Serum sickness in man. Serologic studies in a case due to rabbit serum Fernando O&z, M.D., Madrid, Spain

A case of swam sickness in man is reported, showing the peculiarity of having been produced by int,adermal injections of a small amount of rabbit serum in an ind- klual who had suffered from a typical serum sickness due to horse serum eighteen years previously. Widespread and repeated urticarial rashes were the most prominent feature of the clinical syndrome. The presence of antibodies in the patient’s serum cw.s studied by precipitin (gel diffusion and immunoelectrophresis), hemagglutina- tion (tanned, antigen-coated human rtd cells and antibody-sensitized sheep red cells),

and passive transfer (P. E.) tests, from samples obtain& before the injeotiwn of foreign serum and until eight months afterward. The nature of the antigens in rabbit swum cawing the immune response in this case and of the antibodies re- sponsible for the symptoms are discussed. The results of the serological tests are compared with those reported in the literature for cases of serum sickness in man following the injection of horse serum.

I n a paper from this laboratory, 1 dealing with the suppression of the rcaginic activity of human atopic sera* by precipitation with a rabbit antihuman beta-2A globulin serum, mention is made of t.he development of serum sickness by sensitization to the foreign proteins contained in mixtures of allergic and rabbit sera in the individual on whose skin P. K. tests were performed. These tests were done in order to measure the reaginic activity, if any, remaining after specific precipitation of beta-2A globulin. A more detailed report of that peculiar case of serum sickness in man stems justified because of the infrequent cause, the circumstances leading to its production (dose of foreign serum, route of injection), and the serologic follow-up which was carried out.

CASE REPORT

For the reasons given in the introduction, a human volunteer (the present author) re- ceived, at one time, six intratlermal injections (three on the anterior aspect of each forearm) of mixtures of human allergic sera with a rabbit antiserum, which had been incubated and centrifuged for 1 hour at 1,565 g at 4” C. in order to remove the precipitate formed during incubation. The over-all amount of rabbit strum received by the patient was about 0.35 ml.

From the Instituto de Investigaciones Medicas (Fundacion Jimenez Diaz) . Received for publicat,ion June 15, 1965. “Rera from three patients sensitive to grass pollens (Lolium perenne, Dactylis glomerata),

and one patient scnsitirc to each of the following: a mold (Tilletia), beef, and hog dandruff.

274

.4 marked inflammatory reaction oc~urretl at the injected sites, \vhi,,lc r,~:r,~1,(~,1 it* t,l;(\i,,,,l,,, after 4 to 6 hours and pcrsistcd with dcereasin g intcrrsity until thrs I’oIIo\\ ing ,l:,~. It ,, :\:, attributed to the likely prcscnc~ of voluble antrgcn-antrbotly cornpl~sw in the raIlbit irrlrnr,,l,x serum, which had been previously mixed with human cord hloocl wrutn (as ,lgw*I,it,etl I,> Ortizl) in order to absorb antibodies to proteins other than beta-2.2 globulin. ‘1’11,~ [,retI:~r,~,l sites were challenged with the corresponding allergens (grass pollens, the 11101~l, l,C!Cf, ;rll,l hog dandruff), by the prick method, three days after sensitization. All of the tests vv(~r(~ negative. They were positive (except for hog dandruff) on paired sites of the skin of the same recipient sensitized with the atopic sera. diluted with saline in the same proportion as tll<sJ were in the mixtures of atopic sera and rabbit anti-beta-2A globulin serum.

Five days after the injection (two days after challenge), a confluent, pruriginous urticarial rash appeared on the anterior aspect of the right forearm and spread in the course of one to two hours to cover almost the entire skin of the extremity. A few hours later, a similar eruption appeared on the upper left limb and, on the following days, urticarial bouts of unequal intensity and extension appeared anywhere on the body surface and disappeared within a few hours, only to reappear at the same or a distant place. Cutaneous rashes were accompanied by general malaise and by mild arthralgia affecting mainly the shoulder and knee joints, without inflammatory signs.

Three days after the onset of symptoms, following a severe attack of urticaria involving the whole skin, treatment was started with antihistamines and small doses of corticosteroids (betamethasone, 1.5 mg. the first day, and decreasing doses over the following days, up to a total of 5.0 me;.). The symptoms thereafter subsided and completely disappeared in a couple of days. The clinical picture can be considered as typical of a generalized serum sickness of mild intensity.

