Scand. J. Immunol. 7, 245-249, 1978
Natural and Immune Antibodies to Rabbit Erythrocyte Antigens O. TONDER, B. LARSEN, D. AARSKOG & B. HANEBERG Broegelmann Research Laboratory for Microbiology; The Gade Institute, Department of Microbiology; and Department of Pediatrics, University of Bergen, Bergen, Norway
Tonder, O., Larsen, B.. Aarskog, D. & Haneberg, B. Natural and Immune Antibodies to Rabbit Erythrocyte Antigens. Scand. J. Immunol. 1, 245-249. 1978. Natural agglutinins to rabbit erythrocytes were found in all human sera studied. In the newborn, the antibodies were of IgG class; in the 6-month-old infants they were mainly of IgM class. Older children and adults had both IgG and IgM antibodies. Agglutinins to rabbit erythrocytes were also found in serum from fourteen of fifteen other species studied. The trichioroacetic acid extract from rabbit erythrocyte stromata (TCA-preparation) contains at least three different antigenic determinants: one which we hitherto have found only on rabbit erythrocytes, one which is closely related to human blood group B antigen, and one which is closely related to the 1 antigen. The TCA-preparation did not elicit delayed hypersensitivity skin reactions in humans in spite of high titred agglutinins in serum, bul did so in immunized guinea-pigs. O. Tonder. Broegetmann Re.'iearch Laboratory for Microbiology. MFH-byggei. N-5016 Haukelandsykehus. Bergen. Norway.
Human sera [9], saliva [8], milk [3], and extracts of human faeces [2] contain natural antibodies which agglutinate rabbit erythrocytes. Since these antibodies occur in all humans, they have been used as markers of serum innunoglobulins in other body fluids [1.7]. The origin of the stimulating antigen(s) is unknown. We have obtained various antigen preparations from rabbit erythrocyte stromata and demonstrated the strongest inhibiting activity in a preparation containing sphingoglycolipid [6]. One of the crude antigen preparations has been examined with respect to its ability to inhibit natural human and immune animal antibodies, and its ability to elicit delayed hypersensitivity skin reactions. The results of these studies are presented in this paper. MATERIAL AND
METHODS
Erythrocytes. Rabbit blood was collected in Alsever's solution, and human blood in citrate solution. The 0300-9475/78/0300-0245 $02.00
erythrocytes were washed three times in phosphate buffered saline (0.15 M NaCl-O.015 M phosphate). pH 7.2 (PBS) and finally packed at lOOOg for 10 min (100%). Sera. Pooled human serum (PHS) was a mixture of sera from fifty healthy blood donors. Sera from ninetyfive infants and children of different ages (newborn to 14 years) were obtained from healthy children of hospital employees and from patients admitted to The Children's Hospital, Bergen, with conditions not considered to involve the immune system (undescended testicles, congenital heart diseases, and fractures). Sera from patients with the cold haemagglutinin syndrome or with Mycoptasma pneumoniae infections were selected from sera sent for routine serology. Human IgG (Fraction II) was purchased from AB Kabi. Stockholm. Antisera to human Igs were the same as previously [10]. Blood from four frogs, three chickens, six hedgehogs, four mice, six guinea-pigs, two sheep and one goat was supplied by the animal unit. Blood from one dog. two horses, one cow and one pig was obtained from a local slaughterhouse. Blood from six hagfish was collected at the University's Institute of Marine Biology, and sera from six polar bears were provided by the Institute of Marine Research. Blood from a shrew and a mole was kindly provided by H. Walhovd. Aarhus, Denmark. Serum was collected after centrifugation of the clotted
