CHEMO-IMMUNOLOGICAL STUDIES ON CONJUGATED CARBOHYDRATE-PROTEINS III. Active AND Passive ANAPHYLAXIS WITH SYNTHETIC PUGAR- PROTEINS By WILLIAM §. TILLETT, M.D., OSWALD T. AVERY, M.D..,. anp WALTHER F. GOEBEL, Px.D. (From the Hospital of The Rockefeller Institute for Medical Research) (Received for publication, July 18, 1929) In the two preceding papers of this series Avery and Goebel (1, 2) have reported the results of chemo-immunological studies on con- jugated carbohydrate-proteins. In the first of these communications Goebel and Avery (1) described the chemi- cal methods by means of which the p-aminophenol glucosides were synthesized from glucose and galactose and the diazotized substances bound to protein. The two newly synthesized sugar-proteins differ from one another chemically only in specific rotation and molecular configuration. In the second paper, Avery and Goebel (2) reported the antigenic properties and serological specificity of the con- jugated carbohydrates. They found that the glucosidic radical of the compound antigen endowed the new complex with specific reactivity. This was demon- strated in two ways. First, when the seme glucoside is attached to two different proteins, as serum globulin or egg albumin, the serum prepared by immunization with either one of the antigens is specifically reactive with the other. Second, when the two different glucosides,—glucoside and galactoside—are each com- bined with the same protein, the newly formed compounds are serologically dis- tinct. These facts were found to be true even though the individual glucosides are isomers differing only in the spatial configuration of a single carbon atom. On the other hand the incombined glucosides alone were found to be non- antigenic. They failed to induce antibody formation in the animal body and caused no visible precipitation when added to immune sera in vitro. However, both glucosides possess the capacity of specifically inhibiting the precipitating action of homologous antisugar-protein serum. When galactoside was mixed with serum prepared by immunization with galacto-globulin,! the subsequent addition 1 The terms employed in this paper to represent the sugar-protein compounds are the same as those used by Avery and Goebel (2). 551 5§2 — ‘ CONJUGATED CARBQGHYDRATE-PROTEINS. Ii of galacto-albumin to the mixture did not cause precipitation. Furthermore, when the heterologous glucoside was substituted in the same system, no inhibition of the precipitin reaction occurred. These results demonstrate the specificity of the inhibition phenomenon. Avery and Goebel (2) considered that the uncombined glucosides possess the immunological properties of haptens. The réle of carbohydrate in anaphylaxis has been a subject of recent experimental investigation. Tomcesik (3) working with B. lactis aerogenes, and later Tomcsik and Kurotch- kin (4) employing B. lactis aerogenes, the pneumobacillus, and a yeast, isolated carbohydrate substances which produced anaphylactic shock in guinea pigs passively sensitized with homologous immune serum. Lancefield (5) also ob- tained from streptococci carbohydrate material with which anaphylaxis could be induced in guinea pigs passively sensitized with anti-streptococcus serum. Be- cause of the presence of small amounts of nitrogen in the products, none of these authors felt justified in concluding that the carbohydrate alone was responsible for the shock. Avery and Tillett (6) employing the highly purified polysaccharide of the type-specific pneumococci showed that guinea pigs passively sensitized with homologous anti-pneumococcus rabbit serum were thrown into anaphylactic shock by the subsequent injection of the homologous specific carbohydrate. Guinea pigs could not, however, be actively sensitized with the purified poly- saccharides