Measurement of IgE antibodies by the radioallergosorbent test

radioakrgosorbent II. Analyses Gerald Rochester,

of quantitative

J. Gleich,

M.D.,

test relationships

and Richard

in the test

1. Jones,

B.S.

Ninn.

IgE antibodies have been nuasrred by the radiodlergosorbent test (RAST) aud the relative amozlnts presntt in serwm determined by compati80* wtih refereme 8&ndu~ds. In this study we aazalyzed the qzbantitative aapeots of the b&k&&g of IgE’ antibody to solid-please ragweed a&gen& With the VOtzunreS of albrgio seml)b WWF&I tested, IgE nntibo&es are in exe888 in the fiT8t step of the XAST. The &&i&&y of solid-phase antigens to remove I@ antibody appeared to be due to (an i~@dt quan;tity of antigen on the particles rather than steric interj%rence with b&&g of IgE antibody. Dose-response curves with seruma from several ragwee&sePaPitive subjects were not parallel when plotted on a semilog scale. In GO%tT&, tog-log plots of dose-TespolEYe cu~vc~s with 8erums from ragweed-sensitive 8llbjeots were nearly prcrdlel. ~Jog-log plots of dose-response mrves ?l’ith 8eTwms from SbbjeCt8 w&th ragweed and gross sen8-&vity and tested with the appropriate solid-phctse adigm do were nearly pnmllel. Berxxise of the latter finding, RAST c&d be Stmtdard~~ed using a reference serum in every assay and plotttig the resdts on (c log-log SC&~. Finally, became IgE antibody is in excess in the BAST as it is u&&y performed, the final result rejleots both the quantity and the affinity of the IgE &Gbody.

The introduction of the radioallergosorbent test (RAST) for the measurement of TgE antibodies by Wide, Bennich, and dohansson’ served as a critical stimulus for study of allergies associated with IgE antibodies. In the accompanying paper’ we investigated a number of variables in the measurement of IgE antibodies by RAST, and in this communication we describe experiments designed to quantitate the amount of IgE antibody. Tn their studies, cJohansson and his assoeiatesl.:{ have quantitated I# antibody in terms of refercnee standards either as units or as degrees of positivitg based on comparison of test and control serum, and others have used a similar procedure.+ Ideally, one would like to utilize RAST to obtain weight estimates of IgE antibodies, and in this study we have attempted to utilize the test for this purpose. MA1EWN.S S&d-phase

AND MEWODS ragweed an#@ens

The same solid-phase utilized in most of these

rsglveed stndies.~

antigen Additional

described solid-phase

in

the previous communication ragweed antigens \vere prepared

was by

From the Departments of Medicine, Microbiology, and Immunology, The Allergic Diseeasea Research Laboratory, Mayo Clinic and Mayo Foundation, and the Mayo Me&al Ekbool. Supported by grants from the Food and Drug Administration, Bureau of Hiologics, FDA 73-164, and from the National Institute of AIlergy and Jnfeetions Diseaees, AL-11483. Received for publication May 22, 1914. Reprint requests to : Dr. Gerald J. Gleieh, Mayo Clinic, Rochester, Minn. 55901. Vol.

liti, No. 5, pp. 346-357

VOLUME NUMBER

TABLE

55 5

Radioallergosorbent

I. Coupling Cellulose

of

‘z’l-ovalbumin

activation

Divinylsulfone-phloroglucinol Preparation I Preparation 2 Cyanogen bromide-phloroglucinol Divinylsulfone Cvanoaen bromide ‘40 c”. 23” C.

test.

II

347

to cellulose*

I

% ovalbumin coupled

I

Mg. ovalbumin per Gm. cellulose

34.8 14.9 41.9 17.5

39.4 17.6 49.4 20.7

55.1 41.2

65.1 48.6

“100 mg. of ovalbumin was radiolabeled with 0.8 mCi 1311 employing the chloramine T reactionis Unlabeled ovalbumin. 52 ma.. was added to radiolabeled material. 7 mp.. and reacted with 500 mg. of the appropriat;ly activated cellulose. After thorough war&g with pH 7.4 phosphate-buffered saline and finally with 0.1 N HCl, the number of counts and thus the quantity of ovalbumin bound to the various preparations of cellulose were determined.

