induced in

mice to this infection by immunizing them with an acetylated galactan and shows thatantibody to antigen 5 may be purified by column chromatography using the 0 acetylatedgalactan as the adsorbent. Evidence is also presented indicating that rats which arenaturally resistant to this infection possess natural antibody which is retained on a columnof 0 acetylated galactan.

789

G. D. F. Jackson, D. Rowley and C. R. jenkin

MATERIAL AND METHODSPreparation and acetylation ofpolysaccharidesThe galactan from gum arabic was purified by the method previously reported

(Jenkin et al., 1967). Total acylation of the purified galactans was carried out by themethod of Carson and McClay (1946) using acetic anhydride. The acetyl content of thepolysaccharides was determined by saponification of the acylated material with 041 NNaOH for 2 hours at ambient temperature and back-titrating with 041 N HC1 usingphenolphthalein as the indicator.

Colloidal suspensions of the acetylated polysaccharides were prepared as follows. Onegram of the acylated polysaccharide was dissolved in 25 ml of acetone and the solutionadded dropwise to 200 ml of distilled water during rapid stirring. The resulting suspensionwas filtered through Whatman No. 1 filter paper and the filtrate concentrated by spinevaporation at 600/15 mm. Suspensions of at least 20 mg/ml were stable to standing at 40for at least 6 months. Suspensions were stable only in distilled water, addition of salinecaused varying degrees of aggregation and precipitation.

Column chromatography utilizing acylated polysaccharidesWhatman ashless cellulose flock (9 g) was first moistened with 25 ml acetone and to

this was added 1 g of acylated polysaccharide in 10 ml of acetone. The mixture was driedby evaporation at 600/15 mm and then left at room temperature for 3 hours. The impreg-nated flock, suspended in saline was homogenized in a Waring blender for 30 seconds. Thecolumn (200 x 8 mm) was packed under pressure (10 lb) and allowed to equilibrateovernight with saline. Following equlibration, 0-5-2 ml of serum that had been dialysedovernight against 0 15 M saline was placed on the column and allowed to percolate slowly.Using 0-15 M saline as the first eluent, 5 ml samples were collected from the column untilno further protein could be detected in the eluate as measured by OD at 280 my. Afurther 50 ml saline was then run through and finally 0-2 M acetate buffer at pH 3.7.Fractions collected using the acetate buffer were dialysed overnight against 0f15 Mphosphate buffered saline at pH 7-0. Following dialysis the pH of the fractions waschecked to ensure that this was in the range of 6f8-7-0.

Preparation of antiserumRabbits were immunized intravenously with a living vaccine of Citrobacter (4, 5).

Animals were bled by cardiac puncture at the height of their agglutinin response and theserum separated and stored at -200. Prior to column chromatography the serum wasabsorbed with the minimum amount of an overnight culture of Salmonella abortus equi(4, 12) necessary to remove antibodies to factor 4. Such absorbed serum would not reactat a dilution of 1:2 in a haemagglutination assay with sheep red cells sensitized with thelipopolysaccharide from the absorbing strain.

Blood from an inbred strain of Chocolate-Brown rats which are naturally resistant toSalmonella typhimurium infections was obtained by cardiac puncture, the serum separatedand stored as above.

Haemagglutination and haemagglutination inhibition studiesBasically the methods followed those previously reported by Crumpton, Davies and

Hutchison (1958) and Davies, Crumpton, Macpherson and Hutchison (1958). However,

790

Purification of Specific Antibody Against Antigen 5

in order to sensitize the sheep erythrocytes with lipopolysaccharide from Salmonellatyphimurium C5 without the involvement of alkali treatment the following method wasadopted. A 2S5 per cent (v/v) suspension of sheep erythrocytes in saline containing 50 ugof lipopolysaccharide was incubated at 370 for 2 hours with frequent agitation. Followingincubation the cells were washed three times with saline and made up to a final concen-tration of 2-5 per cent.The inhibition studies were carried out with a rabbit antiserum to Citrobacter (4, 5) that

had been absorbed as above to leave only antibody against 0 antigen 5. One-millilitrealiquots of the absorbed serum were incubated with varying amounts of the 0 acetylatedpolysaccharide at 40 for 10 days with agitation. Following this period the mixtures werecentrifuged at 3000 rev/min for 20 minutes at 40, and the supernatants removed andtested for their haemagglutinating activity against S. typhimurium lipopolysaccharidesensitized sheep erythrocytes.

