Shared allergenic and antigenic determinants in Alternaria and Stemphylium extracts

Shared allergenic and antigenic determinants in Alternaria and Stemph @urn extracts M. K. Agarwal, K~whestcr-.

Ph.D., R. T. Jones,

B.S., and J. W. Yunginger,

M.D.

Minn.

Altemaria and Stemphylium biologic, positi\,ely

extracts were compared by means of various immunochemical and methods. Skin-test responses to Alternaria und Stemphylium in allergic patients \c’err’ correlated, und RAST binding values with the sera of 30 patients utilizing solid-phase

AIM, Alternaria (70% ammonium sulfate-precipitublc ,fraction). und Stemphylium alle,rgt~n.\ rhowed .significant correlation, giving evidence for the presence of common allergenic, determinants among these three extracts. In RAST inhibition assays, Alternaria md Stemphylium extrwts exhibited dose-related inhibition with solid-phase Stemphylium. Altemaria. and AM, confirming the presence of Ait-l us the major shared allergenic, frrrc.tiort if1 the two extracts. In repetitive absorption experiments, both .41t-I and Altemaria solid-phase ullergens absorbed Stemphyliumspec$c IgE antibodies from a l2-person serum pool. Similarly. Stemphylium solid-phase allergen absorbed both Alternaria- and Ah-I--spec$c IyE antibodies. Double-antibody radioimmunoassay for Alt-l showed higher Alt-l activity in Stemphylium than in Alternaria extract. In double-immunodifj%csion e.rperiments with rabbit anti-Ah-1 antibodies, both Stemphylium and Altemaria extracts produced precipitin linrs qt’ idrntit) with Ah-l. The antigenic relationship of the tow crude extracts was further co@rmed b? crossed and c,rossed-line immunoelectrophoresis experiments. Our results showed that Alternaria and Stemphylium extracts contain multiple shared allergenic and antigcnic determinants, includin,q AIM. (J ALLERGY CLIN IMMUNOL 70:437, 1982.)

Airborne fungal spores and fragments are complex bioparticles that play an important role in the etiology of respiratory allergies. Each spore type contains a number of biochemical components, which may possess either antigenic or allergenic properties, or both. Allergenic and antigenic components may be unique to one species or may be shared with related species and genera. This heterogeneity of allergenic and antigenie determinants may be important diagnostically and therapeutically in fungi-sensitive patients. Alternaria is one of the most important airborne fungal allergens, and Alt-I has been isolated and identified as its major allergenic fraction.’ This investigation stud-

ied the allergenic and antigenic relationships between and a morphologically related fungal type,

Alternariu

Stemphylium.

-From the Departments of Pediatrics and Internal Medicine (Allergy), Mayo Medical School, and the Allergic Diseases Research Laboratory. Mayo Clinic and Foundation, Rochester, Minn. Supported by a grant from the National Institute of Allergy and Infectious Diseases (Al-16791) and by the Mayo Foundation. Received for publication May 21, 1982. Accepted for publication July 1, 1982. Reprint requests to: Dr. J. W. Yunginger, Allergic Diseases Research Laboratory. Mayo Clinic, Rochester, MN 55905. 0091-6749/821120437+08$00.80/0

0 1982 The C. V. Mosby Co.

Dry, defatted Alternuriu and Stemphylium allergen powders (source strain Nos. 16086 and 11128, respectively, American Type Culture Collection. Rockviiie, Md.) were purchased from Greer Laboratories, Inc.. Lenoir, N.C. In

Abbre\Wt

ms used

CFA: CIE: CLIE: DARIA: IEP: IFA: P-K: QIE: RAST: PNIJ:

Complete Freund’s adjuvant Crossed immunoelectrophoresis Crossed-line immunoelectrophortiis Double-antibody radioimmunoassay lmmunoelectrophoresis Incomplete Freund’s adjuvant Prausnitz-Kiistner Quantitative immunoeiectrophoresis Radioallergosorhent test Protein nitrogen unii Vol. 70, No. 6, pp. 437-444

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in IFA over the next several months. The antisera were tested by IEP, then pooled and stored at -20” C. Finally, antibodies against the purified PE-I, one of five subfractions of Alt-1 described previously,’ were produced by immunizing rabbits with two subcutaneous injections, I wk apart, of 50 pg (biuret protein) of PE-I fraction in CFA, plus another dose of 50 pg subcutaneously in IFA after I week. After five bleedings at I-wk intervals, an additional booster dose of 50 kg of PE-I was given subcutaneously in IFA. The antisera were tested by IEP, pooled, and stored at -20” C.