Eighteen years previously the patient had had another full-blown clinical episode of serum sickness (fever, lymphadenopathy, arthritis, urticaria) following treatment with diphtheria horse antitoxin. For professional reasons, he had frequently been in contact with rabbits and their sera. There was no previous or family history of other allergies.

SEROLOGIC STUDIES

Material and methods

Patient’s sera. Blood was withdrawn 6, 13, 2 1, 34, 50, 92, 12 1, 153, 183, and 245 days after the intradermal injection of rabbit scr~m1. The scrims was separated in the usual way and stored at -20’ C. until used. Serurn sarnplcs obtained from the same individual 14 months before and similarly kept frozen at -20° C. wcrc used as controls in the serologic tests.

Patient’s gcmnza &Min. Aliyuots of patient’s serum samples giving positive results in precipitin tests were pooled and centrifuged at 10,200 g for 30 minutes at 20’ C., and the supernatant fat and any deposit formed were discarded. The whole volume of serum obtained in this way (about 11.5 ml.) was dialyzed against 18 per cent anhydrous Na,SO, for 3 hours with continuous magnetic stirring. The contents of the dialysis bag were centrifuged at 10,200 g for 15 minutes at 20’ C., and the clear supernate was discarded. The precipitate was resuspended in 5 ml. of 15 per cent Na,SO,, centrifuged as above, and the supernate was discarded. The precipitate was redissolved in 0.5 ml. of distilled water and dialyzed in the cold, first against distilled water and then against M/150 citrate-phoshate buffer solution, pH 5.8. After the excess water taken during dialysis was removed by pervaporation, it was again dialyzed against phosphate-buffered saline solution, pH 7 .2. Finally, the contents of the bag were centrifuged at 3,500 g for 30 minutes at room temperat,ure, and t,hc

276 0762 .T. Allwgy May, 1%x

small deposit and supcrnatant fat discarded. The final product was stored at 4O C. until its use, a few days later.

Rabbit serunt. It was obtained in the usual manner from blood taken from the marginal veins of the ears of nonimmunizcd, adult healthy rabbits.

Rabbit gamma globulin. Gamma globulin was isolated from normal rabbit serum following a procedure similar to that used for the isolation of gamma globulin from the patient’s serum. All the preparations of rabbit gamma globulin obtained by this method appeared in agar gel electrophoresis to be contamina.ted with a trace of beta globulin and significant amounts of alpha-2 globulin (Fig. 1).

Ynssizge hemagglutination. A suspension of human group 0 red cells, washed, and made up to 2.5 per cent in phosphate-buff’ered saline, pH 7.2, was mixed with an equal volume of a 1 :lOO,OOO solution of tannic acid in the same diluent. After 10 minutes of incubation in the water bath at 37O C., the cells were gently washed once and resuspended in the initial volume of pH 7.2 buffered saline. A volurne of tanned cells was mixed with four volumes of a convenient dilution of the antigen in phosphate-buffered saline, pH 6.4; the mixture was incubated at room temperature for 15 minutes, and the coated cells were washed once in pH 6.4 buffered saline and finally resuspended in the initial volume of the same diluent. Serial twofold dilutions of the sera to be tested were made in pH 6.4 buffered saline, in 0.5 ml. volumes, in Perspex agglutination trays. To each dilution, 0.05 ml. of the tanned, antigen-coated red cell suspension was added. Two controls were included with every series of tests: (a) the strongest concentration of test serum plus tanned, uncoated cells, and (b) pH 6.4 buffered saline plus tanned, antigen-coated cells. The trays were gently shaken for about, a minute, and then left undisturbed at room temperature. The results were read after 2 to 4 hours, by the sedimentation pattern of the cells. Titers were expressed as the reciprocal of the highest dilution of the serum in which the cells were evenly spread over the bottom of the well, without the forrnation of a ring or button. Doubtful results, if present, were considered as negative.

Agar-gel diflusion. Ouchterlony tests were carried out in 6 cm. Petri dishes containing 6 ml. of 1 per cent special agar Noble (Difco), in 0.15M NaCl, with 0.05 per cent sodium azide added to prevent microbial growth during incubation, which was carried out at room temperature in a moist chamber.

dgar-gel electrophoresis. This was carried out on 7.5 by 4.0 cm. glass plates covered with 6 ml. of 1.25 per cent special agar Noble (Difco) in ba.rbital-sodium barbital buffer solution of pH 8.6, and ionic strength 0.03. Electrophoresis was conducted at a potential gradient of 2.5 v. per centimeter for 60-70 minutes. At the end of the run, the plates were fixed in 2 per cent acetic acid, dried overnight in the oven at 37O C. under a sheet of filter pa.per, and stained for proteins with bromphenol blue.