Sensitization of sheep erythrocytes with bovine serum albuminThe method employed was that described by Rangel and Repka (1965).

Bacterial strainsThe strains of Salmonella typhimurium used in these studies were S. typhimurium C5 and

S. typhimurium M206 (Jenkin and Rowley, 1965). The lipopolysaccharides from thesestrains were prepared by the method of Westphal, Luderitz and Bister (1952). A strain ofSalmonella adelaide (035) was also used in certain bactericidal assays.

Assay of antibody by bactericidal testsThe method adopted was that described by Michael, Whitby and Landy (1962) using a

constant quantity of guinea-pig complement from which all antibody to the test strainS. typhimurium M206 had been previously absorbed.

RESULTS

PURIFICATION OF ANTIBODY AGAINST ANTIGEN 5 BY COLUMN CHROMATOGRAPHY

As a preliminary experiment various concentrations of the 0 acetylated galactan weremixed with 1-ml amounts of a rabbit anti-5 serum. After 10 days the serum was centrifugedand tested for its residual anti-5 antibody by titration against lipopolysaccharide sensitivesheep red blood cells. The results of this experiment are given in Fig. 1, where it may beseen that relatively small concentrations of the 0 acetylated galactan are sufficient toremove the greater proportion of the haemagglutinating activity. The results expressed inFig. 1 are an average of three separate but similar experiments. Following this experimentcolumns of0 acetyl galactan on cellulose were prepared and 2 ml of specific anti-5 serumfractionated as indicated. The results illustrated in Fig. 2 show that under these conditionsapproximately 70 per cent of the biological activity was retained by the column andeluted by the acetate buffer, whereas more than 95 per cent of the protein passed straightthrough the column. Since the antibody activity of the acetate eluate was measured by abactericidal test, it was essential to establish the specificity of this reaction to ensure thatantibody against other bacterial antigens that might have been present in the rabbit

791

G. D. F. Jackson, D. Rowley and C. R. Jenkin500r

400

:300ci

0'c, 200

E

a

o 1006

0 025 050 2Weight/mg of acetyl galactan/mL antiserum

FIG. 1. The titre of anti-5 antibody remaining in the serum following absorption with varying dosesof 0 acetyl galactan. Antibody titre was measured by a haemagglutination assay using sheep erythro-cytes sensitized with a lipopolysaccharide from S. typhimurium (1, 4, 5, 12).

antiserum and not removed by absorbtion with S. abortus equi had not been held up on thecolumn. The experiment was repeated with the absorbed antiserum to which had beenadded an equal volume of an antiserum to Salmonella adelaide (035). It is evident from Fig.3 (a) and (b) that a clear separation of the bactericidal activity against these two strainshas been accomplished. The main protein peak in the saline eluate contained all the

1l2r

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0 0-8-

c0

° 06

ca)

0

c

0

0s

0

I/ I,I \

I!

T0 T--*T-*-50 100 150 200 250

Volume of eluate (mL)I 300 350

60

0

z

5-5 Noc

0

150 ,

FIG. 2. Recovery of protein and antibody from a rabbit antiserum monospecific for 0 factor 5 followingcolumn chromatography on an 0 acetylated galactan. The antibody was detected by a bactericidaltest involving S. typhimurium (1, 4, 5, 12). The biological activity (- - - -) of the fractions is expressed as

the log of the dilution of the fraction which killed 50 per cent of the bacteria in 90 minutes. The originalserum killed 50 per cent of the bacteria at a dilution of 10 -6. , Protein concentration. Arrowindicates addition of acetate buffer.