. .

. ..

P s.005 .

.:: t-

-I-

Alternaria

. I

I

I

I

CLIN. IMMUNOL. DECEMBER 1982

I

wheal diameter (mm)

Skin tests Puncture tests with the commercial Alternaria and Stemextracts (I : 20 w : v) were performed on 386 consecutive patients to compare the immediate wheal and flare response elicited by the two extracts. The magnitude of skin responses produced by the two extracts in patients showing positive skin reactions were compared by regression analysis.-’

phylium

FIG. 1. Comparison and Stemphylium

of skin-test responses extracts in 56 patients.

to Alternark

addition, Alternuria and Stemphylium extracts ( 1: 20 w : v in 50% glycerinated buffered saline with 47,000 and 50,000 PNU/ml, respectively) were purchased from the same supplier. Solid-phase allergens were prepared from Alt-l, crude Alternariu, and 70% ammonium sulfate-precipitable fraction of Alternaria extract, as described previously.’ Crude Stemphylium extract was prepared by extracting IO gm of powder in 170 ml of 0.05M Tris-HCI buffer (pH 8.0) over a magnetic stirrer for 66 hr at 4” C. The pH was maintained at 8.0 for the first 6 hr by addition of 4N NaOH, and the extract was recovered by centrifugation. The crude extract was then dialyzed in a Spectrapor-3 casing (3500 mol. wt. cutoff; Spectrum Medical Industries, Los Angeles, Calif.) against 0.1 M ammonium bicarbonate (pH 8.4) and was then lyophilized. Fifty milligrams of lyophilized Stemphylium extract were dissolved in 50 ml of borate buffer (pH 8.0) and reacted with 500 mg of cyanogen bromide-activated microcrystalline cellulose (pH 8.2) at 4” C for 48 hr. After centrifugation and repeated washing, the coupled Sternphy/ium was suspended in 500 ml of RAST diluent.Y

Antibodies Rabbit anti-Alrernuria antibodies were produced by two subcutaneous injections, 3 wk apart, of I mg (dry weight) of Alternaria crude extract in CFA, followed by another intramuscular dose of 3 mg of antigen in IFA after 4 mo. After five bleedings at I- to 2-wk intervals, six additional booster doses of 3.0 to 6.6 mg of antigen were given subcutaneously in IFA over the next several months. Antisera obtained after each booster dose were tested for precipitating antibody by IEP, then pooled and purified by ammonium sulfate precipitation (25% w: v) and diethylaminoethylSephadex A-50 column chromatography.Y Rabbit anti-Alt-1 antibodies were produced by giving two subcutaneous injections, I wk apart, of 50 pg (dry weight) of Alt-1 fraction in CFA, followed by another subcutaneous injection of 50 pg of Alt-1 in IFA after I wk. After seven bleedings at I-wk intervals, four additional booster doses of 50 pg of Alt-1 were given subcutaneously

RAST Sera from 30 patients showing positive skin reactions to either Alternaria and Stemphylium extracts were tested for specific IgE antibodies in mini-RAST assays” utilizing solid-phase Alternaria (70% ammonium sulfate-precipitable fraction),’ Alt-I, and Stemphylium allergens. Results were expressed as the percent of total radioactive counts bound to the solid-phase allergen and were compared by multiple linear regression analysis.”

RAST inhibition

studies

To study the possible shared allergens among the two mold genera, each was tested as fluid-phase inhibitors in the Alternaria, Alt-I, and Stemphylium mini-RASTs. For these studies, the IgE serum pool used was from seven nonimmunized patients with positive skin tests to Alternaria.