Immunoelectrophoresis. After an electrophoretic run as described above, a longitudinal trough, 2 mm. wide, was cut and filled as indicated under Results.

Rheumatoid tests. A commercial preparation of latex-human F II was used for the latex agglutination tests. Sensitized sheep-cell agglutination tests were

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Fig. 1 Agar-gel clcctrophor~~is of whole rahhit serum (top) and two diflerent preparations of rablrit gamma globulin. Anode on the left.

carried out by sensitizing mashed sheep red cells with a fourth of an agglutinat- ing dose of a commercial preparation of rabbit antisheep hemolysin. Equal volumes of this sensitized sheep cell suspension and of serial dilutions of the serum to be tested were mixed and incubated for one hour in the water bath at 37’ C. and then overnight in the cold. The titers were expressed as the reciprocal of the greatest dilution of the serum giving an agglutination visible with the aid of a hand lens when the tubes were gently shaken to resuspend the cells.

In a modification of this test, sheep erythrocytes, sensitized as indicated above, were washed three tirnes in saline in order to remove rabbit proteins not fixed to the cells. The agglutination reaction was t.hcn performed by adding 0.05 ml. of the washed cell suspension to serial dilutions of the test sera in Perspex trays, similarly to the way described for passive hemagglutination tests.

Passive transfer (P. K.) tests. Skin sites on the anterior aspect of the forearm of a healthy human volunteer were sensitized by the intradermal injection of 0.1 ml. of the undiluted serum. Two days afterward, the sites were challenged by pricking with a sharp needle through a drop of the antigen (undiluted rabbit serum). Sn unsensitized site of skin of the same individual was tested in the same way. R.cadings of the wheal and flare reactions were made 20 to 30 minutes after challenge.

Direct cutaneous tests on the patient’s skin were also carried out by the prick method through a drop of the antigen (undiluted rabbit serum).

Results

Precipitin tests. Ouchterlon~ plates were set up by filling a cchntral ~~~11 with normal rabbit serum and placing the several samples of patient’s serum in peripheral wells. The formation of at least a line of precipitate could bc serll with sera taken between 6 and 50 days after the injection of foreign scrmn, the density of the precipitin lines bein, 0‘ maximal with the scrurn taken on the thirteenth day. No prclcipitation occur& with SCYIII~ taken bcforcl the irljtction or with scra taken 92 days or more after it.

In order better to assess the nunibt~r 01’ lines obtainrd by this mt~thotl at1(1 the relations bctwcen them, gamma globulin, isolated and concentrated from those scra which had given ;I positive result, in the preceding test, was matl~ to diffuse in another Ouchterlony plate against two separate> antigc>ns : whol(~ ral)l)it

378 ortiz J. Allrry,y IMay, 1966

serum and rabbit gamma globulin which, although relatively purified, contained an appreciable amount of alpha-2 globulin, as indicated in Methods. Three lines of precipitation were formed with whole rabbit serum and two with rabbit gamma globulin (Fig. 2). The main line obtained with both antigens clearly showed an identity type of reaction. The other line formed with rabbit gamma globulin as antigen did not appear to be related to any of the two additional lines formed with whole rabbit serum. It is not unlikely, however, that by varying the quantitative relationships of t.he reagents it might have been seen to fuse with the line nearest to the whole rabbit serum reservoir; this type of test could not be done, because of the small amount of patient’s gamma globulin available.

To get a deeper insight into the nature of the rabbit serum antigens involved in this reaction, whole rabbit serum and rabbit gamma globulin were analyzed by immunoelectrophoresis using the patient’s gamma globulin as a source of antibody in the longitudinal trough. Two precipitin lines appeared with whole rabbit serum, corresponding in electrophoretic mobility to the albumin and alpha-2 fractions of an electrophoretic separation of rabbit serum proteins run simul- taneously in the same agar plate. No precipitin line was present on the rabbit gamma globulin side, even after 10 days of incubation. Thus immunoelectropho- resis proved to be less sensitive than double two-dimensional diffusion in agar gel for the detection of the antibodies present in the patient’s serum, under the experimental conditions used.

