84

ARTIFICIAL ANTIGENS WITH AGAR, GUM ACACIA ANDCHERRY GUM SPECIFICITY.

S. M. PARTRIDGE* AND W. T. J. MORGAN.

From the Biochemical Department of the Lister Institute, London.

Received for publication March 3, 1942.

THE conversion of a bacterial polysaccharide into an antigenic complex thatwill induce the formation of immune-body complex specific for the polysac-charide component of the complex has been reported in earlier papers (Morganand Partridge, 1940a, -1941a, b; Partridge and Morgan, 1940). The method ofcombining bacterial polysaccharide and protein components in formamidesolution has now been extended to include the formation of antigenic com-plexes that contain as the serological specific haptens polysaccharides- ofvegetable origin, and in the present paper the conversion of agar-agar, gumacacia and cherry-gum into antigenic complexes is described. A preliminaryaccount of the work with the artificial agar antigen has already appeared(Morgan and Partridge, 1940b).

EXPERIMENTAL.The experiments were made with the polysaccharides, agar-agar, kanten,

gum acacia and cherry gum. In each instance the commercial material wassubjected to a simple purification procedure that removed the gross impuritiespresent and reduced considerably the nitrogen content of the polysaccharidepreparations.

The agar antigenic complex.A commercial sample of agar was freed from gross impurities and inorganic

matter by solution in anhydrous formamide. The clear viscous solution wasfractionated by precipitation with alcohol, and the main fraction that separatedbetween the alcohol levels 50 per cent. and 60 per cent. was again dissolvedin formamide. The fraction was precipitated from solution by 60 per cent.alcohol, and thoroughly washed with 60 per cent. alcoholic formamide and finallywith absolute alcohol. The agar was then dissolved in boiling water to make a0-1 per cent. solution and after cooling, dialysed for several days against dis-tilled water at 00. The purified agar was recovered by the addition of alcoholto the dialysed solution, the material being subsequently washed with alcoholand dried in vacuo. The substance contained 0-8 per cent. N and was tested

* Beit Memorial Research Fellow.

ARTIFICIAL ANTIGENS.

for antigenicity in rabbits (Table I, animals 140, 141, 170 and 171). Twoanimals received eight doses of 0 05 mg. and two others ten doses of 02 mg.,but in no instance were agar precipitins subsequently detected in the sera ofthese animals. For convenience, kanten, a breakdown product of agar, wasused as hapten in the precipitation tests.

A portion of the agar (60 mg.) was dissolved in 3 ml. of anhydrous forma-mide (m.p. + 20) and a sample of conjugated protein (40 mg.) prepared fromthe specific somatic antigen of Bact. shigae was added as a dry powder. Theconjugated protein was prepared according to the method described by Morganand Partridge (1940a, 1941a, b) and contained 11-4 per cent. N. The proteinslowly passed into solution. The mixture was then allowed to stand at 370for 2 hours, and, after increasing the volume by the addition of 10 ml. offormamide, was kept at 0-2° for 48 hours. The agar-protein complex wasrecovered by the addition of alcohol to the formamide solution, the precipitatewas thoroughly washed with alcohol, was taken up in 10 ml. of boiling waterand, after cooling, was evaporated to dryness from the frozen state. Samplesof the material (10 mg.) were prepared for animal inoculation by suspensionin 10 ml. of sterile saline and boiling for a few secorfds to ensure solution orfine dispersion. The opalescent, colloidal solution that was formed, however,was unstable on cooling, and a sediment of fine particles appeared on standingat room-temperature for several hours.

The whole suspension was given to rabbits Nos. 142 and 143 (Table I).Three doses each of 0 05 mg. failed to induce the formation of specific precipitins,but after a rest period of one week and a further course of five 0-2 mg. doses,the serum of both animals gave precipitation when mixed with an equal volumeof 1 in 104 or 105 dilution of kanten. Definite, though slight, precipitationwas also given by the kanten preparation at a dilution of 1 in 106.