©1978 Blackwell Scientific Publications 245
246
O. Tdnder et al.
blood al 1000 g for 30 min. Sera from immunized hedgehogs were the same as described previously [5]. All sera were heated for 30 min at 56°C to inactivate complement. They were stored at — 25°C. Saliva. Saliva was collected from sixteen healthy adults and from sixteen infants and children ranging in age from 6 months to 12 years. The specimens were frozen overnight, thawed and centrifuged to remove cell debris, then stored frozen. Preparation of erythrocyte antigen. The rabbit erythrocyte antigen preparation was identical to that described previously [6]. Briefly, erythrocyte stromata were extracted repeatedly with 10% trichloroacetic acid; the supernatants were pooled, dialysed against distilled water and lyophilized (TCA-preparation). Digestion with enzymes. Human IgG was digested with papain (2 x crystallized. Sigma Chemical Co., St Louis, Mo., USA) or pepsin (2 x crystallized, Sigma) at an enzyme to protein ratio of 2:100 (w/w) for 18 h at 37°C. Papain digestion was performed in 0.05 M Tris-HCl (tris{hydroxymethyl)aminomethane) -f 0.002 M EDTA (ethylene-diamine-tetraacetate), pH 7.0, and pepsin digestion in 0.1 M acetate buffer, pH 4.1. Pepsin digestion was stopped by raising the pH to 7.2, and papain digestion was stopped by adding 1/10 volume of 1.25% N-ethyl-maleimide (BDH Chemicals Ltd, Poole, England). Both preparations were then dialysed against PBS. Absorption of sera. Serum diluted 1 in 4 was mixed with an equal volume of the packed erythrocytes and left al room temperature for 20 min. The mixture was then centrifuged and the supernatant (absorbed serum) was used in agglutination experiments. Immunization of guinea-pigs. Two guinea-pigs were injected intraperitoneally with 1.2 mg of the TCA-preparation in complete Freund's adjuvant, and 18 days iater 1.0 mg without adjuvant. The animals were bled on day 24, and on day 27 they were tested for delayed hypersensitivity to the TCA-preparation. Testing for delayed hyper.sensiiiviiy. Guinea-pigs were given intracutaneous injections of 0.1 ml of (a) TCApreparation, I mg/ml; (b) TCA-preparation, 0.1 mg/ml; and (c) saline. Humans were tested by the scratch method using a TCA-preparation containing I mg/ml and by intracutaneous injections using 0.05 ml of the TCA-preparation. The reactions were read every twelfth hour for four successive days. Prior to testing on humans, the TCA-preparation was heated in a boiling water bath for 5 min. Other methods. The agglutination and the agglutination inhibition tests were performed as described previously [6]. The titre of agglutination is given as the reciprocal value of the highest dilution of serum giving agglutination. One agglutinating unit is defined as the amount of serum corresponding to the titre. Tests for antiglobulin activity and for inhibition of agglutination by antisera to human Igs were performed as previously described [10]. The following sugars were used as 10% solutions to inhibit the agglutination of rabbit erythrocytes by serum from immunized guinea-pigs; D-I-Glucose, D-t-Galactose, D-t-Mannose, D —Fructose, D + M a l tose, a- and ^-Lactose, L —Fucose, D-I-Glucosamine and N —acetyl-D-glucosamine. Experiments with cold agglutinins were performed by
inhibition at 0°C (ice-water bath) and at 37°C. Treatment with 2-mercaptoethanol (ME) was performed by incubating a mixture of equal volumes of serum diluted 1 in 2 and 0.2 M MEfor 30min at 37°C. Fractionation of serum by filtration on a Sephadex G-200 column was performed as previously [8]. The TCA-preparations were treated with enzymes and periodate as previously described [6].
EXPERIMENTS AND RESULTS
Time of appearance of agglutinins in humans The appearance of agglutinins to rabbit erythrocytes in humans was studied with sera from children of different age-groups (Fig. 1). Notably, agglutinins were present in all sera tested. MEresistant agglutinins were present at time of birth. Serum from 6-month-old infants contained mainly ME-sensitive agglutinins. The titres of both ME-sensitive and ME-resistant agglutinins from then on rose with the age of the child. To ascertain the Ig class of the agglutinins, all thirtythree sera from the infants were assayed in antiglobulin and agglutination inhibition tests using antisera to IgG, IgM and IgA. As expected, Ihe ME-resistant agglutinins were always of IgG and the ME-sensitive of IgM class.
TiUes (13)
4096
(5) (16)
102A
(U)
(13) (9)
(11) (11)
(5)
256 (5)
64
(9) 16 4 < 2 •-
T
New
i'
-H M-
H I
born 1- 4 - 7- 12- 24- 3- 6- 9- 12- AduUs 3 6 11 23 35 5 8 11 14 Months Years Age
FIG. 1. Agglutination of rabbit erythrocytes by human sera. Mode titres of sera from ini"ants and children of different age compared to sera from adults. •—», Untreated sera; • — •, sera treated with ME. Vertical bars indicate titre range. Number of sera in each age group given in parentheses.