Passive hemagglutination. The hemagglutination titers of the several samples of patient’s serum for tanned cells coated with whole rabbit serum (diluted 1 :lOO) and for tanned cells coated with rabbit gamma globulin (diluted 1500) are shown in Table I.

The same results have been represented by a graph in Fig. 3. Both CLWVCS

show a certain parallelism, with the following differences. The antibodies detected by means of tanned red cells coated with whole ra.bbit serum had a low but measurable titer before immunization. A decrease was seen 6 days after the injection, followed by a sharp increase; maximal levels wcrc maintained between

WRS RGG

0 0

the thirteenth and thirty-fourth days, and a slow decline ensued, initial lcvc~ls being reached 5 months after the antigen injection.

The antibodies detected by the agglutination of tanned red ~11s coated with rabbit gamma globulin, on the other hand, showed a marked increase by the sixth day and maintained maximal levels only between 13 and 21 days after stimulation. The decrease was somewhat quicker than in the preceding casc~,

uoys after injection

Preimmunieation 6

13 21 34 50 92

121 153 183 245

I Hemagglutinatio~n titers for

Whole rabbit serzlm 1 Rabbit gamma, globudin .-

40 Less than 10 10 640

2,560 2,560 2,560 2,560 2,560 1,280

640 640 320 160 320 40

40 20 80 “0 40 “0

. . . Ab to whole rabbit serum

280 07’tiz J. Allergy May, 1966

and a low but detectable titer remained at the end, as opposed to the complete absence of such antibodies before the injection of foreign serum.

IZheumatoid tests. In order to ascertain the role that rabbit gamma globulin itself could have played as an antigen in these reactions, rheumatoid tests were done with some of the patient’s serum samples. Both the latex and the sensitized sheep cell agglutination tests were negative with serum taken 21 days after the injection, at a time when the precipitin reaction was markedly positive, and passive hemagglutination titer was at its maximum with both antigens used. The negative result of the former was to be expected, as the antigen which sensitized latex particles was human, not rabbit gamma globulin; as for the negative results of the latter, it could be explained by neutralization of the antibodies in the patient’s serum by rabbit gamma globulin remaining free in t,he mixture of sheep red cells and rabbit antiserum. For that reason, the test was repeated using the modification described in Methods, consisting of the t,horough washing of sheep red cells after sensitization with rabbit antibody. The test carried out in this fashion with the serum of the patient taken 3 months after the injection, when hemagglutination titers were still fairly high, was also negative.

Xl&n tests. Direct skin test carried out with rabbit serum on the day following the onset of symptoms, that is to say, 6 days after the injection of foreign serum, caused the appea,rance of an enormous wheal (15 by 6 cm.), surrounded by a large flare. Direct skin tests on the patient were not repeated, so that no changes would be made in the shape of the antibody curves.

The passive transfer (P. K.) test with serum taken 6 days after the injection, challenged with whole rabbit serum, gave a slightly positive reaction. Further I’. K. tests were not done.

DISCUSSION

Clinical as well as serologic data allow the present case to be considered as one of true generalized serum sickness. Ratner2 reported the observation by various workers of a phenomenon to which the name of local serum sickness was given, occasionally due to the injection of small amount,s of rabbit serum by the intradermal route. In such cases, urticaria and erythema were the prominent features of the clinical syndrome, but, at variance with what was seen in the present case, the recurring urticarial bouts remained confined to the extremity where the injection was made and, although they could reach areas of the limb distant from the injection point, they failed to involve the whole skin. Another differential point in the cases referred by Ratner would be the failure to demonstrate prccipitins in the sera of the patients, although this cannot bc given much weight, in view of the relative inefficiency of methods used at t>hat time, as compared to present methods to detect precipitins. The loca: character of such cases is attributed to the small dose of antigen and t,o thr peculiar routr used for its injection. The prcscnt, case, mild in character and with urticarial rashes as the main symptom, can be in some way considered as intermediate betmccn those examples of local serum sickness and the typical

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generalized cases with full-blown clinical picture, and its miltlness may wcxll bc related to the small amount of antigen which caused it.