The gelatinous character of a solution of the antigenic complex suggestedthat the preparation contained an excess of uncombined agar. The materialwas therefore dissolved in boiling water, allowed to cool slowly and to standfor some hours until the formation of the sparingly soluble floccules was com-plete. The suspension was then centrifuged and the deposit washed withsmall quantities of boiling water. The residue was dehydrated with alcoholand dried in vacuo. The material contained 8-9 per cent. N and 22 per cent.agar, estimated according to the method of S0rensen and Haugaard (1933).A specimen of the agar purified by formamide treatment was used for colori-metric comparison. The protein preparation gave only a trace of colour withorcinol and sulphuric acid, under the conditions of estimation employed. Ifone may judge from the limited number of animals used, the removal of theexcess agar enhanced the antigenicity of the agar protein complex. Threeintravenous doses of 0 05 mg. now gave rise to powerful kanten precipitinsin each of the 3 rabbits inoculated (Table I, animals 182, 183 and 184).

An antigenic complex was also prepared from agar and the conjugatedprotein obtained from the " 0 " antigen of Bact. typhosum. The same methodfor the formation of the complex was employed, and the resulting agar-proteincomplex was very similar in general appearance to that derived from agarand the " Shiga " protein component. The material again showed itself tobe antigenic, and although three doses of 0 05 mg. proved insufficient to elicit

7

85

S. M. PARTRIDGE AND W. T. J. MORGAN.

demonstrable kanten or agar precipitins, a further four similar doses gave riseto strongly precipitating sera (Table I, rabbits 185 and 186).

In view of the claim made by Zozaya (1932) that agar could be renderedantigenic by adsorption on collodion, the agar preparation employed in theexperiments described above was treated with a preparation of collodion(Schering-Kahlbaum) particles prepared according to Zozaya's technique.The particles were readily agglutinated with anti-agar immune serum, thusindicating that an agar-collodion adsorption complex had been formed. Theagar-coated collodion particles were given intravenously to two rabbits (TableI, animals 176 and 177). Four doses each of 0*2 mg. gave no demonstrableanti-agar immune-body. After a week's rest a further course of four dosesof 0 5 mg. was given, but there was no evidence that even weak agar precipitinshad been formed.

Preparation of kanten for use as test hapten.The anti-agar sera from rabbits immunized with the agar-protein complex

were found to give heavy precipitation with a preparation of partially degradedagar called kanten. The preparation and properties of the substance have beendescribed in detail by Takahashi and Shirahama (1934) and by Pirie (1936).Kanten lends itself more readily to purification than agar since it is readilysoluble in cold water or saline yielding clear mobile solutions, and for thisreason it has been found very convenient to use kanten in place of agar astest hapten. A 1 per cent. solution of agar was heated at 1000 for 2 hours, setaside to cool slowly and left overnight at 00. The liquid which separated fromthe agar-gel was collected and after concentration was evaporated to dryness.The crude kanten obtained in this manner contained 1-7 per cent. N, showed anegative rotation, [o]M5461-747, and dissolved readily in cold water to yield aclear solution. The material could be recovered from aqueous solution byprecipitation with acetone, but precipitation was not complete even at 80 percent. acetone level. A convenient method for the removal of nitrogenousimpurities, however, was found and was based on the insolubility of kantenin 90 per cent. phenol solution. After three extractions with 90 per cent. phenolthe nitrogen remaining in the kanten fell to 0*3-0 4 per cent. This materialwas used as test substance in place of agar in all experiments recorded inTable I.

The gum acacia antigen.A method of purification of the commercial gum was chosen which was

designed to avoid the use of strongly acid or alkaline reagents or heat treat-ment in order to reduce the risk of any degradation of the polysaccharidetaking place. A sample of commercial gum B.P. (1 gm.) was dissolved informamide (50 ml.), the small residue which remained undissolved wasremoved and the clear solution was treated with alcohol. The bulk of thematerial precipitated between the alcohol levels 40-50 per cent. Only a smallquantity of substance was thrown out of solution above 50 per cent. alcoholand this was rejected. The main fraction was washed thoroughly with alcohol,

86

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dissolved in water and dialysed against repeated changes of distilled water.The acid polysaccharide was recovered by addition of an equal volume ofethyl alcohol after acidification with 1 ml. glacial acetic acid. The substancewas redissolved in distilled water and again precipitated from solution byaddition of an equal volume of alcohol. The dried preparation contained0-23 per cent. N and 0 4 per cent. ash.

A group of 6 rabbits was inoculated with varying doses of the purified gumacacia as set out in Table II (animals 178, 179, 199, 200, 205 and 206), but noprecipitins specific for gum acacia were detected in any, of the sera examined.Uhlenhuth and Remy (1933, 1934) showed that certain commercial prepara-tions of gum arabic are antigenic, but that after purification to remove anitrogenous contaminant, the resulting gum no longer gave rise to immune-body. Similarly, Seideman (1940) showed that the serum of animals immu-nized with gum arabic preparations that gave positive protein reactionsresponded with the formation of immune-bodies reactive against a purifiednon-antigenic gum arabic.