792

_ I

Purification of Specific Antibody Against Antigen 5

1I

C

IL)

0-0

.

n)

793

* (b)

JO-6 1-3

Dilution of fraction

FIG. 3. Recovery of specific antibody to: (a) S. typhimurium (1, 4, 5, 12), and (b) S. adelaide (035) from amixture of a rabbit antiserum to S. adelaide (035) and a rabbit antiserum monospecific for 0 factor 5following chromatography on a column of0 acetyl galactan. The antibody was detected by a bacteri-cidal test using S. typhimurium (1, 4, 5, 12) in (a) and S. adelaide (035) in (b). The number of bacteriasurviving was measured at the end of90 minutes. 0, Original serum; 0, acetate eluate; A, saline eluate.

activity against S. adelaide and a little against the S. typhimurium strain M206, whilst theacetate peak contained activity only against S. typhimurium M206 and none against S.adelaide.

Further evidence for the selectivity of the column was obtained by passing through it arabbit antiserum to bovine serum albumin. The data given in Fig. 4 shows that antibodyto bovine serum albumin as measured by a haemagglutination assay passed straightthrough the column.

6

czi0to(Nj00

c

0

0

00Tu

.20

C-

200Volume of eluate (ml)

300

1700

600 :

0

c

500 _

C'ca400 E

300 "o

z

FIG. 4. Recovery of protein and antibody to bovine serum albumin from a rabbit antiserum to bovineserum albumin following chromatography on an 0 acetylated galactan. The antibody was detected by ahaemagglutination assay using sheep erythrocytes that had been sensitized with bovine serum albumin.Original serum possessed 800 haemagglutinating units. , Protein concentration-haemagglutinating activity. Arrow indicates addition of acetate buffer.

G. D. F. Jackson, D. Rowley and C. R. JenkinUnder the above conditions using an antibacterial serum about halfof the total biological

activity seemed to be always associated with the main protein peak, and it was possiblethat this activity might be due to antibodies reacting with antigens other than antigen 5that were not shared with the absorbing strain. To test for this possibility the mainprotein peak containing the activity was concentrated by dialysis against sucrose, andfollowing dialysis against saline, re-chromatographed. The results of this experimentindicated that this activity was due to antibody against antigen 5 since approximately 50per cent of the activity that had originally passed through with the main protein peakwas retained by the column. The total amount of specific antibody against antigen 5 inthe absorbed serum was 360 pg/ml as determined by the quantitative precipitin reactionusing lipopolysaccharide from S. typhimurium. The amount of protein held back by thecolumn and eluted by the acetate buffer was very small. Based on the distribution ofbactericidal activity, the fractions were pooled and concentrated by dialysis againstsucrose. The total concentration of protein in the eluate was 1160 pg. Protein concentra-tions in both cases were determined by OD at 280 my and compared to a standard curveof human y-globulin. Since 720 pg of specific antibody were applied to the column, itwould suggest that the eluted material could be 65 per cent pure antibody.As a further test for specificity the eluate obtained from the column with the acetate

buffer was concentrated and 1-ml aliquots of the concentration absorbed with varyingamounts of the 0 acetylated galactan. The mixtures were incubated at 40 for 24 hoursand finally the galactan removed by centrifuging at 2000 rev/min for 10 minutes. Thesupernatants were then tested for their bactericidal activity against S. typhimurium M206.The results given in Table 1 indicate that at concentrations of the 0 acetylated galactangreater than 1 mg/ml, all the bactericidal activity had been adsorbed.