The production of serum sickness with such small doses of antigen is by no means frequent. According to Arbrsman, Kantor, Rose, and Wit&sky,” tho incidence of serum sickness is proportional to the amount of foreign serum injected. Cell4 also calls attention to the fact that serum sickness in man does not result from minor immunizations. It is also uncommon that rabbit strum he the cause of the disease, as it is now rarely used for prophylactic or thcrapentic lmrposes, or that the route of injection be intradermal, except in the above cited cases of local serum sickness.

The latent period between antigen injection and onset of symptoms was in this case markedly shorter than usual. Although the length of the la,tent period varies according to the antigen causing the disease and, at least in experimental serum sickness in the rabbit, is shorter when the antigen is gamma globulin than when it is albumini in the more common cases of human serum sickness from horse serum the symptoms usually do not appear until 10 to 14 days after injection.” In the present ease, the early start of symptoms must be related to the fact that the patient had had a typical serum sickness several years before, caused by the therapeutic injection of horse antitoxin. A certain degree of cross antigenicity exists between horse and rabbit serum proteins,” so that the strum of a patient with a high titer (512,000) of hemagglutinating antibodies to horse serum may show at the same time a moderate titer (1,600) for rabbit serum. It is then logical to suppose that the a.ntibody response in this case was of the secondary or anamnestic type, and therefore accelerated, the patient having been primed by the previous immunization with horse serum. This would explain not only the shorter latent interval, but also the production of serum sickness with doses usually insufficient in this respect. Reisman, R.ose, and Arbesman” reported a case of serum sickness from bovine antitoxin in an individual who, 18 years before, had had another episode of serum sickness of eyuinc etiology; the latent period in this cast was very short, 4 days, and cross-reactivity between sera of both species was shown by an increase, albeit slight and transitory, of the titer of antibody to horse serum proteins during the second at.tack, cansctl

by bovine serum. In the case here reported, no data were available concerning antibody levels to horse serum during the first serum sickness, neither were their possible fluctuations during the second one studied.

Changes in two kinds of antibody, precipitating and hemagglutinating, were followed for several months. As is known for other antigen-antibody systems and has also been observed in cases of serum sickness in man,” precipitin tests turned out to be far less sensitive than hemagglutination, the former being positive only with serum samples giving hemagglutination titers of 640 or more. As far as the hemagglutination test is concerned, it is remarkable that the titer of the serum taken the day following the onset of symptoms was nlnch higher for rabbit gamma globulin than for whole rabbit serum, despite the fact that all the antigens present in the gamma globulin preparation had to bc contained also in the latter. Such discrepancy can be explained by assuming

282 ortiz J. Allergy May, 1966

that, in whole serum, scvcwl prvtcins cornpcte with each other for their fisation to the surfacc~ of tanned rctl ~~~11s. The l)rciinmnnizatioll titer found with wl~olc rabbit scrurn could rcflwt, prcvions scbnsitization to horse serum, and bc due to the cxist.ence of cross-reactivity between rabbit and horse sera, as pointed out above. The longer duration of hemagglutination titers with whole rabbit serum, as opposed to that with the gamma globulin preparation used, must be attributed to antibodies reacting with antigens present in the former and absent in the latter.

Hemagglutination titers are in the present case much lower than those reported in the literature for similar cases of sernm sickness of different causes. Reisrnan, Rose, Witebsky, and Arbesman’ found titers higher than 10,000 in all their cases of equine serum sickness, and in almost all of them the titers before the injection of foreign strum were higher than 200. The lower titers found in the present ca,sc can bc attributed to the different etiology, the mildness of the attack, or to minor differences in the technique used for the detection of antibodies by hernagglutination. In our case, twenty-five fold changes in the concentration of the antigens used for coating tanned cells had no effect on the serum titers. Although initial as well as maximal levels of hemagglutinating antibody were lower, the increase experienced by the antibodies in relation to their initial titer was of the same order of magnitude as reported by others. Furthermore, the evolution of the disease in relation to antibody production was as usually described in serum sickness, symptoms beginning in the initial phase of the antibody response and subsiding before the maximal level was reached. The duration of the response can also be considered as normal for the amount of antigen injected. It is very unlikely that the antibody response could have been in any way affected by the smdl doses of steroids given after antigenic stimulation, at the acme of clinical symptoms.