Antibodies to Rabbit Erythrocytes Agglutinins were also present in saliva from the sixteen cliildren; the youngest child was 6 months old. The titres ranged from 4 to 32. Generally the titre was lower in saliva from children than from adults. Results of antiglobulin and agglutination inhibition tests confirmed [8] that the salivary agglutinins were of IgA class. Agglutinins in sera from other species
In the present study sera from all animals tested (hagfish, frog, chicken, hedgehog, shrew, mole, mouse, guinea-pig, goat, sheep, cow. horse, pig, dog and polar bear) agglutinated rabbit erythrocytes, except serum from hagfish; titres ranged from 2 to 256, but for most species from 4 to 16. Inhibition studies with the TCA-preparation Untreated and ME-treated PHS, partly purified human IgM (first peak after gel filtration of PHS on Sephadex G-200), isolated human IgG (Fraction II) and normal human saliva was added to dilutions of the TCA-preparation. The preparation was more efficient in inhibiting tgM antibodies than IgG antibodies; 50 //g inhibited 8 agglutinating units, but only 2 or 4 agglutinating units of IgG antibodies (Table I). Enzymedigested human IgG was also studied in the inhibition test. Two agglutinating units of papaintreated IgG was inhibited by 25 p% of TCA-preparation, while 6.25 pg was sufficient to inhibit 2 agglutinating units of pepsin digested IgG. This indicated that the preparation inhibited a true antigen-antibody reaction, although a possible additional inactivation of antibody by Fc-binding to the TCA-preparation cannot be ruled out. TABLE 1. Agglutination of rabbit erythrocytes. Number of agglutinating units of human antibodies from different sources inhibited by various amounts of TCA-preparation.
/igof TCA-preparation Antibody source
50
12.5
3.13
PHS untreated PHS ME-treated Partly purified IgM IgG (Fraction II) Saliva
8 2 8 4 4
4 1 4 0 2
2 0 2 0 0
PHS: Pooled human serum.
247
A few of the animal sera were also studied in the inhibition test. Of these, 2 agglutinating units of serum from chicken, polar bear, guinea-pig and sheep were inhibited by 25 /ig or less of TCA-preparation. Serum from a horse and from a pig were not inhibited by this amount, possibly indicating different specificities of the natural antibodies to rabbit erythrocytes. Sera from six blood donors of blood group A were absorbed with rabbit erythrocytes. The titres against group B erythrocytes (16 to 128) dropped to 4 or less. On the other hand, absorption of the sera with group B erythrocytes did not reduce the titre against rabbit erythrocytes. In the inhibition test using TCA-preparation and group B erythrocytes, 6.25 pg or less of the preparation inhibited 2 agglutinating units of anti-B antibodies. Apparently an antigen partly identical to the blood group B antigen is present on rabbit erythrocytes and in the TCA-preparation. Sera from six patients suffering from the chronic cold haemagglutinin syndrome and from six patients with Mycoplasma pneumoniae infections were used in the inhibition experiments. The TCA-preparation inhibited the agglutination of both human and rabbit erythrocytes by all twelve sera, indicating the presence of the I antigen in the TCA-preparation. In general, the antigen preparation was more effective per weight unit in inhibiting the agglutination at 4°C of human erythrocytes of blood group O than rabbit erythrocytes. Peak-II material from Sephadex G-200 filtration of sodium-dodecyl-sulphate treated TCA-preparation [6] also inhibited the cold agglutinins, indicating that the I antigen was present in this purified TCA-preparation. Immunization of hedgehogs with rabbit or sheep erythrocytes leads to formation of antibodies to the serum sickness (ss) antigen [5]. Inhibition experiments were performed with the TCA-preparation and these antisera. As much as 100/;g of TCA-preparation did not inhibit 1 agglutinating unit of the various hedgehog antisera, indicating that the ss-antigen was not present in the TCA-preparation. In vivo response to the antigen The two guinea-pigs that had been given injections of TCA-preparation intraperitoneally both developed a delayed hypersensitivity reaction when they were tested intracutaneously with the TCA-preparation 4 weeks after the contact with