The sample of " Shiga " conjugated protein used to prepare the gum acaciaantigen was obtained from the bacterial antigen isolated by the phenol method(Morgan and Partridge, 1941b). The purification ofthe conjugated protein con-sisted of a single precipitation with acetic acid from solution in cold, diluteNaOH solution. The sample contained 112 per cent. N, 1-07 per cent. P, andshowed [cx]546l-48 ± 3°. The finely powdered protein (20 mg.) was mixed withthe purified gum acacia (30 mg.) and formamide, m.p. + 20, (1 ml.) was added.The mixture was thoroughly stirred and allowed to stand at 0-20 overnight.Solution was not quite complet9; the small residue was rejected, however, andthe clear supernatant was transferred to another centrifuge tube and 4 ml.alcohol added. The precipitate of protein-gum acacia complex was washedthree times with alcohol to effect the complete removal of formamide, and thesubstance was suspended in 10 ml. of 0 9 per cent. NaCl solution. An opales-cent suspension of the complex was obtained which slowly settled on standing.The suspension was centrifuged and the clear supematant discarded. Theinsoluble material contained 8-9 per cent. polysaccharide. It was againtaken up in 10 ml. of 0-9 per cent. NaCl solution and finely dispersed by meansof a glass tissue grinder. The protein content of the suspension was estimatedand the volume adjusted so that the suspension contained 1 mg. of proteinper ml. The dose given to the animals (Table II, animals 217, 221 and 227)was O 5 ml.

A sample of the conjugated protein component of the " 0 " antigen ofBact. typhoaum was also used in place of the " Shiga " conjugated protein asthe second component in the gum acacia antigenic complex. The proteinwas prepared from a diethyleneglycol primary extraction product of a cultureof the " 0 " strain " 901 " and contained 11.5 per cent. N. Details of itspreparation and properties will be given in a later paper. The technique usedto accomplish the combination was the same as that already described. Thecomplex proved to be a powerful antigen, and three intravenous doses of0 05 mg. (calculated as polysaccharide) only were sufficient to induce theformation of strong gum acacia precipitins in the two animals immunized(Table II, animals 197 and 198).

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Cherry-gum antigen.A specimen of cherry-gum (0-75 mg.) kindly supplied by Prof. J. R. Marrack

was dissolved in water (25 ml.). A small insoluble residue was removed, andthe gum was precipitated from solution by the addition of 50 ml. alcohol con-taining a few drops of glacial acetic acid. The material was thoroughlywashed with absolute alcohol and dried in vaCuo. The gum contained 0-52per cent. N and was further purified by extraction with 90 per cent. phenolsolution. The powder swelled to a clear gelatinous mass but did not dissolve.After three extractions the phenol was removed from the gum by triturationwith alcohol. The material after drying in vacuo weighed 0-46 g. and wasfree from N. The antigenic complex was prepared exactly as described abovefor the gum acacia antigen. The complex after precipitation from formamidewith alcohol was taken up in saline, vigorously shaken and- centrifuged. Thedeposit was again shaken with saline and finely dispersed with the aid of aglass tissue grinder. The resulting suspension was not stable, but slowlysettled out on standing overnight in the cold room. Four rabbits were givensix intravenous doses of the purified cherry-gum (Table III, animals 235, 243,257 and 272), and three animals (Nos. 232, 262 and 263) were given the equi-valent amount of gum in the form of cherry-gum protein complex. It willbe seen from the results given in Table II1 that the hapten alone failed toinduce the formation of specific cherry-gum precipitins, whereas the complexcaused the production of specific immune-body after three doses only.

Cross Precipitation Tests.In view of the overlapping specificity shown by the partial hydrolysis

products of certain vegetable gums with anti-pneumococcal serum (Type II)(Marrack and Carpenter, 1938), it was considered of interest to investigatethe reactions of the anti-sera described above with the pneumococcal specificpolysaccharides (Types I and II) and with the heterologous gums. Theundiluted immune serum was mixed with an equal quantity of a dilution ofthe gum or polysaccharide preparation, incubated for 2 hours at 370 andexamined after standing at 0° for 18 hours. The results of these tests aregiven in Table IV. Gum acacia failed to give any precipitate when mixed

TABLE IV.-Showing the Specificity of the Anti-polysaccharide Immune Sera.Precipitation test with-

Test hapten. Anti-agar serum. Anti-gum acacia serum. Anti-cherry gum serum.Dilution of test hapten. Dilution of test hapten. Dilution of test hapten.