TABLE 1

ABSORPTION OF THE ACTIVE ACETATE FRACTION WITHVARYING CONCENTRATION OF THE 0 ACETYLATED

GALACTAN

Weight of 0 acetylated Percentage bactericidalgalactan/mg/ml activity removed

0-5 751.0 752-0 1005*0 100

10-0 100

EFFECT OF THE ACETATE ELUATE ON THE PHAGOCYTOSIS OF S. typhimurium CS BYPERITONEAL MACROPHAGES

Previous studies have indicated that on a quantitative basis antibody to antigen 5is more important than antibodies to antigens 1, 4 and 12 in determining the fate ofvirulent S. typhimurium within phagocytic cells. The above experiments indicated that theacetate eluate was biologically active in an in vitro bactericidal test involving complementbut they gave no information as to the importance of this antibody in the phagocytosis andsubsequent inactivation of the parasite (Jenkin, 1964; Archer and Rowley, personalcommunication 1968).

794

Purification of Specific Antibody Against Antigen 5

The virulent S. typhimurium C5 was opsonized at a concentration of 5 x 109 organism/mlby the method of Jenkin and Rowley (1961) with the acetate eluate at a concentrationequivalent to a 1:1000 dilution of the original serum placed on the column. Followingopsonization, 0-2-ml aliquots of a dilution containing 104 organisms were injected into theperitoneal cavities of normal mice and the fate of the bacteria followed. Control normalmice received similar numbers of unopsonized bacteria. The results given in Fig. 5 showthat bacteria opsonized with the acetate eluate are killed within the peritoneal cavitiesof normal mice, a result contrasting with that observed if the bacteria were unopsonized.

100

80 -

a 60

*O40 _- 0

20 -

430 60 90Time (minutes)

FIG. 5. Survival of S. !yphimurium C5 (virulent) in the peritoneal cavities of normal mice followingopsonization with the acetate eluate from a column of 0 acetyl galactan after passing through an anti-serum monospecific for 0 factor 5. o, Unopsonized bacteria; *, opsonized bacteria.

From the results on the amount of anti-5 antibody placed on the column and the totalamount ofprotein eluted from the column, one can, by making some assumptions, calculatethe total number of molecules associated with each opsonized bacterium. Since the serumwas from a hyperimmunized rabbit, one can assume that the bulk of the antibody was

IgG in nature with a molecular weight of 160,000 (Rowley and Turner, 1966). Thus,1-2 yg of protein was sufficient to opsonize 5 x IO' bacteria. The number of molecules ofprotein per bacterium is given by:

602 x I" x 120 901-6 x 105 x 106 x 5Tx109(minutes)

This figure is comparable with the 2200 molecules of specific IgG antibody per bacterialcell determined by Rowley and Turner (1966) for the promotion of phagocytosis of S.adelaide by mouse peritoneal macrophages.

795

G. D. F. Jackson, D. Rowley and C. R. jenkin

ABSORPTION OF ANTIBODY BY COLUMN CHROMATOGRAPHY FROM NORMAL RAT SERUM

Previous investigations have shown that sera from rats naturally resistant to S. typhimur-ium C5 was highly opsonic for this strain and led to a degree of protection if mice were chal-lenged with these pre-opsonized bacteria (Jenkin and Rowley, 1959). It was of interest tosee, therefore, if this opsonic activity could be retained on a column of the 0 acetylatedpolysaccharide. The technique required some modification with regard to elution since itwas consistently found that after passing through the column none of the fractions wereactive nor could activity be eluted using the previous acetate buffer. Two millilitres of ratserum were dialysed overnight against 0-01 M phosphate buffer at pH 6'3. Followingdialysis the serum was placed onto the column and a buffer at a similar molarity and pHused for the initial elution. Fractions (25 ml) were collected and assayed for protein. At atime when little protein was demonstrable in the fraction the buffer was changed to 1M NaCl in 0f02 M phosphate at pH 6f3. Two further fractions of 10 ml were collected andall fractions were then tested for their ability to opsonize and promote killing of S. typhim-urium C5 as described above. The results given in Table 2 indicate that the opsonicactivity could be clearly separated from the main protein peak and could be eluted fromthe column under the above conditions.