alone. Since the materials used in those experiments were protein- free, and in the case of the Type II and Type IIT substances also nitrogen free, the results conclusively demonstrate the capacity of complex sugars to induce anaphylactic shock in animals passively sensitized with antibacterial sera. The immunological specificity of pneumococcus polysaccharides has a close analogue in the serological specificity exhibited by gluco- protein and galacto-protein. The immunologic behavior of the syn- thesized sugar-proteins led to their use in sensitization ‘experiments. The results, reported in this paper, on anaphylaxis with artificially prepared carbohydrate-proteins confirm and extend the serological findings previously reported. The production of both active and passive anaphylaxis was attempted in order to determine the sensitiz- ing properties of the synthetic antigens and to demonstrate the speci- ficity of the reactions. Gluco-globulin represents phenol 6-glucoside-azo-globulin. Gluco-albumin represents phenol f-glucoside-azo-albumin. Galacto-globulin represents phenol -galactoside-azo-globulin. Galacto-albumin represents phenol é-galactoside-azo-albumin. The globulin was prepared from horse serum, and the albumin from egg white. W. S, TILLETT, 0. T. AVERY, AND W. F. GOEBEL 553 EXPERIMENTAL Guinea pigs weighing 240 to 275 grams were employed. The sensitizing dose, whether serum or sugar-protein, was always injected intraperitoneally. The shocking dose was uniformly injected intravenously into a superficial vein of the hind leg. For details concerning the chemical procedures involved in the synthesis of the materials, the reader is referred to the article by Goebel and Avery (1). The serological characteristics of the serum employed and the method of prepara- tion are described by Avery and Goebel (2). Passive Sensitization A. Results obtained with sugar-protein compounds Eight guinea pigs were injected intraperitoneally with the pooled serum of three rabbits immunized with gluco-globulin. Five guinea pigs each received 5 cc. of serum, one received 3 cc., one received 1 cc., and one 0.5 cc. As previously men- tioned, serum of this character possesses the capacity to precipitate complex anti- gens composed of heterologous protein conjugated with the homologous glucoside. The antigluco-globulin sera used in these experiments had an average titre of specific precipitins, as determined by tests made with gluco-egg-albumin, of 1 to 80,000. By reason of the fact that horse globulin alone when used as antigen does not elicit antibodies reactive with egg-albumin, the high precipitin titre of these sera is obviously dependent upon the conjugated glucoside radical. Twenty-four hours after the administration of gluco-globulin antiserum, each pig received intravenously 1 cc. of gluco-albumin. From Table I it can be seen that the five pigs sensitized with 5 cc. of serum all died with typical symptoms of anaphylactic shock. The animals passively sensitized with 3 cc. and 1 cc., respectively, of antigluco-globulin serum had definite and typical symptoms imme- diately following the injection of 1 cc. of gluco-albumin but recovered. Pig No. 6, which received 0.5 cc. of serum, exhibited only a slight reaction. A similar experiment was carried out using antigalacto-globulin serum for sensitization and galacto-albumin as the toxigenic antigen. Sera obtained from three rabbits immunized with galacto-globulin were pooled; ’ the precipitin titre, as determined with galacto-albumin, averaged 1 to 80,000. Eight guinea pigs were injected intraperitoneally as follows: 5 animals received 5 cc. of serum each, one received 3 cc., one received 1 cc., and one, 0.5 cc. The results given in Table IT are equally as definite as those shown in Table I. All 554 TABLE J Passive Anaphylaxis with Anti-Gluco-Globulin Serum CONJUGATED CARBOHYDRATE-PROTEINS. IT Reactions induced by the use of the homologous glucoside conjugated with a heterologous protein—Gluco-Albumin Anti-ghu paterval : Guinea pig No. globulin” injection of Shocking Symptoms Result serum ip. | serumand | C%!