JOURNAL oF BACTERIOLOGY, Jan., 1966Copyright © 1966 American Society for Microbiology

Vol. 91, No. 1Printed in U.S.A.

Patterns of Human Antibody Reactions inCoccidioidomycosis'

Y. SAWAKI, M. HUPPERT, J. W. BAILEY, AND Y. YAGI

Mycology Research Laboratory, Veterants Administrationz Hospital, Sani Fernanzdo, Californiia, and Departmelntof Biochemistry Research, Roswell Park Memorial Institute, New York State Department of Health,

Buffalo, New York

Received for publication 7 September 1965

ABSTRACT

SAWAKI, Y. (Veterans Administration Hospital, San Fernando, Calif.), M.HUPPERT, J. W. BAILEY, AND Y. YAGI. Patterns of human antibody reactions incoccidioidomycosis. J. Bacteriol. 91:422-427. 1966.-At least two antigen-antibodysystems in coccidioidomycosis have been demonstrated by immunodiffusion andimmunoelectrophoresis, including the use of Il3t-coccidioidin. Sera which were posi-tive in the conventional complement-fixation (CF) test, but negative in the tubeprecipitin (TP) test, yielded only one line of precipitate in the immunodiffusiontest. In immunoelectrophoresis, also, there was only one precipitate arc which ap-

peared in the -y2-globulin region. On the other hand, sera which were positive inboth CF and TP tests yielded two different lines in the immunodiffusion t, st. Inimmunoelectrophoresis, also, there were two precipitate arcs: one in the -y,-globulinregion and the other in the 'yl-globulin area. Radioimmunoelectrophoresis demon-strated that the antibody activity in IG-G globulin, roughly parallels the CF titer.A similar correlation existed between the IG-M antibody activity and the TPreactivity. Antibody activity was present in the IG-A globulin but it did not reflecteither CF or TP activity.

Serological studies are important diagnosticand prognostic aids in coccidioidomycosis. Cur-rently, the tube precipitin (TP) test and the com-plement-fixation (CF) test are employed, andthese are highly specific. Smith and his colleagueshave demonstrated a relationship between theclinical stages and serological patterns (7, 8).Resolution of the various antigen-antibody sys-tems involved in coccidioidomycosis was facili-tated with the application of immunodiffusiontechniques. The results suggested that this pro-cedure is useful as a screening test for the disease(3). Huppert and Bailey (4) demonstrated the suc-cessful separation of the antigens involved in theCF and TP test for coccidioidomycosis. These twoantigen-antibody systems were detected as dis-tinctly different in the agar-gel immunodiffusiontest.The work reported here is concerned with the

identification, by means of immunoelectrophore-I This research was supported in part by the

Tuberculosis and Health Association of Kern County,Calif., and by the Tuberculosis and Health Associa-tion of California. Part of this work was performedat the California College of Medicine, Los Angeles.

sis and radioimmunoelectrophoresis, of antibodycomponents in patients' sera which are involvedin these reactions.

MATERIALS AND METHODS

Human sera. Blood specimens were obtained fromfasted patients with a confirmed diagnosis of cocci-dioidomycosis. After separation of the serum, aqueousMerthiolate was added to a final concentration of0.01%, and the sera were stored at 5 C until used.

Antisera againist humant serum components. Goat orrabbit antiserum for various protein fractions ofhuman serum was purchased from Hyland Labora-tories, Los Angeles, Calif. Antiserum specific tohuman IG-G, IG-A, or IG-M showed only a singleprecipitate arc in immunoelectrophoresis when testedwith normal or patients' sera (IG-G for yG-globulin,7S y-globulin; IG-A for yA-globulin, YIA-globulin,32A-globulin; IG-M for yM-globulin, ytM-globulin,32m-globulin).CF and TP tests. CF and TP tests were performed

according to the methods described by Smith et al.(7). For the CF test, the procedure of overnight fixa-tion at 5 C was used.