TABLE 2FRACTIONATION OF THE OPSONIC ACTIVITY IN RAT SERUM TO A STRAIN OF S. typhimurium C5 BY COLUMN CHROMATOGRAPHY

USING AN 0 ACETYLATED GALACTAN AS THE ABSORBENT

Eluate volume Percentage recovery of Total concentration of proteinBuffer Fraction No. (ml) opsonizing activity (mg)

0-01 M phosphate 1 25 0 130buffer at pH 6 3 2 25 0 10

3 25 0 44 25 0 25 25 0 136 25 0 0-5

1 M Nacl in 0-01M 7 10 50 5phosphate buffer pH 6 3 8 10 0 1 6

DISCUSSION

The immunodominant group responsible for the specificity of0 factor 5 has been shownto be an 0 acetyl galactose, the 0 acetyl group probably residing on the C2 of the galac-tose (Kotelko et al., 1961). Considering the structure of the galactose in the 0 antigen ofS. typhimurium it is reasonable to assume that there may be some cross-reaction betweenantigen 5 and the acetyl galactose under study (Fig. 6). One obvious difference is that theadjacent sugars are different and the linkages in the main chain of the bacterial polysac-charide are a compared with f in the plant polysaccharide. Nevertheless, substitutionsin the galactose of both molecules are comparable. In the plant polysaccharide thegalactose is substituted in the 1, 3 and 6 positions (Aspinal, Hirst and Nicolson, 1959),whilst the galactose in the bacterial polysaccharide is substituted in the 1 position withmannose and either position 4 or 6 with glucose (Luderitz, Staub and Westphal, 1966).

796

Purification of Specific Antibody Against Antigen 5 7976 6

Gu OCH ROCH2I 0 ~~~~~~~5 0

0 ~~~~~~~Ho4 0-Man 4 O OR

032 ~~~~~~~32

Rha? OCOCH R OH(a) (b)

FIG. 6. Comparison of the substitution of the galactose in: (a) the lipopolysaccharide from S. typhimurium(1, 4, 5, 12) and (b) the galactan from gum arabic. Glu, Glucose; Rha, rhamnose; Man, mannose,R. other sugars of the polysaccharide.

Thus, if position 2 in the latter polysaccharide is acetylated, the linkage to rhamnose, theadjacent sugar, is likely to be through position 3. From the structures proposed for gumarabic it may be seen that either positions C2 or C4 are free for acetylation (Fig. 6).

Ideally, it would be advantageous to specifically acetylate at position 2, but as both areof approximately equal activity this is not feasible. However, even if position 4 were alsosubstituted it is not certain that this would decrease to any great extent the cross reactionbetween the two structures. Indeed, from the results of the present experiments in which afully acetylated polysaccharide is used, there are good indications of cross-reactivity.The data expressed in this paper show that antibody to antigen 5 may be purified by

column chromatography using the polysaccharide from gum arabic after 0 acetylationas the absorbent. The studies with rat serum indicate that these animals, naturallyresistant to S. typhimurium infections, have in their serum an antibody which may be speci-fically absorbed onto the 0 acetylated galactan. In view of our previous speculations onthe importance of antibody to antigen 5 in determining resistance to this infection itwould be of importance to know if the differences in susceptibility between mice and ratsto this infection reside in the rate and magnitude of the antibody response to this particularantigen (Jenkin and Rowley, 1963a, 1965). Certainly it would appear on the basis of pastexperiments that the natural resistance of rats to this infection is not dependent on anypeculiar property of the phagocytic cells (Rowley and Jenkin, 1962).

ACKNOWLEDGMENTS

This work was supported by a grant from the Australian Research Grants Committee.We are grateful to Mrs Margaret Gibberd for her excellent technical assistance.

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