Absorption

studies

The cross-allergenicity of Alternaria and Stemphylium was further investigated by repetitive absorption of IgE antibodies by different solid-phase allergens. The IgE antibody pool used for these studies was from 12 nonimmunized subjects highly reactive by skin test and RAST to both Alternaria and Stemphylium. Five sets of seven tubes each were serially numbered. All tubes in each set received 2 ml each of one of five allergens linked to microcrystalline cellulose, either crude Alternaria extract, 70% ammonium sulfate-precipitable Alternaria, Alt-1 fraction, crude Stemphylium extract, or bovine serum albumin. The solid-phase allergens were washed three times to remove the RAST diluent, and were then capped and stored at 4’ C. On day 1, 0.5 ml of the 12-person serum pool and 1.5 ml of Sorensen’s buffer with 0.5% human serum albumin were pipetted into one tube of each washed solid-phase allergen and tumbled end-over-end overnight. On day 2, these five tubes were centrifuged, and the supernatant was carefully transferred to another tube with washed homologous solid-phase

90 0 Alternaria

80

(1 20 W/V)

A Stemphylium

(1:20

tests

in

WI!?

W/V)

70

= .o 3 c

60

.c if

40

50

30

-i

20

14

1.20

10

ii00

1500

12400

t-12500

Serum dilution

0 01

1

1

Allergen

.-

10

(p)

16

la

Skin tests

414

10

.Ol

001

.l

1.20

g

1.2500

1 12500

(@)

C

- & AN-1 (10 mglml) 0 Affemana A

1500

6

Serum dilution

1

Allergen

1.100

with

Skin tests with AH-I (5.78 pgB/ml)

Sternphyliwn

30 20 10

001

1

01

Allergen

1

(p)

FIG. 2. RAST inhibition curves with Alternaria, Stemphylium, and Alt-l extracts using solid-phase Alternaria (70% ammonium sulfate-precipitable fraction) (A), Stemphylium (B), and Alt-l (C) allergens.

allergen and tumbled overnight. This process was repeated on the next 5 days, and the final five supematants (each absorbed seven times) from each solid-phase allergen were tested for specific IgE antibodies to crude Alternaria extract, AIt-1 fraction, and crude Stemphylium extract by performance of P-K tests on healthy nonallergic subjects. Absorbed sera were diluted in Sbrensen’s buffer to obtain

14

1.20

1:100

1:500

12500

t 12500

Serum dilution FIG. 3. P-K test results with A&maria (500 PNlJimi) (Al, Stemphylium (500 PNUlml) (61. and Alt-I (5.78 @ml) (C) using unabsorbed 12-person serum pool (0) or serum pool absorbed (seven times) with various solidphase allergens, including crudeAltemari@ (01, Alternaria (70% ammonium sulfate-precipitable fraction) (a), Stemphylium (A), Alt-l (A.), or bovine serum albumin (a). 1:20, l:lOO, 1:500, 1:2500,and 1:125OOv:~ finaldilutions. Similar dilutions were also prepared with unabsorbed serum pool as a control. A total of 36 sites each were prepared on three healthy nonallergic subjects by intradermal injection of 0.1 ml of each dilution of absorbed and nonab-

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(1

A

1

10

Allergen

FIG. 4. Dose-response w:v, and Stemphylium Al&l.

W/V)

20

W/V)

Stemphylwm (1

1

20

CLIN. IMMUNOL. DECEMBER 1982

100

@I)

curves with Ah-l, Alternaria 1 : 20 1: 20 w: v extracts in DARIA for

sera. The sites on each subject were challenged after 1 to 5 days with one of the three test allergens, i.e., commercial crude Alternariu extract (500 PNU/ml), commerextract (500 PNU/ml), and Ah-1 cial crude Stemphylium fraction (5.88 pg/ml). Test allergens, along with Stirensen’s buffer and histamine (10 pg/ml) control solutions, were injected intradermally into sensitized and nonsensitized sites in sufficient quantities to produce an initial 5 by 5 mm wheal. The resultant wheal and flare diameters were measured at 15 min. The solid-phase allergen from each absorption step was washed three times with 2 ml of RAST wash, after which were added 0.2 ml of izJI-anti-IgE (40 ng) and 0.8 ml of fetal calf serum diluent.2 After an overnight incubation, the solid-phase allergen was washed as before and then counted in a gamma scintillation counter. sorbed

DARIA for Alt-l The Alt-I contents of the two fungal extracts were measured by DARIA. Ah-1 fraction (200 pg dry weight) was radiolabeled with i3’I by the chloramine-T method7; radiolabeled Ah-1 was separated from unreacted iodine by Sephadex G-25 filtration. Void-volume tubes were pooled and dialyzed against 0.15M saline at 4” C. Rabbit anti-Ah-I serum (1 :20 dilution, 0.1 ml), radiolabeled Ah-1 (10 ng, 0.1 ml), and various quantities of Ah-I, crude Alternariu, and crude Stemphylium extracts were incubated overnight at 4’ C, after which normal rabbit serum (1 :20 v:v, 0.05 ml) and an excess of burro anti-rabbit IgG (0.2 ml) were added to precipitate all rabbit IgG. Dose-response curves obtained with each test extract were plotted in a logit-log fashionR to permit comparison of the slopes of each curve by analysis of covariance4 using a programmable Hewlett-Packard 9845B computer.