-V- shock dose «|. | goa 1 5 24 1 cc. Typical 134 minutes 2 5 24 1cc. Typical +34 minutes 3 5 24 1 ce. Typical {24 minutes 4 3 24 1ce. Marked scratch- | Definite symp- ’ ing, bucking, toms followed coughing, respir-| by recovery atory distress 5 1 24 icc. Violent typical Definite symp- symptoms toms followed by recovery 6 0.5 24 I cc. Occasional No reaction scratching “1 galacto-egg- . albumin 7 5 24 lee. None No reaction gluco-egg- albumin . Same animal 4 - ~ 1lcc. | Typical {4 minutes hrs. later golacio-ege- albumin 8 5 24 lee. None No reaction lsc 7 “ibumin Same animal 4 - - icc. | Typical {4} minutes hrs. later i } ¢ Death of animal. ip. = intraperitoneal. i.v. = intravenous. W. S. TILLETT, 0. T. AVERY, AND W. F. GOEBEL 555 pigs sensitized with homologous immune serum in amounts from 1 to 5 cc. reacted typically and fatally to the intravenous injection of 1 cc. of galacto-albumin; 0.5 cc. of serum was insufficient to sensitize. TABLE II Passive Anaphylaxis with Anti-Galacte-Globulin Serum Reactions induced by the use of the homologous galactoside combined with a heterologous protein—Galacto-Albumin Interval Anti | between Guinea pig No. «| globulin | injection of | Shocking Symptoms Result serum i.p. | serum and ™ shock dose o. hrs. salgcio-et6- 1 5 | 24 1cc. | Typical {3 minutes 2 5 24 1cc. Typical +8 minutes ‘3 5 24 1 cc. Typical $34 minutes 4 3 24 lec. | Typical {3 minutes 5 1 24 lce. Typical 134 minutes 6 0.5 24 tcc. Slight scratching | Very mild reac- and coughing tion with re- covery I S ‘albumin 7 5 24, lee None No reaction alacto-egg- \clbumine Same animal 1 - - 1ce. Typical $34 minutes hr. later shuco-ege- 8 5 24 lec. None No reaction alacto-egg- solacto-ese Same animal 1 - - lee. | Typical 3 minutes hr. later . t Death of animal. i.p. = intraperitoneal. i.v. = intravenous. In Tables I and II it is also shown that the reactions in guinea pigs induced with sugar-proteins and anti-sera are strictly specific. 556 ‘CONJUGATED CARBOHYDRATE-PROTEINS. III Animals No. 7 and No. 8 af Table I received antigluco-globulin serum and 24 hours later were injected intravenously with galacto-albumin. No reaction occurred. Four hours later, the introduction of gluco-albumin induced typical fatal shock. Similarly pigs No. 7 and No. 8 of Table Il, sensitized with anti- galacto-globulin serum, were unharmed by gluco-albumin; the subsequent ad- ministration of the homologous galacto-albumin antigen caused anaphylactic death of these animals. The animals also serve to demonstrate the fact that gluco- albumin and galacto-albumin are not primarily toxic. 'B. Results obtained with uncombined glucosides The specific inhibitory effect exerted by the glucosides on tk precipitin reaction of sugar-protein and anti-sera has been describe: in detail by Avery and Goebel (2) and has been previously commented upon in this paper. It, therefore, seemed of interest to determine whether anaphylactic shock could be elicited by glucosides alone in passively sensitized guinea pigs, and if not, whether the inhibition which these substances have on the precipitin test would also be evident in the anaphylactic reaction. As shown in Table III, two pigs (Nos. 1 and 2), sensitized 24 hours previously with antigluco-globulin serum, were injected intravenously with 1 cc. of the un- combined homologous glucoside. No reaction occurred. Two hours later 1 cc. of gluco-albumin injected into the same animal caused prompt anaphylactic death. In guinea pigs Nos. 3, 4, and 5, the introduction of glucoside