Antigen from Coccidioides immitis. Antigens weremanufactured by a modification (4) of the toluenelysis technique of Pappagianis (6). Briefly, this con-

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sisted of growing 24 strains of C. immitis separately ina 2%7o glucose and 1% yeast extract dialysate broth ona rotary shaking machine at 30 to 33 C. The fungusgrowth was separated by Buchner filtration. Merthio-late was added to the filtrate to a final concentrationof 0.01%cc, and then was sterilized by filtration througha Selas 02 candle. The sterilized filtrate was concen-trated by ultrafiltration behind a 6%o Parlodion mem-brane to approximately one-sixteenth of the originalvolume. This concentrated antigen was standardizedfor optimal activity against known positive humansera.

lodin1ationi of anitigeni with I131. odination of theantigen (coccidioidin) was carried out by a modifica-tion of the method described by Hunter and Green-wood (2). A 0.2-ml amount of concentrated coccidioi-din (16 times), containing 210 jig of protein (expressedas rabbit a-globulin by Lowry's reaction) was mixedwith 0.05 ml of I'3l (1.9 mc), 0.05 ml of 5 X 10-4 MKI, and 0.10 ml of Chloramine-T (141 j,g) at pH 8.0.After 5 min at room temperature, 0.10 ml of NaHSO3(240 jAg) was added to reduce any remaining iodineand oxidant. A 0.1-ml amount of KI (1 mg) wasadded after a few minutes, and the mixture (0.6 ml)was passed through a column of Sephadex G-25(2.5 g of fine bead form, 1.1 by 11 cm). All the abovesolutions were made with borate buffer (pH 8.0), andthe column was equilibrated with the same buffer. Theportions corresponding to the peak of protein (elu-tion volume, 5.5 to 9.5 ml) were pooled and used asI'31-coccidioidin. About 380 jic of I131 was found inthis fraction. A preliminary experiment with the samecolumn indicated that the separation of the cocci-dioidin and I131-iodide was almost complete, and theportion collected above contained about 57% of theapplied material based on optical density at 280 my.

Immunodiffusion. The immunodiffusion procedurewas a slight modification of the method described byHuppert and Bailey (3). A 1.5% lonagar No. 2 solu-tion was prepared in saline containing 0.067 M Soren-sen phosphate buffer and with 0.01%o aqueous Mer-thiolate as preservative. Final pH was 8.2. Plastic petridishes (12 by 50 mm) were charged with 5 ml ofmolten agar and allowed to gel. A seven-well patternwas cut out of the agar, consisting of a center wellsurrounded by six equally spaced peripheral wells. Thedimensions were as follows: center well, 4.5 mm indiameter; peripheral wells, 3 mm in diameter; wall-to-wall distance between center and peripheral wells,5 mm. The wells were numbered from 1 to 6 in aclockwise direction, and, routinely, a known positivecontrol serum was placed in wells 1 and 4. The serato be tested were placed in wells 2, 3, 5, and 6. In thismanner, any reactions of the unknown sera could becompared with the control positive sera for reactionsof identity, partial identity, or nonidentity.

Reverse immunoelectrophoresis. Immunoelectropho-resis was performed with an LKB 6800 A instrument.lonagar No. 2 (1%) in Veronal buffer (pH 8.2) wasused on microscope slides as a layer 2 mm thick. Acentral well for the sample to be tested was 1.5 mm indiameter (about 0.004 ml capacity). Electrophoresiswas done at 225 v for 1 hr at room temperature, andthen two parallel troughs were made at a distance of

3.1 mm from the central well on each side. One troughwas charged with antiserum against a fraction of hu-man serum, and the other contained antigen fromC. immitis. Diffusion occurred overnight in a moistchamber at 23 C.

Radioimmunioelectrophoresis. I13l-coccidioidin wasused either for forming precipitate arcs directly withpatients' sera or for identifying the arcs of humanantibody components produced by antiserum againsthuman globulins. In both cases, the pattern differedby having a single trough down the center separatingtwo wells to allow comparison of two serum speci-mens simultaneously.