248
O. Tdnder et al.
the antigen. Before immunization they showed no reaetion. The animals were killed on day 39 and the sera used in inhibition experiments. The untreated and ME-treated sera agglutinated rabbit erythrocytes to titres of 1024 and 128 respectively (preimmunization titres 8 and
Natural and Immune Antibodies to Rabbit Erythrocyte Antigens O. TONDER, B. LARSEN, D. AARSKOG & B. HANEBERG Broegelmann Research Laboratory for Microbiology; The Gade Institute, Department of Microbiology; and Department of Pediatrics, University of Bergen, Bergen, Norway
Tonder, O., Larsen, B.. Aarskog, D. & Haneberg, B. Natural and Immune Antibodies to Rabbit Erythrocyte Antigens. Scand. J. Immunol. 1, 245-249. 1978. Natural agglutinins to rabbit erythrocytes were found in all human sera studied. In the newborn, the antibodies were of IgG class; in the 6-month-old infants they were mainly of IgM class. Older children and adults had both IgG and IgM antibodies. Agglutinins to rabbit erythrocytes were also found in serum from fourteen of fifteen other species studied. The trichioroacetic acid extract from rabbit erythrocyte stromata (TCA-preparation) contains at least three different antigenic determinants: one which we hitherto have found only on rabbit erythrocytes, one which is closely related to human blood group B antigen, and one which is closely related to the 1 antigen. The TCA-preparation did not elicit delayed hypersensitivity skin reactions in humans in spite of high titred agglutinins in serum, bul did so in immunized guinea-pigs. O. Tonder. Broegetmann Re.'iearch Laboratory for Microbiology. MFH-byggei. N-5016 Haukelandsykehus. Bergen. Norway.
Human sera [9], saliva [8], milk [3], and extracts of human faeces [2] contain natural antibodies which agglutinate rabbit erythrocytes. Since these antibodies occur in all humans, they have been used as markers of serum innunoglobulins in other body fluids [1.7]. The origin of the stimulating antigen(s) is unknown. We have obtained various antigen preparations from rabbit erythrocyte stromata and demonstrated the strongest inhibiting activity in a preparation containing sphingoglycolipid [6]. One of the crude antigen preparations has been examined with respect to its ability to inhibit natural human and immune animal antibodies, and its ability to elicit delayed hypersensitivity skin reactions. The results of these studies are presented in this paper. MATERIAL AND
METHODS
Erythrocytes. Rabbit blood was collected in Alsever's solution, and human blood in citrate solution. The 0300-9475/78/0300-0245 $02.00
erythrocytes were washed three times in phosphate buffered saline (0.15 M NaCl-O.015 M phosphate). pH 7.2 (PBS) and finally packed at lOOOg for 10 min (100%). Sera. Pooled human serum (PHS) was a mixture of sera from fifty healthy blood donors. Sera from ninetyfive infants and children of different ages (newborn to 14 years) were obtained from healthy children of hospital employees and from patients admitted to The Children's Hospital, Bergen, with conditions not considered to involve the immune system (undescended testicles, congenital heart diseases, and fractures). Sera from patients with the cold haemagglutinin syndrome or with Mycoptasma pneumoniae infections were selected from sera sent for routine serology. Human IgG (Fraction II) was purchased from AB Kabi. Stockholm. Antisera to human Igs were the same as previously [10]. Blood from four frogs, three chickens, six hedgehogs, four mice, six guinea-pigs, two sheep and one goat was supplied by the animal unit. Blood from one dog. two horses, one cow and one pig was obtained from a local slaughterhouse. Blood from six hagfish was collected at the University's Institute of Marine Biology, and sera from six polar bears were provided by the Institute of Marine Research. Blood from a shrew and a mole was kindly provided by H. Walhovd. Aarhus, Denmark. Serum was collected after centrifugation of the clotted