Something can be conjectured about the antigens of rabbit serum which acted as antigenic stimuli. These were at least three, as judged from the results of agar precipitin tests, using the concentrate of gamma globulin isolated from the patient’s serum. The main line of precipitation must have been due to an alpha globulin of rabbit serum, as this protein was also present in significant amounts in the rabbit gamma globulin preparations used. It is not likely that gamma globulin had had the predominant role, as the agglutination tests with sheep cells sensitized with rabbit antibody were consistently negative. On the other hand, rabbit gamma globulin might be responsible for the weak line far distant from the rabbit gamma globulin reservior, whose relationship to the weak line nearest the whole rabbit serum reservoir could not be assessed. Could the modified sensitized sheep cell agglutination reaction have been also done with sera showing maximum antibody titers, perhaps some positive results would have been obtained; antibody to rabbit gamma globulin might have been present at low titer and only for a short time. Finally, the precipitin line corresponding to rabbit serum albumin as seen in immunoelectrophoresis must be the counter- part of the third line seen in the agar plates between whole rabbit serum and the patient’s gamma globulin; this is supported by the great distance of this line from the antigen reservoir and the lack of a similar line of precipit,ation

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between the pat,ient’s gamma globulin and t,he rabbit gamma global in prepara- tion, which, as shown by clcctrophoresis, was essentially free of albumin. The above intcrprctations arc consistent with data in the literature concerning tht antigens in foreign sera active in human serum sickness,“, *, !’ some of which underline the predominant role of alpha globulin.

It is not possible to reach any conclusion about which of these several antigen-antibody systems was responsible for the patient’s symptoms. The possible pathogenic role of skin sensitizing antibodies, as pointed out by Rose,5 should not be forgotten, especially in cases such as the present one, in which the symptoms were, for the most part, easily attributable to such a class of reaginic antibodies which, on the other hand, could not be detected by the in vitro tests used. Although passive transfer (P. K.) tests, the only ones capable of detecting skin sensitizing antibodies, gave only slightly positive results in the present case, the direct cutaneous test was strongly positive and had an immediate and evanescent character which would prevent it from being interpreted as an Arthus-type reaction, caused by precipitating antibody present at the same time. Such dissociation between the results of direct skin tests and those of passive transfer tests in human serum sickness has also been noticed by R,eisman, Rose, Witebsky, and Arbesman,7 and can be readily explained if the skin-sensitizing antibody present in the serum is either the small amount of newly synthesized antibody in transit from the antibody-forming cells to the tissues to which it becomes fixed, or the excess remaining free in the circulation after all the tissues able to fix that antibody have been “saturated” with it.

REFERENCES

1.

2.

3.

4.

5.

Ortiz, F.: The Specific Precipitation of Reagins With an Anti-Beta-2A Globulin Serum, Acta Allergol. In press. Ratner, B.: Allergy, Anaphylaxis and Immunotherapy, Baltimore, 1943, The Williams & Wilkink Company;h: 423.- -

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Arbesman, C. E., Kantor, S. Z., Rose, N. R,., and Witebsky, E.: Serum Sickness. Serologic Studies Following Prophylactic Tetanus Antitoxin, J. ALLERGY 31: 257, 1960. Gell, P. G. H. : Serum Sickness, in Gell, P. G. H., and Coombs, R. R. A., editors: Clinical Aspects of Tmmunology, Oxford, 1962, Blackwell Scientific Publications, p. 382. Rose, N.: Discussion, ill. Grahar, P., and Miescher, P., editors: Mechanism of Cell and Tissue Damage Produced by lmmune Reactions, Base1 and Stuttgart, 1962, Benno Schmabe & co., p. 92. Reisman, R. E., Rose, N. R., and Arbesman, C. E.: Immunologic Studies of Serum Sick- ness From Bovine Anti-Tetanus Toxin, J. A. M. A. 176: 1004, 1961. Reisman, R. E., Rose, N. R., Witebsky, E., a.nd Arbesman, C. E.: Serum Sickness. II. Demonstrat,ion and Characteristics of Ant,ibodies, J. ALLERGY 32: 531, 1961. Rose, N. R., Reisman, R. E., Witebsky, E., and Arbesman, C. E.: Serum Sickness. III. Characterization of Antigens, J. ALLERGY 33: 250, 1962. Barnett, E. V., Stone, G., Swmher, S. N., and Vaughan, J. II.: Serum Sickness and Plasma- cytosis. A Clinical, Immunologic and Hematologic Analysis, Am. J. Med. 35: 113, 1963.