In the direct precipitation procedure, the I'll-labeledcoccidioidin, diluted in 10%70 normal rabbit serum(carrier), was used in the trough. The final concentra-tion of I'3l-coccidioidin as protein was 5 ,ug/ml. Afterelectrophoresis of the serum specimen and overnightdiffusion with antigen, the slides were washed, dried,and stained with 0.5% Amidoschwartz in methanol-water-acetic acid (5:5: 1). The dry stained slides wereused for radioautography with Kodak Industrial TypeKK X-ray film.

For identification of antibody components, a two-step procedure, similar to that used previously forragweed-sensitive patients' sera (9), was used. Afterelectrophoresis of the serum specimen, the trough wasfilled with appropriate antihuman serum. After over-night diffusion, the slides were washed for 30 hr with

FIG. 1. (upper) Typical immuniodiffusion results withsera which were complement-fixation positive but tubeprecipitin negative. (lower) Characteristic immuno-electrophoresis result with sera which were comple-ment-fixation positive but tube precipitin negative. Uppertrough contained coccidioidin, lower trough aniti-IG-Gserum, and ceniter well the patient's serum.

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SAWAKI ET AL. J. BACTERIOL.

RESULTS

Direct precipitation ofcoccidioidin with patients'sera in agar layer. Coccidioidin and patients' serawere reacted in agar by Ouchterlony's immuno-diffusion test and by reverse immunoelectro-phoresis. Patients' sera formed specific precipi-tate arcs with coccidioidin in both cases. However,there was a distinct difference between the serawhich differed in the TP test regardless of theirCF titer. TP-negative CF-positive sera showed asingle arc of precipitate in immunodiffusion. In

TABLE 1. Serological and radioimmunoelectro-phoresis (direct precipitation) reactions ofsome representative sera from patients

with coccidioidomycosis (see alsoFig. 3)

FIG. 2. (upper) Typical immunodiffusion results withsera which were positive by both complement-fixationand tube precipitin tests (wells 2, 3, and 5). (lower)Characteristic immunoelectrophoresis pattern with serawhich were positive by both complement-fixation andtube precipitin tests. Upper trough contained coccidioi-din (note two arcs of precipitate), lower trough anti-IG-M serum, and center well the patient's serum.

Radioimmunoelectro-

Patient u .. Tube phoresisC3a precipitinOt 'Yi 72

B. J. P. 1:64 + ++ +V. S. 1:16 4i ++M. E. 1:256 +++L. J. 1:32 +++A. C. 1:4 _ 4± 4

*:......,, +

* Longbut withregion.

continuous arc covering bOth regionsmaximal intensity in the 72-globulin

a i 3#S t,,w , .... : . ... . , .,W, .. ...,s, .... ' t. ' ,,,.:.:,: £F. . , '.iss'.,: .e. X t.... ;. x.,.,s ..... ass XM. . . s, . s.... ..... .- S,

*:::: : . s ..* :.i s : , : ,,* i; W: .n. .s'<X':'S l:: q

5. :<it [ a

..

.,FIG. 3. Precipitate arcs revealed by radioautography

after immunoelectrophoresis. Direct precipitation tech-nique was used. Normal serum (negative control) is onthe extreme right. The remaining sera were comple-ment-fixation positive. The first and third specimens(from the left side) were also tube precipitin positive.

frequent changes of buffered saline (pH 8.0) to removesoluble proteins. Then, the trough was filled with amixture of 131-coccidioidin and normal goat or rabbitserum (5 ,ug of antigen per ml of serum). After diffu-sion overnight, slides were washed, dried, stained withAmidoschwartz, and used for radioautography asabove.

FIG. 4. Representative specimens demonstrating thatantibody activity is present in all the immunoglobulins.The two-step radioimmunoelectrophoresis procedurewas used, and the above reproductions were made fromthe radioautographs. The serum in the left well ofeachslide had a complement-fixation titer of 1:32 and wastube precipitin positive. The corresponding reactions forthe serum in the right well of each slide were 1:64 andnegative. (a) Anti-IG-G. (b) Anti-IG-A. (c) Anti-IG-M.