alone was followed immediately by an injection of gluco-albumin. Except for slight scratching, no response was elicited. From these results it may be seen that the injection of glucoside into a sensitized animal exerts a definite but transitory protection against the shocking capacity of material which otherwise would be fatal. That the protective action of the glucoside is specific is demon- _ strated by guinea pigs Nos. 6, 7, and 8. These animals were injected with the heterologous galactoside; when, immediately thereafter they were given gluco-albumin no protection resulted and they died promptly with typical anaphylactic shock. Table IV presents the results obtained with guinea pigs, which, after having been sensitized with antigalacto-globulin serum, were protected by the galactoside from the toxigenic effect of galacto- albumin. TABLE UI Passive Anaphylaxis with Anti-Gluco-Globulin Serum Effect of Uncombined Glucoside ip. = intraperitoneal. iv. = intravenous. Am a ati- | Detween Interval between’ | Injection uco- | injection | Injection of iniecti a panes obulin of un usoside Result shuctldeand sug ‘poten Symptoms Result ip. phylactic Ve test ot. ‘hrs. glucoside hrs. sluco-ege- “4Y 5. 24 ice. No reaction 2 1cc. | Typical {4 minutes. 2 5 24 1 cc. No reaction 2 1cc. | Typical {4 minutes 3 5 24 1ce, No reaction | Followed imme-| 1cc. | Scratches slightly, no | No reaction diately by other symptoms 4 § 24 lec. No reaction | Followed imme-| 1cc. | Scratches slightly, no | No reaction ‘ diately by other symptoms 5 5 24 1 cc. No reaction | Followed imme- | icc. | Scratches slightly, no | No reaction diately by other symptoms salactoside | __ : 6 5 24 Ice. No reaction | Followed imme- | 1cc. | Typical 17} minutes diately by 7 5 24 1 ce. No reaction | Followed imme-j} 1cc. | Typical 134 minutes diately by . 8 5 24 le. No reaction | Followed imme- | icc. | Severe symptoms, falls | Severe shock followed diately by on side. Apnoea; by recovery on feet f Death of animal. THEHOO ‘A ‘M ONV ‘AMHAV “L ‘O ‘LLETIIL *S *M LSS TABLE IV Passive Anaphylaxis with Anti-Galacto-Globulin Serum Effect of Uncombined Galactoside ip. = intraperitoneal. i.v. = intravenous, Anti- | betwcen nu- . Injection pine ee o cron of rcoside Result injection of glacoakde of suger Symptoms Result serum | and ana- iv. and sugar-protein Ly. ip, phylactic at test ce. hrs, salactoside | hrs. salacto-egs- 1 5 24 1 cc. No reaction 2 1cc. | Marked scratching, | Definite symptoms coughing, back- followed by re- ing, respiratory covery distress 2 5 24 tcc. No reaction 24 lec. | Typical 4 minutes ’ 3 5 24 1 ce. No reaction 24 lec. | Typical {34 minutes 4 5 24 lec. No reaction | Followed immedi- | 1 cc. None No reaction ately by ‘ 5 5 24 1 cc. No reaction | Followed immedi- | 1 cc. None No reaction ately by 6 5 24 1 cc. No reaction | Followed immedi-| icc. | None No reaction ately by glucoside 7 5 24 1 cc. No reaction | Followed immedi- | 1 cc. Typical {23 minutes ately by . 8 5 24 1 ce. No reaction | Followed immedi- | 1 cc. Typical 13 minutes ately by , T Death of animal. "SNIALOWd-ALVEGAHOMAVO AELVOOEINOD gcs m TABLE V Desensitisation Induced in Passively Sensitized Guinea Pigs by Injection of Glucoside and Homologous Sugar-Protein galacto-egg-albumin Interval ‘ Previous tests. between . Guines ““emiston Injections macy hrs. after Result peyend Material injected Symptoms Result _ iv. desensitiza-~ tion tests : krs, 1 |5 cc. anti-gluco- | 1 cc. glucoside followed im- | No reaction 4} 1 cc. gluco-egg- | None No reaction ‘globulin serum mediately by 1 cc. gluco- al albumin , egg-albumin 2 |5 cc. anti-gluco- | 1 cc. glucoside followed im- | No reaction 4} 1 cc. gluco-egg- | None No reaction globulin serum mediately by 1 cc. gluco- albumin egg-albumin 3 |5 cc. anti-gluco- | 1 cc. glucoside followed