lmmunodiffusion Double immunodiffusion in agarose gel was performed9 with rabbit anti-crude Alternaria, anti-PE-I sera, and exStemphylium, Ah-I, and PE-I fractions. tracts OfAlternaria,

FIG. 5. Results of immunodiffusion studies using: A, rabbit antibodies to crude Alternaria (X) and extracts of Alternaria (1: 20 w:v), Stemphylium (1: 20 w: v), and Ah-l (0.4 mglml) (wells 7, 2, and 3, respectively) and B, rabbit antibodies to PE-I fraction (X) and extracts of Alternaria (w : v 1: 201, Stemphylium (1 : 20 w: v), Alt-l (1.0 mglml), and PE-I (0.5 mglml) (wells 7, 2, 3, and 4, respectively).

cm CIE and CLIE methods”‘* ” were used to study the shared antigenic determinants in Alternaria and Stemphylium extracts. Rabbit antisera raised against crude Alternaria extract, Ah-1 fraction, and PE-I fraction were used for these studies. QIE gels were prepared from 1% w : v agarose (Induboise Agarose, L’Industrie Biologique Francaise, Clichy, France) in barbital buffer (pH 8.6) containing 0.024M barbital, 0.073M Tris, 0.0004M calcium lactate, and

O.OO3M

sodium

azide.

First-

and

second-dimension

‘VOLUME NUMBER

70

Alternarla

5

TABLE I. Reduction in IgE antibody levels during absorption -_% Counts

bound

study with

the

and Stemp~yltwn

~--._--.. solid-phase

allergen

441

_.... --.. ._-.._

used

Alternaria No. of days absorbad*

Crude

I 2 3 4 5 6 7

6.04 4.28 3.90 3.64 3.67 3.18 2.59

Net reduction in counts hound from day 1

58%

70%(NH&S04 fraction

Alt-l

7.31

16.46 6.86 2.68 2.61

6.10 5.25 4.35 3.53 2.79 2.15

2.10 1.93 1.72

71%

90%

*A 0.5 ml volume of la-person Alternan’a-sensitive serum pool was absorbed overnight with 2 mg of solid-phase allergen. Supematants from each step were transferred to unabsorbed solid-phase allergen, and the process was repeated for 7 days

electrophoreses were performed in 0.15 cm- thick gel at 10 V/cm for 25 to 35 min and 1 to 2 V/cm for 15 to 18 hr, respectively. The plates were washed, pressed, dried, and stained with Coomassie Brilliant Blue stain.

RESULTS skin tests

Of the 386 consecutive patients simultaneously tested with Alternaria and Stemphylium extracts, 56 gave positive skin responses to one or both extracts. The mean wheal diameters produced by Alternaria and Stemphylium extracts in each of the 56 patients were significantly correlated (r = 0.39, F,, 55= 9.95, p < 0.005: see Fig. 1). RAST Of the 30 patients’ sera tested, levels of IgE antibodies to Alt-I, Alternaria, and Stemphylium extracts were highly correlated (p < 0.001). RAST inhibition

studies

Both the Alt-I fraction and crude Alternaria produced significant and dose-related inhibition Stemphylium RAST (Fig. 2, B). Similarly, phylium extract produced significant inhibition Alternaria (70% ammonium sulfate-precipitable tion) and Alt-I RASTs (Fig. 2, A and C). Absorption

extract of the Stem-

of the frac-

studies

The absorbed IZperson serum pool was serially diluted and assayed for the remaining specific IgE antibodies by P-K test using Alternaria (Fig. 3, A), Stemphylium (Fig. 3, B), and Alt-I allergens (Fig. 3, C). The serum pool absorbed with crude Alternaria