Kanten . . . . 4 4 1 . 1 0 0 . 0 0 0Gum aoaoia. . . . 0 0 0 . 4 4 2 . 0 0 0Cherry gum. . . . 0 0 0 . 1 2 1 . 3 4 2Gum tragacanth. . 0 0 0 0 0 0 0 0 0Hyaluronic acid .0 0 0 0 0 0.0 0 0PneumococcueTypeI I1 I 0 0 0 0 - 0 0 0specific poiy-}yeI. O 1saeohafidP jy-Type II 0 0 0 - 0 0 0 - 0 0 0sacharinde

GEastric mucin 'A" substance . O O O . O O O . 0

90

ARTIFICIAL ANTIGENS.

with an anti-cherry-gum serum, whereas cherry-gum gave distinct, althoughweak, precipitation with anti-gum acacia serum. Preparations of kanten,gum tragacanth, hyaluronic acid, the specific polysaccharides ofpneumococcus(Types I and II) and the blood group " A " polysaccharide isolated from piggastric mucin failed to give any precipitation. The slight precipitation givenby the Type I pneumococcus hapten with the anti-agar serum was probablydue to agar substance in the preparation of the specific substance.

Complement-Fixation Test8.Complement-fixation tests were carried out with anti-sera prepared against

agar, gum acacia and cherry gum and the corresponding polysaccharidehaptens. The soluble degradation product of agar, kanten, was employedas hapten in the tests with anti-agar serum, but owing to its strong anti-com-plementary action up to a dilution of 1 : 100,000 the tests have been considereduntrustworthy. Similarly, with the cherry-gum-anti-cherry gum system thetests were rendered unsatisfactory owing to the anticomplementary action, upto a dilution of 1: 200, shown by anti-cherry gum serum. On the other handneither the gum acacia at 1: 1000 dilution nor its homologous serum at 1: 20dilution showed any anticomplementary action, and the complement-fixationtests showed that 1 M.H.D. of complement (0*2 ml.) was fixed by as little as1 part of gum in 1: 10,000,000 parts of saline (0x2 ml.) in the presence of a1: 20 dilution of the immune-body (0 2 ml.). Anti-sheep cell serum, 3-5M.H.D., was used as amboceptor throughout the tests. Owing to the unsatis-factory nature of the agar and cherry gum complement-fixation tests no cross-reactions within the three systems were investigated.

Anaphylaxis Experiments.Groups of guinea-pigs (300-450 g.) were passively immunized with anti-

agar, anti-gum acacia and anti-cherry gum rabbit serum by receiving an intra-peritoneal injection of 2-3 ml. of the immune serum. After 24-36 hours eachanimal was given an intravenous dose of the homologous hapten and observedfor symptoms of anaphylaxis. Three animals immunized with anti-agarserum showed severe anaphylaxis after a dose of 1 mg. kanten; one animaldied after 2 minutes. Two other animals which received 0.1 mg. of kantenshowed definite symptoms of anaphylaxis, but subsequently recovered.Five guinea-pigs passively sensitized with 2 ml. of anti-gum acacia serum andshocked with 1 mg. of the homologous gum showed severe anaphylaxis. Three-of the animals died in 2-3 minutes after receiving the injection; the remainingtwo animals slowly recovered from shock during the following 2-3 hours.Similarly, a group of guinea-pigs, each of which had received an intraperi-toneal injection of 3 ml. of anti-cherry gum rabbit serum likewise showed allthe usual symptoms of shock when given an intravenous dose of the homo-logous gum 24 hours after the serum. Owing to the limited amounts ofimmune sera that were available no anaphylaxis tests that involved the useof heterologous haptens to induce shock were made.

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92 S. M. PARTRIDGE AND W. T. J. MORGAN.

DISCUSSION.

The method of combining bacterial polysaccharide and protein componentsin formamide solution to form antigenic complexes has now been extended toinclude the formation of antigens in which polysaccharides of vegetable originfunction as haptens. In this manner the polysaccharides agar-agar, gumacacia and cherry gum have been combined with the conjugated protein com-ponents of the " 0 " somatic antigens of Bact. shigae and Bact. typhosum andpowerful antigenic complexes have been formed.