im- | No reaction 4} 1 cc. gluco-egg- | None No reaction globulin serum mediately by 1 cc. gluco- , , albumin egg-albumin 4 15 ce, anti-galacto- | 1 cc. galactoside followed | No reaction 1 1 cc. galacto-egg-| None No reaction globulin serum | immediately by 1 cc. albumin galacto-egg-albumin / 5 |S cc. anti-galacto- | 1 cc. galactoside followed | No reaction 2 1 cc. galacto-egg- | None No reaction globulin serum immediately by 1 cc. albumin galacto-egg-albumin 6 |S ce, anti-galacto-- | 1 cc. galactoside followed | No reaction 34 1 cc. galacto-egg- | None No reaction globulin serum immediately by 1 cc. albumin i.p. = intraperitoneal. i.v. = intravenous. THEIOOD “f ‘M ANV ‘AMTAV “L ‘O “LLATIIL 'S *M 560: CONJUGATED CARBOHYDRATE-PROTEINS. II The results are, in every respect, identical with those given in Table III both with regard to the transitory nature of the phenomenon and to its specificity. . ‘The mechanism of the protection afforded by the glucosides is not as yet understood. The fact that the protective effect is no longer demonstrable after two hours indicates that “‘desensitization,”’—if such has occurred—is transitory. Instances of what appears to be true desensitization have been observed in these experiments and are recorded in Table V. Guinea pigs Nos. 1, 2, and 3, passively sensitized with antigluco-globulin serum, were protected from the shocking effect of 1 cc. of gluco-albumin by a pre- vious injection of homologous glucoside. Four and one-half hours later the same pigs received a second injection of 1 cc. of gluco-albumin. No reaction occurred. The absence of shock following the second injection of whole antigen seems to be dependent upon the first dose of gluco-albumin. That the glucoside alone plays no direct part in the refractory state is demonstrated by its ineffectiveness when in- jected singly, two hours prior to the shocking dose (Table III). Pigs Nos. 4, 5,and 6 of Table V demonstrate the same principle, the difference being that antigalacto- globulin serum was used for sensitization, and galactoside and galacto-albumin were employed to complete the test. C. Resulis obtained with uncombined protein Avery and Goebel (2) have shown that the serum of rabbits immu- nized with synthetic sugar-proteins (gluco- or galacto-globulin) pos- sesses two distinct antibodies; 1) the specific precipitin so intimately _ associated with the carbohydrate radical of the compound; 2) the “common” precipitin, reactive with globulin alone. Passive ana- phylaxis experiments were, therefore, carried out, using pure horse globulin as the toxigenic material. For sensitization, one pig received intraperitoneally 1 cc. of serum prepared by immunization with gluco-globulin; a second pig received 1 cc. of serum derived from a rabbit immunized with galacto-globulin. Twenty-four hours later each received 12 mgms. of globulin. Both animals died in typical anaphylactic shock (Table VI). A normal pig receiving the same dose of globulin gave no reaction. X W. S. TILLETT, 0. T. AVERY, AND W. F. GOEBEL 561 TABLE VI Passive Anaphylaxis with Anti-Gluco-Globulin and Anti-Galacto-Globulin Sera Reactions induced by the use of horse globulin pene | Sestie intaral} — SBOMGE dove | Symptoms | Result hrs. 1 1 cc. anti-gluco- | 24 | 1cc. horse-globulin} Typical | {3 minutes globulin serum (12 mgms.) 2 1 cc. anti-galacto- | 24 | icc. horse-globulin| Typical {| {2} minutes globulin serum , 3 Normal control , — | 1cc. horse-globulin| None No reaction f Death of animal. i.p. = intraperitoneal, i.v. = intravenous. Active Anaphylaxis For purposes of testing the capacity of the synthetic sugar-proteins to produce active sensitization, 10 guinea pigs were injected intra- peritoneally with 5 cc. of gluco-globulin and 10 other animals were similarly inoculated with 5 cc. of