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ANTIBODY PATTERNS IN COCCIDIOIDOMYCOSIS 425

TABLE 2. Clinical and immunological status of three representative cases of coccidioidomycosis*

Antibody component

++

+

Clinical status

On admission: pneumonic infil-trate and pleural effusion.Gradual improvement.

History: erythema nodosum andpleural effusion for 1 month.Residual nodular lesions bi-lateral.

Treated with amphotericin B andimproved. Therapy stoppedand clinical condition de-teriorated. Continues un-

stable.

* The antibody components were determined by radioimmunoelectrophoresis with the use of the two-step method.t CF = complement-fixation titer; TP = tube precipitin test; ID = immunodiffusion test; (2) = two arcs of precipitate.

TABLE 3. Relationship between antibody components and complement fixation (CF) test or tubeprecipitin (TP) test*

Relative antibody activity in eachi IGt

Determination IG-G IG-M IG-A

_ ++ +++ - + ++ - + ±++CF titert

2-8......................... 0 3 1 0 2 1 1 1 3 016-32......................... 0 0 4 3 3 0 4 1 5 164-256.. 0 0 0 5 2 2 1 0 4 1

TP testtINegative ....................... 0 3 0 9 7 4 1 3 9 0Positive ........................ 0 1 5 2 2 1 5 1 6 1

* The number of sera belonging to each category is listed.t Antibody activity in each immunoglobulin was estimated by comparing the radioactivity of the

corresponding arc by radioautography. The activity in each component may be compared betweendifferent sera. However, the comparison of the activity between antibodies of different classes is notvalid.

$ Twenty specimens shown for TP test include 16 specimens assayed for CF titer. The titer rangeindicated is expressed as the reciprocal.

immunoelectrophoresis, a single arc was seen inthe 72-globulin region (Fig. 1). TP-positive CF-positive sera in most cases showed two precipitatearcs in immunodiffusion. In immunoelectro-phoresis, two independent arcs were seen, one

short arc in the yl-globulin region and the otherarc extending from 71- to 'Y2-globulin region (Fig.2). The presence of the two arcs indicates thattwo independent antigens and the correspondingantibodies are involved in these cases.

Similar results could be obtained by the use ofI'3l-labeled coccidioidin in immunoelectrophore-

sis. Precipitate arcs were seen more clearly on

the radioautographs (Fig. 3). TP-negative sera

showed a long single arc with maximal intensityin the 7Y2-globulin region. TP-positive sera showeda short characteristic arc in the yl-globulin regionalong with, or without, another arc in the 72-

globulin region. The data on some representativepatients' sera are summarized in Table 1.

Identification of antibody components in pa-tients' sera. Antibody active components in pa-tients' sera were identified by radio immuno-electrophoresis. Antisera specific to each of the

VOL. 91, 1966

Serological testt

Patient

B. J. P.

L. J.

M. E.

TP

+++

IG-A IG-MDiagnosis

Primarypulmonarycoccidioido-mycosis

Chronic pul-monary cocci-dioidomycosis

Disseminatedcoccidioido-mycosis

.0_

0

Eo

25

81012

4101220

CF

1:321:641:2

1:641:641:32

1:2561:161:16Trace

ID

+ (2)+ (2)+

+ (2)

IG-G

++

++

++

+++

++

+++

+

+

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SAWAKI ET AL.

known immunoglobulins, IG-G, IG-A, andIG-M, were used to form immune precipitate arcsafter electrophoresis of each patient's serum. Theprecipitate arcs were stained with I'3l-labeledcoccidioidin (for antibody activity) and dye (forprotein). The antibody activity in each componentwas estimated by the density of arcs revealed onthe radioautographs.The radioautographs showed that the antibody

activity is present in all the immunoglobulins(Fig. 4). The data are summarized in Table 2. Allthe sera contained antibody of the IG-G class.The antibodies of IG-A and IG-M class werefound in some of the sera. Studies with antiserumagainst human globulins indicated that only thearcs of immunoglobulins fix I'3l-labeled coccidi-oidin. A number of other arcs seen on the stainedslides failed to fix the antigen, indicating thelack of antibody activity in serum componentsother than immunoglobulins.