Agar, gum acacia and cherry gum do not possess the property of formingsoluble complexes with the conjugated protein in aqueous solution in the sameway as certain other bacterial and tissue haptens (Morgan and Partridge,1941b; Morgan, 1941). It is now shown that when these gums are mixedin formamide solution with a sample of the bacterial protein, the proteinfirmly retains part of the polysaccharide, yielding a complex that cannot bealtered greatly in composition by repeated washing with water or dilute saltsolution. Although the polysaccharide content of the association complexis in each instance low compared with that of the natural bacterial antigens,the complexes are nevertheless found to induce the formation of powerfulanti-gum immune sera on injection into rabbits. It is now well known thatproteins when combined with mono- and di-saccharides, polysaccharides andmany simple organic compounds (see Landsteiner, 1936; Marrack, 1938)acquire a new immunological specificity, and when the complexes are sub-sequently injected into animals they induce the formation of immune bodythat is largely specific for the attached group. It is, however, frequentlydifficult to secure a chemical linking of protein with the more complex andlabile polysaccharides such as the specific acetyl polysaccharide of the pneumo-coccus (Type I) or the " A " specific blood group polysaccharide of gastricmucin or saliva, without damaging the polysaccharide molecule and thusmodifying its immunological specificity. For this reason a method of formingan antigenic polysaccharide-protein complex that involves a procedure nomore violent than solution of the components in anhydrous formamide atroom temperature is likely to prove of considerable value in future investiga-tions.

Some years ago Sordelli and'Mayer (1931) found that the immune-sera ofhorses that had been immunized with bacteria grown on agar medium developedagar precipitins. Utilizing this observation, Morgan (1936) showed that itwas possible to detect, by means of anti-agar horse serum, agar and agarbreakdown products in a. number of specific polysaccharide preparations..The potency of these anti-agar horse sera, however, is not high even after thehorses have received a prolonged course of immunization lasting over a periodof several months. Furthermore, such sera arise only occasionally, and mustbe considered as accidental by-products in the manufacture of therapeuticsera for clinical use. The potent anti-agar rabbit serum, on the other hand,produced by means of the artificial agar-protein complex has proved of con-siderable value in following the removal of agar and kanten from antigenicmaterial isolated from agar grown bacteria. Indeed, the precipitation reaction

ARTIFICIAL ANTIGENS.

of agar or kanten with its homologous immune-body is so sensitive that materialwhich fails to yield any of the colour reactions in the tests elaborated by Pirie(1936) for the detection of agar will nevertheless give definite precipitationwhen tested with anti-agar serum. The strong anti-complementary pro-perties of kanten suggest that the anti-complementary nature of certainbacteria grown on an agar medium may, in part, be due to a surface coatingof kanten. Since the original observation of Sordelli and Mayer no explana-tion for the conversion of agar into an antigenic complex has been forthcoming,but in view of our observations it seems likely that agar or its breakdownproducts combines with certain protein compon'ents of the bacterial cell andin this manner is transformed into a full antigen.

The anti-gum acacia and anti-cherry gum immune sera were consideredsuitable for studying the serological relationships that might exist betweenthese sera and other heterologous haptens, such as the specific polysaccharidesof pneumococcus (Types II and III), thus contributing to the valuable obser-vations of Marrack (1937) and of Marrack and Carpenter (1938) on the crossreactions shown by the acid hydrolysis products of vegetable gums and pneumo-coccus (Types II and III) immune-body (see also Morgan, 1937). Heidelberger,Avery and Goebel (1929) showed that the native gum acacia reacted onlyoccasionally, and then very weakly, with Type II and III anti-pneumococcusserum, and that it was necessary to partially hydrolyse the gum before itshowed any pronounced cross-reactions with the pneumococcus Types II andIII immune-body. These workers suggested that even the slight activity ofthe original gum might be satisfactorily accounted for on the assumption thattraces of the specifically reacting material were formed during a period ofexposure in the process of refining the commercial product or by enzymeaction. Indeed, it is clear from our experiments that there is no cross pre-cipitation reaction between the specific polysaccharide hapten of the pneumo-coccus (Type II) and gum acacia and gum cherry immune-body. It seemsimprobable therefore that the aldobionic acid structure, which plays such animportant part in the cross reactions shown by the partially hydrolysed gumsand the pneumococcus (Type II) immune-body, exists as a serologicallyimportant configuration in the native gum-acacia and cherry-gum. Thisconclusion is supported by the observation that glucuronic acid fails to inhibiteither of the homologous gum-anti-gum precipitation reactions. Galacturonicacid is also inactive in similar inhibition tests. Furthermore, virulent culturesof pneumococcus Types II and III are not agglutinated by the gum acaciaand cherry gum immune-body.