galacto-globulin. In the preparations employed, 5 cc. of sugar-globulin contained 50 mgms. of protein. It is estimated (1) that 15 per cent by weight of this complex represents chemically combined glucoside. Consequently each pig received approximately 7.5 mgms. of the synthesized sugar-protein. The 20 pigs were tested 21 days later for active sensitization. Asin the experiments on passive anaphylaxis, the sensi- tizing dose consisted of material in which the glucoside was joined to a protein heterologous to that used for sensitization. As previously mentioned, this pre- caution was taken in order to eliminate the possibility of protein-antiprotein reactions entering into the results. In Table VII, the results of active sensitization obtained by the use of gluco-proteins are given. Pigs Nos. 1, and 2, previously sensitized with gluco-globulin, were injected in- travenously with 1 cc. of gluco-egg-albumin. Each promptly succumbed with typical symptoms. Pigs Nos. 3 and 4 of Table VII demonstrate the specificity of the sensitization; both of the animals when tested with 1 cc. of galacto-albumin showed no reaction. However, when, 3 hours later, 1 cc. of gluco-albumin was introduced, they reacted fatally. Pigs Nos. 5, 6, 7, and 8 were used to determine the influence of homologous uncombined glucoside on the reaction. In these tests the same relations were found to exist as described in the experiments on passive TABLE VII Active Anaphylaxis in Guinea Pigs Sensitized with Gluco-Globulin ip. = intraperitoneal. iv. = intravenous. Interval Santt oases Int 1 eae Gui Sensitizing between Ist Injection Interval between 2nd Injection between 3rd Injection Pig No. dose sensitizing Material tat ea 2nd Mater 1 2nd and Vien cal * Pp. ateria, | vp and test | iv. Result ow“ iv. Result | injection | iv. | Result Liein days sluco-cee- min. hrs. min, hrs, min. 1 5 ce. 21 1ce. 15 - _ _ _ - _ 2 5 ce. 21 Ice. 134 - _ —_ - _ _ salacto-cgg- sluco-ege- albumin bunsin 3 5 cc. 21 lec. No reaction 3 1 cc. 3 - - - 4 5 ce. 21 lee. No reaction 3 1 ce. Severe symp- - - - toms. Re- covery shucoside 5 5 cc. 21 1 cc. No reaction 14 1 cc. t3 - - - 6 5 ce. 21 1c. No reaction 2 1 ce. {23 . - - _ 7 5 cc. 21 1 ce. No reaction 2 ice. {44 - ~ _ 8 5 cc. 21 lec. No reaction | Followed im- lee. No reaction _ -_ = mediately relate tt Aiea 982: se 9 Sc. 21 1 ce. No reaction 4 lee. No reaction 4 tcc. 44 t Death of animal. 79S "SNIDLOUd-ALVAACAHOTAVO GaLvon[Nood mm Active Anaphylaxis in Guinea Pigs Sensitized with Galacto-Globulin TABLE VIII I al : eo gs : sags Guinea | Sensitisi between 1st Injection Interval between 2nd Injection faterval 3rd Injection Pig No. dose sensitizing Material 1st and 2nd Material 2nd and Mated tp | anatest | iv. Result tnection we Result injection | wiv! | Result eee days salacto-ege- min. hrs. min, hes, . mins, 1 5 cc, 21 1 ce. 134 _ - - _ - - 2 5 cc. 21 1 ce. ' 14 - ~ _ _ _ _ , I - alacto-egg- ‘banca ‘ albumin 3 S cc. 21 ~Lee. No reaction 23. 1 cc. 14 - - _ 4 5 cc. 21 1cc. | No reaction 2 lc. "13 sobactoside 5 § ce. 21 1 cc. No reaction 14 1 ce. {44 6 |: Scc. ai ice. No reaction 3 1 cc. 3 7 5 ce. a1 1 cc. No reaction 3 1 ce. {2 8 5 cc. 21 1ce. No reaction | Followed im- 1cce. Very slight mediately symptoms. by Recovery . glucoside sluco-cee- calacto-eee- 9 5 cc. 21 1 cc, No reaction 4 1 ce. No reaction 1 1ce. ts 10 5 cc, 21 -Lee. No reaction 4 1c. No reaction 1 1 cc. 134 t Death of animal. i.p. = intraperitoneal. i.v. = intravenous. JATAOO “A °“M AONV ‘'AEHAV °L *O SLIATIIL ‘S °M £9s 564 CONJUGATED CARBOHYDRATE-PROTEINS. It anaphylaxis. When glucoside was injected immediately before gluco-albumin, complete inhibition of anaphylaxis resulted. However, when glucoside was in- jected one and one-half to two hours before the shocking dose (Pigs Nos. 5, 6, and 7), no protection occurred. Pig