solid-phase allergen not only showed decreased P-K reactivity to both crude Alternaria and AIt- fraction but also an appreciable reduction in P-K reactivity to Stemphylium extract, providing evidence of shared allergenic components in the two extracts. Similarly, the serum pool absorbed with Stemphvlium solidphase allergen showed a decreased P-K response not only to the Stemphylium extract but also to Alt-I fraction and crude Alternuria extract, further confirming the presence of shared allergenic components. The fact that Alt-I-specific antibodies could be removed by absorption with either crude Alternaria or Stemphylium extracts provided evidence that Ah-1 determinants are contained in both extracts, The solidphase fungal allergens from each absorption stage, after washing and reaction with radiolabeled anti-IgE, showed progressively decreasing binding, and after seven consecutive absorptions the net re&ction varied from 54% to 90% in the origmal binding depending on the solid-phase allergen used for absorption (see Table I). DARfA for Ah-l

Both Alternaria and Stemphylium extracts produced dose-related inhibition in DARIA for Alt-I (Fig. 4). The slope of the inhibition curve produced by Stemphylium extract was parallel to those obtained with Alt-I fraction and crude Ahernariu extract. On the basis of the amounts of Ah-1 and the test extracts required for 50% inhibition in the DARIA, it was calculated that Stemphylium extract contained a higher quantity of Alt-I than did the crude Alrernaria extract (84 and 30 kg/ml in 1: 20 w : v extracts, respectively).

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FIG. 6. CIE analysis ofA/ternaria (300 pg) (A) and Stemphylium (300 pg) (B) extract against rabbit antidlternaria antibodies (300 PI), and Altemaria (300 wg) (C) and Stemphylium (300 pg) (D) extract against rabbit anti-PE-I antibodies (30 ~1). The anode is to the left in the first-dimension electrophoresis and to the top in the second-dimension electrophoresis.

vow&E NUMSM

70 6

f

Rabbit antiserum to crude Alternariu showed bands of identity between Alternuria and StemphyLium and a band of partial identity .between Alt-I fraction and Stern~hy~~urn (Fig. 5, A). Rabbit antiserum to the I%-I fraction showed bands of identity among P&I ft-action, Alt-I fraction, cntde A4ternaria and Stemphy&um extracts (Fig. 5, B).

In CIE using rabbit antibodies to crude Alternaria extract (Fig. 6, A and B), Alt-I fraction (not shown), and PE-I fraction (Fig. 6, C and D), preeipitin bands were obtained with both Alternaria and Stem@rylium extracts, giving further evidence of shared antigenic determinants in the two extracts. However, crude Alrernak extract produced a larger number of precipitation bands against rabbit anti-crude AEternariu antiboclies (Fig. 6, A), indicating the presence of additional specitic amigenic determinants in Alternaria extract. Stemplaylium extract showed several precipitation bands with the antibodies to crude Alternaria extract (Fig. 6, B) but only one precipitation band with antibodies to Ah-1 (results not shown) as well as with antibodies to PE-I fraction (Fig. 6, D). CLIE using SremphyEium extract in the intermediate gel also showed several bands of identity between Alternaria and Stemphylium (Fig. 7).

Using fungal materials from one commercial supplier, we found a positive correlation between the skin-test responses to Alternaria and Stemphylium extracts, suggesting the presence of shared allergenic determinants in the two extracts. This observation was supported by the RAST results with solid-phase Alt-1, Alternaria, and Stemphylium performed with sera from 30 patients. These results suggested the presence of shared allergenic fractions in the two extracts. However, these findings could also be explained by coexistent hypersensitivity to the two extracts. The RAST inhibition studies ruled out this possibility. Fluid-phase crude Alternaria and Alt-I produced doserelated RAST inhibition not only with solid-phase Alternaria and Alt-1 but also with the solid-phase Stemphylium RAST. Similarly, fluid-phase Stemphylium elicited dose-related RAST inhibition with solid-phase Stemphylium, crude Alternaria, and Alt-I. It was interesting to note that both Stemphylium and Alt-I produced dose-related inhibition with both solid-phase Stemphylium and Alt-1 allergens. Hoffman and Kozak’* found that the sera showing cross-reactivity by RAST with Alternaria and Stemphylium recognized an antigen distinct from Alt-I. However, our

FIG. 7. CLE analysis of A&ma+ a&, m BXtracts. Antigen wdl containedA&~~& a~~M718D &; Stemphyiium extrsct {O.&J mg) *s iticovpora&d in the intermediate gel, and the .secoOd-&mansion gel contained 300 ~1 of rebbit anti-crud4 Akwnaria arteitidjes. The anode is to the left in the first-dimww&n elactrophoresis and to the top in the aecoffd-dime&i& electrophoresis.