Table 3 shows the relationship between the anti-body activity in each immunoglobulin and the CFtest or the TP test on a larger number of sera. Theresults indicate that the titer roughly parallels theactivity of IG-G class antibody. Furthermore, itwas noted that most sera containing significantamounts of IG-M class antibody were positive inthe TP test and showed a distinct direct precipi-tation arc in the yl-globulin region in immuno-electrophoresis and two precipitate arcs in im-munodiffusion (Table 2). These observationswould indicate that the IG-M antibody is re-sponsible for the positive TP test and that theIG-G antibody is responsible for the positive CFtest.

DIscussIoN

Resolution of various antigen-antibody systemsinvolved in coccidioidomycosis has been demon-strated. As in other diseases (1, 9, 10), antibodyactivity appears in at least three components ofpatients' sera: the IG-A, IG-M, and IG-G globu-lins. This was demonstrated clearly with theradioimmunoelectrophoresis technique.The sera used in these studies were obtained

from patients in various clinical stages of coc-cidioidomycosis; primary infection, chronic, anddisseminated stages. Antibody of IG-G class waspresent in all cases as demonstrated by radio-immunoelectrophoresis. Antibody of IG-A classwas found also, but the presence and activity ofthis component were more variable and lessmarked than those of the IG-G antibody. Theantibody of IG-M class was found most clearly inserum specimens which yielded a positive TP test.In coccidioidomycosis, the latter test is positiveonly in the early stage and after exacerbation ofpre-existing disease.

Immunodiffusion and immunoelectrophoresisindicated the presence of two independent systemsof antigens and corresponding antibodies, asrepresented by two independent precipitate arcs.The antigen responsible for the TP test seems tobe associated mainly with the antibody of IG-Mclass. Sera containing higher amounts of IG-Mantibody showed positive results in the TPtest. A short characteristic arc of precipitate inthe 'y-globulin region, observed by direct pre-cipitation reaction of coccidioidin and serum(after electrophoresis), seems to represent thisantigen-antibody system. Two of the sera gavepositive results in the TP test, but did not containIG-M antibody (Table 3). IG-G antibody in thesesera may have been directed against the aboveantigen.On the other hand, some sera of high CF titer

containing no IG-M antibody (e.g., L. J. inTables 1 and 2) were negative in the TP test, inspite of strong antibody activity in IG-G globulin.These sera gave a single arc of direct precipita-tion in the Y2-globulin region upon reverse im-munoelectrophoresis. This arc is probably due toanother antigen which hardly precipitates withthe antibody in the test tube. The antibody againstthis antigen appears to be mainly IG-G globulin.Since the CF titer roughly parallels the antibodyactivity in IG-G globulin, regardless of the resultsin the TP test, this antigen-antibody system seemsto be mainly responsible for the fixation of com-plement. These findings are similar to those re-ported recently by Pappagianis et al. (5).The successful separation and identification of

the C. immitis antigens, corresponding to the ac-tivity in CF and TP tests, has made it possible tostandardize each antigen for optimal activity andto use the antigens during the course of the dis-ease in humans (4). Further studies on the rela-tion of clinical status and serum antibodies are inprogress. In this regard, the disseminated case ofcoccidioidomycosis (patient M.E.) is interesting(Table 2). The 12-month specimen from this pa-tient yielded a positive CF test but a negative TPtest. The immunodiffusion and immunoelectro-phoresis tests on the same specimen producedprecipitate arcs indicating the presence not onlyof IG-G antibody, but also of IG-M antibody,which seems to be the reactive serum componentin the TP test. It has been our experience that theTP test is less sensitive than the immunodiffusiontest and the immunoelectrophoresis test withI3l-coccidioidin. Furthermore, this patient hadbeen started on amphotericin B therapy at thetime of the 10-month specimen. He showed anexcellent clinical improvement, and the therapywas stopped shortly before the 12-month speci-men was obtained. Thereafter, his clinical status