No. 9 of Table VII is further evidence of the specificity of active sensitization; in this animal attempts to inhibit shock with heterologous galactoside and to desensitize with galacto-albumin were ineffectual since the subsequent injection of gluco-albumin produced characteristic death. TABLE IX Active Anaphylaxis in Guinea Pigs Sensitized with Gluco-Globulin and . Galacto-Globulin Reactions induced by the use of horse globulin—the protein common to both antigens caine | Sensitifitg serum interval| nae oO | Symptoms Result days 1 5 cc. gluco-globulin | 21 | 1cc.horse-globulin| Typical {34 minutes (18 mgms.) 2 5cc. gluco-globulin 21 | O.5cc. horse-glob- | Typical $14 minutes , ulin (9 mgms.) 3 Scc. galacto- 21 | 1cc. horse-globulin | Typical +3 minutes globulin (18 mgms.) 4 5 cc. galacto- 21 | 0.5 cc. horse- Typical {74 minutes globulin globulin (9 mgms.) 5 Normal control — | tcc. horse-globulin| None No reaction (18 mgms.) ¢ Death of animal. i.p. = intraperitoneal. iv. = intravenous. In Table VIII, a similar group of experiments was carried out employing galacto-globulin for sensitization instead of gluco-globulin. Galacto-albumin was the toxigenic agent; galactoside was injected for inhibition tests. Results comparable in every respect to those recorded in Table VII were obtained. Consequently a detailed description need not be given. . Table IX presents the results obtained in guinea pigs actively sensitized with gluco-globulin (animals Nos. 1 and 2) or with galacto- globulin (Nos. 3 and 4) and subsequently injected with horse globulin. W. S. TILLETT, 0. T. AVERY, AND W. F. GOEREL 565 All the animals gave typical reactions. Active sensitivity in these pigs was in all probability induced by the uncombined globulin present in the sensitizing material. DISCUSSION The experiments reported in this paper demonstrate the capacity of artificially prepared Sugar-proteins to produce both active and passive anaphylaxis.. The tests were devised and carried out in such a manner as to emphasize the significance of the carbohydrate radical. The fact that guinea pigs, passively sensitized with antigluco-globulin serum, or actively sensitized with gluco-globulin, can be subsequently shocked with gluco-albumin, demonstrates that the antigen-antibody specificity in these instances is directly dependent upon the carbo- hydrate fraction of the antigenic compounds, The introduction of the sugar radical into the protein molecule endows the new complex with a sharply defined specific antigenicity. This fact is brought out by experiments in which galactoside was substituted for glucoside in the Preparation of sugar-proteins used for sensitization. The same specific relations hold in the production of anaphylaxis with galacto-proteins as that described for gluco- proteins. Attempts to incite anaphylactic shock with heterologous material were ineffectual. The results of the anaphylactic experi- ments conform to the results anticipated by the serological findings of Avery and Goebel (2). Landsteiner (7), employing complex antigens, has reported experi- ments on anaphylaxis of a similar character to those presented in this report. He found that guinea pigs sensitized with one azoprotein could be shocked by the injection of a second compound containing the same azo-groups attached to a different protein. In addition to the new specificity which the carbohydrate radical confers upon the conjugated proteins, the uncombined glucosides by themselves also exert a definite influence on the reactivity of sensitized animals. When sensitized pigs are injected with the homologous glucoside immediately before the introduction of the toxigenic sugar- protein, they are completely protected from shock. However, the protection afforded by the glucoside alone is apparently only