results gave definite evidence that both crude Aliernaria and Stemphylium extracts contained Alt-1 as one of the major shared allergenic components. In cross-absorption studies ‘the serum pool repeatedly absorbed with solid-phase crude AEternaria or Alt-1 allergens showed decreased P-K reactivity to homologous allergens as well !as Stern~i~~rn extracts. Similarly, the serum pool: repeatedly absorbed with solid-phase Stemphylium allergen showed reduced P-K reactivity to crude Stemphyficam and Alternaria extracts as well as to Alt-I fraction. These results confirmed the presence of shared allergenic components in crude Afternaria extract, its Ah-1 fraction, ard Stemphyl~um extract. In double immunodiffusion in agarose gel using rabbit anti-Alternaria and anti-PE-I sera, crude Alternaria extract, Alt-I, and Stem&&urn extract produced precipitin bands of identity, showing t&t Afternuria and Stemphylium extracts also contain shared antigenic determinants. The presence of common antigenic determinants in the two crude extracts snd the Alt-1 fraction was further supported by the results of DARIA for Alt-I, where both Alternariu and Stem-

444

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extracts produced dose-relatedinhibition. In CIE studiesStemphylium extract produced precipitin bandswith rabbit antisera to crude Alternaria, AN-I, or PE-I fractions of Alternaria. Stemphylium extract produced at least 10 bands of identity with rabbit anti-crude Alternaria sera in the CLIE experiment. Alternaria extract, however, produced several additional bands indicating the presenceof many specific antigenic determinants in addition to the ones shared with Stemphylium. Collectively, these findings provide definite evidence that crudeAIternaria andStrmphylium extracts possesssharedallergenic as well as antigenic determinants. In addition, the in vivo and in vitro tests employed recognized Ah-1 as the major sharedallergenic and antigenic component in the two extracts. phylium

We acknowledgewith gratitudethe technicalassistance of Mary Jo E. Dahlbergand the secretarialassistance of Marian Bortolon. We alsothankCheryl Adolphsonfor her editorialassistance in preparing the manuscript. REFERENCES I. Yunginger

JW, Jones RT, Nesheim ME, Geller M: Studies on allergens. III. Isolation of a major allergenic fraction (Alt-1). J ALLERGY CUN IMMUNOL 66: 138, 1980.

Alterrwriu

J. ALLERGY

CLIN. IMMUNOL. DECEMBER 1982

of IgE antibodies by the 2 Gleich GJ, Jones RT: Measurement radioallergosorbent test. I. Technical considerations in the performance of the test. J ALLERGY CLIN IMMUNOL 55:334. 1975. 3 Harboe N, lngild A: Immunization, isolation of immunoglobulins, estimation of antibody titer. Stand J lmmunol Z(Suppl. 1):161, 1973. 4 Dixon WS, Massey FJ Jr: introduction to statistical analysis. New York, 1969, McGraw-Hill Book Co., Inc., p. 189. 5 Gleich GJ. Adolphson CR, Yunginger JW: The mini-RAST: comparison with other varieties of the radioallergosorbent test for the measurement of immunoglobulin E antibodies. J ALLERGY 0-1~ IMMUNOI. 65:20, 1980. 6 Zar JH: Multiple regression and correlation, in Biostatistical analysis, 1974, Prentice-Hall, Inc., p. 252. Greenwood FC, Hunter WM. Glover JS: The preparation of ““I-1abelled human growth hormone of high specific radioactivity. Biochem J 89: 114, 1963. Rodbard D: Statistical quality control and routine data processing for radioimmunoassays and immunoradiometric assays. Clin Chem 20: 1255, 1974. Macy NE, O’Sullivan MB, Gleich GJ: Comparison of sensitivities of lmmunodiffusion methods (33204). Proc Sot Exp Biol Med 128:1098, 1968. Weeke B: Crossed immunoelectrophoresis. Stand J Immunol 2(Suppl. 1):47. 1973. Krgll J: Crossed-iine immunoeiectrophoresis. Stand J Immunol Z(Suppl. 1):79, 1973. Hoffman DR, Kozak PP: Shared and specific allergens in mold extracts. J ALLERGY CUN IMMUNOL 63:213, 1979. (Abst.)