©1978 Blackwell Scientific Publications 245
246
O. Tdnder et al.
blood al 1000 g for 30 min. Sera from immunized hedgehogs were the same as described previously [5]. All sera were heated for 30 min at 56°C to inactivate complement. They were stored at — 25°C. Saliva. Saliva was collected from sixteen healthy adults and from sixteen infants and children ranging in age from 6 months to 12 years. The specimens were frozen overnight, thawed and centrifuged to remove cell debris, then stored frozen. Preparation of erythrocyte antigen. The rabbit erythrocyte antigen preparation was identical to that described previously [6]. Briefly, erythrocyte stromata were extracted repeatedly with 10% trichloroacetic acid; the supernatants were pooled, dialysed against distilled water and lyophilized (TCA-preparation). Digestion with enzymes. Human IgG was digested with papain (2 x crystallized. Sigma Chemical Co., St Louis, Mo., USA) or pepsin (2 x crystallized, Sigma) at an enzyme to protein ratio of 2:100 (w/w) for 18 h at 37°C. Papain digestion was performed in 0.05 M Tris-HCl (tris{hydroxymethyl)aminomethane) -f 0.002 M EDTA (ethylene-diamine-tetraacetate), pH 7.0, and pepsin digestion in 0.1 M acetate buffer, pH 4.1. Pepsin digestion was stopped by raising the pH to 7.2, and papain digestion was stopped by adding 1/10 volume of 1.25% N-ethyl-maleimide (BDH Chemicals Ltd, Poole, England). Both preparations were then dialysed against PBS. Absorption of sera. Serum diluted 1 in 4 was mixed with an equal volume of the packed erythrocytes and left al room temperature for 20 min. The mixture was then centrifuged and the supernatant (absorbed serum) was used in agglutination experiments. Immunization of guinea-pigs. Two guinea-pigs were injected intraperitoneally with 1.2 mg of the TCA-preparation in complete Freund's adjuvant, and 18 days iater 1.0 mg without adjuvant. The animals were bled on day 24, and on day 27 they were tested for delayed hypersensitivity to the TCA-preparation. Testing for delayed hyper.sensiiiviiy. Guinea-pigs were given intracutaneous injections of 0.1 ml of (a) TCApreparation, I mg/ml; (b) TCA-preparation, 0.1 mg/ml; and (c) saline. Humans were tested by the scratch method using a TCA-preparation containing I mg/ml and by intracutaneous injections using 0.05 ml of the TCA-preparation. The reactions were read every twelfth hour for four successive days. Prior to testing on humans, the TCA-preparation was heated in a boiling water bath for 5 min. Other methods. The agglutination and the agglutination inhibition tests were performed as described previously [6]. The titre of agglutination is given as the reciprocal value of the highest dilution of serum giving agglutination. One agglutinating unit is defined as the amount of serum corresponding to the titre. Tests for antiglobulin activity and for inhibition of agglutination by antisera to human Igs were performed as previously described [10]. The following sugars were used as 10% solutions to inhibit the agglutination of rabbit erythrocytes by serum from immunized guinea-pigs; D-I-Glucose, D-t-Galactose, D-t-Mannose, D —Fructose, D + M a l tose, a- and ^-Lactose, L —Fucose, D-I-Glucosamine and N —acetyl-D-glucosamine. Experiments with cold agglutinins were performed by
inhibition at 0°C (ice-water bath) and at 37°C. Treatment with 2-mercaptoethanol (ME) was performed by incubating a mixture of equal volumes of serum diluted 1 in 2 and 0.2 M MEfor 30min at 37°C. Fractionation of serum by filtration on a Sephadex G-200 column was performed as previously [8]. The TCA-preparations were treated with enzymes and periodate as previously described [6].
EXPERIMENTS AND RESULTS
Time of appearance of agglutinins in humans The appearance of agglutinins to rabbit erythrocytes in humans was studied with sera from children of different age-groups (Fig. 1). Notably, agglutinins were present in all sera tested. MEresistant agglutinins were present at time of birth. Serum from 6-month-old infants contained mainly ME-sensitive agglutinins. The titres of both ME-sensitive and ME-resistant agglutinins from then on rose with the age of the child. To ascertain the Ig class of the agglutinins, all thirtythree sera from the infants were assayed in antiglobulin and agglutination inhibition tests using antisera to IgG, IgM and IgA. As expected, Ihe ME-resistant agglutinins were always of IgG and the ME-sensitive of IgM class.
TiUes (13)
4096
(5) (16)
102A
(U)
(13) (9)
(11) (11)
(5)
256 (5)
64
(9) 16 4 < 2 •-
T
New
i'
-H M-
H I
born 1- 4 - 7- 12- 24- 3- 6- 9- 12- AduUs 3 6 11 23 35 5 8 11 14 Months Years Age
FIG. 1. Agglutination of rabbit erythrocytes by human sera. Mode titres of sera from ini"ants and children of different age compared to sera from adults. •—», Untreated sera; • — •, sera treated with ME. Vertical bars indicate titre range. Number of sera in each age group given in parentheses.