transi- tory; for, when the interval between introduction of glucoside and 566 = CONJUGATED CARBOHYDRATE-PROTEINS. II shocking agent was as long as two hours, the injection of homologous sugar-protein produced prompt and typical anaphylactic death. That the temporary protection just mentioned is specific, was ' demonstrated by the experiments in which uncombined carbohydrate of heterologous type was shown to exert no such protective action. The transitory, specific protection afforded by the glucosides alone is not yet understood. Landsteiner (7) found in experimaits on ana- phylaxis with azoproteins that the azo-component, when injected one hour before the conjugated azoprotein, inhibited shock. He con- sidered that a state of anti-anaphylaxis had been induced. In the tests with glucosides and sugar-proteins, sufficient evidence has not been obtained to interpret the mechanism other than to say that the inhibitory effect of the glucosides disappears in at least two hours. Active and passive anaphylaxis has also been elicited with uncom- bined globulin. Whether animals were passively sensitized with anti- gluco-globulin or antigalacto-globulin serum, the toxigenic action of . globulin was equally effective. Since the sera employed contained , anti-globulin antibodies, these results were to be expected. Guinea pigs actively sensitized with either gluco-globulin or galacto-globulin, were found to be equally sensitive to uncombined globulin. Since the material used to produce active sensitization contained free globulin, the subsequent intoxication with horse globulin is obviously based on a simple protein- anti-protein reaction. CONCLUSIONS 1. Guinea pigs passively sensitized with the serum of rabbits immunized with an artificially prepared sugar-protein (gluco-globulin) exhibit typical anaphylactic shock when subsequently inoculated with gluco-albumin; the serum of rabbits immunized with a second syn- thetic sugar-protein (galacto-globulin) similarly sensitizes guinea pigs to galacto-albumin. The reactions, in each instance, are specific and depend for their specificity on the carbohydrate component, and not on the protein fraction of the synthesized sugar-protein. 2. Guinea pigs actively sensitized with gluco-globulin or galacto- globulin are similarly subject to anaphylactic shock’, when injected, after 21 days, with sugar-proteins containing carbohydrate identical with that present in the sensitizing antigen, regardless of the kind of protein with which it is combined. W. §. TILLETT, 0. T. AVERY, AND W. F. GOEBEL 567 3. The unconjugated glucosides, although themselves not capable of inducing shock, inhibit the anaphylactic reaction when injected immediately prior to the introduction of the toxigenic sugar-protein. The protective action of the glucosides disappears within two hours after injection. In order to elicit the phenomenon, the carbohydrate must be the same as that combined in the sugar-protein complex. 4. Anaphylactic shock may be induced by uncombined globulin in guinea pigs passively sensitized with either antigluco-globulin serum or antigalacto-globulin serum; globulin is similarly effective in animals actively sensitized with gluco-globulin or galacto-globulin. Thereac- tions elicited by globulin alone are dependent upon the common pro- tein present in the antigens, and exhibit only species specificity. * BIBLIOGRAPHY - Goebel, W. F., and Avery, O. T., J. Exp. Med., 1929, 50, 521. . Avery, 0. T., and Goebel, W. F., J. Exp. Med., 1929, 50, 533. - Tomesik, K., Proc. Soc. Exp. Biol. and Med., 1927, 24, 812. . Tomesik, K., and Kurotchkin, T. K., J. Exp. Med., 1928, 47, 379. . Lancefield, R. C., J. Exp. Med., 1928, 47, 843, . Avery, O. T., and Tillett, W. S., 7. Exp. Med., 1929, 49, 251. . Landsteiner, K., J. Exp. Med., 1924, 39, 631. SAO hw bd Be