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ANTIBODY PATTERNS IN COCCIDIOIDOMYCOSIS

deteriorated, but his CF titer continued to anessentially negative reaction in 20 months. Subse-quent specimens from this patient have shown areturn of the positive CF reaction to a titer of1: 16. Additional studies on patients not reportedhere appear to indicate that the immunodiffusionand immunoelectrophoresis results reflect theclinical condition of the patient more accuratelythan does the CF test, as has been illustrated inthe case of patient M. E.The recognition of at least two of the C. immitis

antigens involved in known serological reactionsof coccidioidomycosis offers the opportunity tostudy the changing patterns of immunologicallyactive serum proteins as they occur during thecourse of the disease. Isolation and study of addi-tional antigens may reflect even more accuratelywhether a patient's clinical condition will improveor deteriorate. Such information would serve asan objective guide in the clinical management ofcases of coccidioidomycosis.

ACKNOWLEDGMENTThe senior author wishes to acknowledge the helpful

advice and cooperation of Ross A. Hampson in ob-taining serial specimens from selected patients.

LITERATURE CITED

1. CHAPMAN, J. S., J. BAUM, AND M. SPEIGHT. 1964.19S and 7S antibodies in the sera of patientswith sarcoidosis. Am. Rev. Respirat. Diseases90:309.

2. HUNTER, W. M., AND F. C. GREENWOOD. 1962.Preparation of iodine131-labeled human growthhormone of high specific activity. Nature 194:495-496.

3. HUPPERT, M., AND J. W. BAILEY. 1963. Immuno-diffusion as a screening test for coccidioido-mycosis serology. Sabouraudia 2:284-291.

4. HUPPERT, M., AND J. W. BAILEY. 1965. The useof immunodiffusion tests in coccidioidomy-cosis. II. An immunodiffusion test as a substi-tute for the tube precipin test. Am. J. Clin.Pathol. 44:371-375.

5. PAPPAGIANIS, D., N. J. LINDSEY, C. E. SMITH,AND M. T. SAITO. 1965. Antibodies in humancoccidioidomycosis: immunoelectrophoreticproperties. Proc. Soc. Exptl. Biol. Med. 118:118-122.

6. PAPPAGIANIS, D., C. E. SMITH, G. S. KOBAYASHI,AND M. T. SAITO. 1961. Studies of antigensfrom young mycelia of Coccidioides immitis.J. Infect. Diseases 108:35-44.

7. SMITH, C. E., M. T. SAITO, R. R. BEARD, R. McF.KEPP, R. W. CLARK, AND B. U. EDDIE. 1950.Serological tests in the diagnosis and prog-nosis of coccidioidomycosis. Am. J. Hyg.52:1-21.

8. SMITH, C. E., M. T. SAITO, AND S. S. SIMONS.1956. Pattern of 39,500 serologic tests in cocci-dioidomycosis. J. Am. Med. Assoc. 160:546-552.

9. YAGI, Y., P. MAIER, D. PRESSMAN, C. E. ARBES-MAN, AND R. E. REISMAN. 1963. The presenceof ragweed-binding antibodies in the beta-2A,beta-2M, and gamma-globulins of the sensitiveindividuals. J. Immunol. 91:83-89.

10. YAGI, Y., P. MAIER, D. PRESSMAN, C. E. ARBES-MAN, R. E. REISMAN, AND A. R. LENNOR. 1963.Multiplicity of insulin-binding antibodies inhuman sera. Presence of antibody activity inGamma-, beta-2A and beta-2M-globulins. J.Immunol. 90:760-769.

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