Cretcher and Butler (1928) and Butler and Cretcher (1929) showed thatby mild acid hydrolysis gum acacia loses, its arabinose and rhamnose com-ponents very readily. Under these conditions of hydrolysis it is known fromthe work of Heidelberger, Avery and Goebel (1929) that a new serologicalspecificity develops and that the resulting partially degraded gum now reactswith pneumococcus (Types II and III) antibody. These observations suggestthat the serological specificity of the native gum acacia might be due in partto the specific configuration of the arabinose and rhamnose molecules, espe-cially if the ring structure of the complex acidic nucleus brought forward byNorman (1937) is accepted. In such a structure for the native gum the

93

94 S. M. PARTRIDGE AND W. T. J. MORGAN.

arabinose and rhamnose units are joined to the nucleus by glucosidic linkage,and thus, by masking the reactive surface of the aldobionic acid molecule theymight be expected to play a dominant role in determining the serological speci-ficity of the native gum. It has been found, however, that d-arabinose,I-rhamnose and d-fucose fail to inhibit the acacia-anti-acacia or the cherry-gum-anti-cherry gum precipitation reactions. There is, therefore, no evidencebased on specific inhibition that the pentose and methyl pentose componentsof the natural gums play any dominant part in determining their immuno-logical specificity. We have had no opportunity of investigating further manypoints of considerable interest arising out of this work.

SUMMARY.1. The formation of antigenic complexes from the non-antigenic poly-

saccharides, agar, gum acacia and cherry gum and the conjugated proteincomponent of the specific somatic antigens of Bact. sIhgae and Bact. typlhosumare described.

2. The artificial antigens induce the formation of immune-body specific forthe polysaccharide components of the artificial antigenic complexes.

3. The homologous gum-anti-gum precipitation reactions are not inhibitedby glucuronic or galacturonic acid and the anti-gum sera fall to agglutinatevirulent pneumococci (Types II or III).

REFERENCES.BUTLER, C. L., AND CRETCHER, L. H.-(1929) J. Amer. chem. Soc., 51, 1519.CRETCHER, L. H., AND BuTLER, C. L.-(1928) Science, 68, 116.HEIDELBERGER, M., AVERY, 0. T., AND GOEBEL, W. F.-(1929) J. exp. Med., 49, 847.LANDSTEINER, K.-(1936) 'The Specificity of Serological Reactions.' Baltimore.MARRACK, J. R.-(1937) ' Proc. 2nd Intemat. Congr. for Microbiol.' London.-(1938)

'The Chemistry of Antigens and Antibodies.' London.Idem AND CARPENTER.-(1938) Brit. J. exp. Path., 19, 53.MORGAN, W. T. J.-(1936) Biochem. J., 30, 909.-(1937) J. Hyg., 37, 372.-(1941)

Chem. Ind., 60, 722.Idem AND PARTRIDGE, S. M.-(1940a) Biochem. J., 34, 169.-(1940b) Chem. Ind.,

59, 849.-(1941a) Ibid., 60, 722.-(1941b) Biochem. J., 35, 1140.NORMAN, A. G.-(1937) 'The Biochemistry of Cellulose, the Polyuronides, etc.'

London.PARTRIDGE, S. M., AND MORGAN, W. T. J.-(1940) Brit. J. exp. Path., 21, 180.P1E, N. W.-(1936) Ibid., 17, 269.SEIDEMAN, R. M.-(1940) J. Immunol., 38, 237.SORDELLi, A., AND MAYER, E.-(1931) C. R. Soc. Biol., Pari8, 107, 736.TAKAILSHI, E., AND SHIRAHAMA, K.-(1934) J. Fac. Agric. Hokkaido Univ., 35, 101.UHLEKNHUTH, P., AND REMY, E.-(1933) Z. ImmunForsch., 79, 318.-(1934) Ibid.,

82, 229.ZOZAYA, J.-(1932) J. exp. Med., 55, 325.