Antibodies to Rabbit Erythrocytes Agglutinins were also present in saliva from the sixteen cliildren; the youngest child was 6 months old. The titres ranged from 4 to 32. Generally the titre was lower in saliva from children than from adults. Results of antiglobulin and agglutination inhibition tests confirmed [8] that the salivary agglutinins were of IgA class. Agglutinins in sera from other species
In the present study sera from all animals tested (hagfish, frog, chicken, hedgehog, shrew, mole, mouse, guinea-pig, goat, sheep, cow. horse, pig, dog and polar bear) agglutinated rabbit erythrocytes, except serum from hagfish; titres ranged from 2 to 256, but for most species from 4 to 16. Inhibition studies with the TCA-preparation Untreated and ME-treated PHS, partly purified human IgM (first peak after gel filtration of PHS on Sephadex G-200), isolated human IgG (Fraction II) and normal human saliva was added to dilutions of the TCA-preparation. The preparation was more efficient in inhibiting tgM antibodies than IgG antibodies; 50 //g inhibited 8 agglutinating units, but only 2 or 4 agglutinating units of IgG antibodies (Table I). Enzymedigested human IgG was also studied in the inhibition test. Two agglutinating units of papaintreated IgG was inhibited by 25 p% of TCA-preparation, while 6.25 pg was sufficient to inhibit 2 agglutinating units of pepsin digested IgG. This indicated that the preparation inhibited a true antigen-antibody reaction, although a possible additional inactivation of antibody by Fc-binding to the TCA-preparation cannot be ruled out. TABLE 1. Agglutination of rabbit erythrocytes. Number of agglutinating units of human antibodies from different sources inhibited by various amounts of TCA-preparation.
/igof TCA-preparation Antibody source
50
12.5
3.13
PHS untreated PHS ME-treated Partly purified IgM IgG (Fraction II) Saliva
8 2 8 4 4
4 1 4 0 2
2 0 2 0 0
PHS: Pooled human serum.
247
A few of the animal sera were also studied in the inhibition test. Of these, 2 agglutinating units of serum from chicken, polar bear, guinea-pig and sheep were inhibited by 25 /ig or less of TCA-preparation. Serum from a horse and from a pig were not inhibited by this amount, possibly indicating different specificities of the natural antibodies to rabbit erythrocytes. Sera from six blood donors of blood group A were absorbed with rabbit erythrocytes. The titres against group B erythrocytes (16 to 128) dropped to 4 or less. On the other hand, absorption of the sera with group B erythrocytes did not reduce the titre against rabbit erythrocytes. In the inhibition test using TCA-preparation and group B erythrocytes, 6.25 pg or less of the preparation inhibited 2 agglutinating units of anti-B antibodies. Apparently an antigen partly identical to the blood group B antigen is present on rabbit erythrocytes and in the TCA-preparation. Sera from six patients suffering from the chronic cold haemagglutinin syndrome and from six patients with Mycoplasma pneumoniae infections were used in the inhibition experiments. The TCA-preparation inhibited the agglutination of both human and rabbit erythrocytes by all twelve sera, indicating the presence of the I antigen in the TCA-preparation. In general, the antigen preparation was more effective per weight unit in inhibiting the agglutination at 4°C of human erythrocytes of blood group O than rabbit erythrocytes. Peak-II material from Sephadex G-200 filtration of sodium-dodecyl-sulphate treated TCA-preparation [6] also inhibited the cold agglutinins, indicating that the I antigen was present in this purified TCA-preparation. Immunization of hedgehogs with rabbit or sheep erythrocytes leads to formation of antibodies to the serum sickness (ss) antigen [5]. Inhibition experiments were performed with the TCA-preparation and these antisera. As much as 100/;g of TCA-preparation did not inhibit 1 agglutinating unit of the various hedgehog antisera, indicating that the ss-antigen was not present in the TCA-preparation. In vivo response to the antigen The two guinea-pigs that had been given injections of TCA-preparation intraperitoneally both developed a delayed hypersensitivity reaction when they were tested intracutaneously with the TCA-preparation 4 weeks after the contact with
248
O. Tdnder et al.
the antigen. Before immunization they showed no reaetion. The animals were killed on day 39 and the sera used in inhibition experiments. The untreated and ME-treated sera agglutinated rabbit erythrocytes to titres of 1024 and 128 respectively (preimmunization titres 8 and