coupling short ragweed extract to cyanogen bromide (CNBr) activated Sepharose 2B or microcrystalline cellulose using either partially purified ragweed extract2 or lyophilized crude short ragweed extract (Lot No. 00802 FD) generously provided by Center Laboratories, Port Washington, New York. In order to determine whether ragweed antigens might be better able to react with IgE antibodies if the antigens were physically further from the cellulose support, we prepared solid-phase cellulose ragweed in which the antigen was separated from the particle by spacers. These solid-phase antigens were prepared using the reactions described by Porath and Sundbergs in which phloroglucinol and epichlorohydrin (both obtained from Fisher Scientific Company, Chicago, Illinois) are reacted with microcrystalline cellulose and ragweed antigen is coupled to the substituted cellulose after activation with CNBr or with divinylsulfone (DVS) (Research Chemical Corporation, Sun Valley, California). We also reacted cellulose directly with DVS at pH 11 and, after mashing, exposed the cellulose to ragweed antigen. This method of covnlently linking antigens to polymers has been used by Sundberg and Porath.6 1 II preliminary experiments we investigated the binding of radiolabeled ovalbumin (Schwarz/Mann, Orangeburg, New York) to cellulose activated by various procedures. For the preparation of the epichlorohydrin-phloroglucinol cellulose, 1 Gm. of cellulose was washed once with 0.1 M HCl and thrice wit,11 distilled water. The cellulose was suspended in 25 ml. of 1 N NaOH containing 4.2 Gm. phloroglueinol, and 2 ml. of epichlorohydrin was added. The capped reaction vessel was heated in a water bath at 60” C. for 2 hours, after which the cellulose was washed thrice with water and divided into two aliquots. One 500.mg. aliquot was suspended in 10 ml. of 1 M pH 11 Na,CO, containing 1.0 ml. of DVS, and the pH was maintained at 11 by addition of 4 M NaOH. The other 500.mg. aliquot was suspended in 10 ml. of 0.1 M NaHCO, and reacted at pH 11 with 2 Gm. CNBr dissolved in 2 ml. dimethylfornmmide.7 After washing, both aliquots were reacted with ovalbumin, 59 mg. (see Table I), the DVS-activated cellulose in 0.1 M pH 9.1 NaHCO,, and the CNBr-activated cellulose at pH 8 in borate saline.2 Ovnlbumin was also coupled directly to cellulose activated with either DVS or CNBr. The quantities of radiolabeled ovalbumin bound to the various celluloses are listed in Table I. Solid-phase

grass

antigen

A grass pollen extract was prepared by mixing 10 Gm. of June grass pollen (HollisterStier Laboratories, Spokane, Washington, Lot No. RM710695) in 90 ml. of water for 8 hours with magnetic stirring. The resulting extract contained 44.6 mg. of dry weight per milliliter. Solid-phase grass antigen was prepared by reaction of 11.2 ml. of extract, 500 mg. dry weight, with 1 Gm. of CNBr-activated cellulose. Twenty per cent of total 280 nM absorbance adhered to cellulose.

848

Oleich

and

J. ALLERGY

Jones

CLIN.

IMMIJNCIL. MAY 1975

FIG. 1. Effect of variation in amount of solid.phase ragweed and umount of anti-IgE on the quantitation of IgE antibody. The results are expressed as the number of counts bound to the ragweed antigen-polymer complex (ARC) divided by the number added x 100. In A, varying quantities of allergic serum (see footnote to Table I in Ref. 2 for a description of the particular serum used) were reacted with either 0.5 mg. or 5 mg. of solid-phase ragweed, and 2.6 ng. of anti-IgE was employed in the second step. In B, varying quantities of allergic serum were reacted with 0.5. mg. of solid-phase ragweed, and either 2.6 or 26 ng. of anti-lgf was tested in the second step of the RAST.

Performance This overnight we varied

of the RMT

test was usually performed incubation in both steps, the amount of solid-phase

Measurement

as described previously2 with 0.5 mg. solid-phase and 26 ng. anti-IgE in Step 2, although in certain antigen or the amount of a&-IgE.

antigen, studies

of IgE

IgE bound to solid-phase results were analyzed by logit caleulator.~

ragweed antigens was measured by transformation using a programmable

radioimmurcoaasay.s Hewlett-Pa’ekard

The W310

RRSULTS

In our earlier studies, we measured IgE antibodies by the uptake of radiolabeled anti-IgEl and expressed the results as the number of counts bound to the solid-phase allergen-IgE complex divided by the total counts added times 100. These percentages were taken as estimates of the quantity of IgI3 antibodies bound to the ragweed antigens, and the results were analyzed using nonparametric statistical methods. In these experiments the percentages of counts bound to the solid-phase ragweed antigens were not directly prvlMrtioual to the quantity of serum utilized in the test. Plots of per cent of counts bound versus volume of serum tested were ourvilinear and approached a maximum value with increasing quantities of serum. This observation suggested that either the number of antigenie sites available for reaction with IgE antibodies or the quantity of anti-IgE in the second step of the RAST was limiting. Aeeordin&v, we tested the effect of varying the concentration of polymer in the first step of the RAST and varying the concentration of anti-&E in the second step of the

VOLUME NUMBER

55 5

Radioallergosorbent

TABLE II. Quantitation

Serum

FCS Normal Allergic

tested

(pl)

IgE antibodies

Solid-phase antigen lmg.)

0.1 2: 5 :i 50

FCS Normal Allergic

of

50 50 5 ii 50

I

2.0

t

by

test.

II

349

RAST* Per cent

By serum

of counts

bound

By supematant

IgE bound to solid-phase antigen (rag.)

0.28 0.40 26.98 28.74 33.19 34.38

21.24 33.61

1.14 0.89 4.84 5.17 5.59 6.30

0.90 1.17 32.66 31.09 38.20 42.12

0.42 0.45 5.46 1.91 10.21 15.08

1.06 0.78 IO.71 12.66 12.31 14.60

FCS: fetal calf serum. *In this experiment varying volumes of allergic serum were tested with two amounts of solidphase ragweed. Solid-phase ragweed antigen was prepared by coupling 100 mg. of lyophilized short ragweed extract to 500 mg. CNBr-activated cellulose. After the first step, each supematant was retested by RAST to determine whether all IgE antibodies were removed. Finally after the first step of the RAST, one of the duplicate tubes containing the solidphase ragweed-IgE complex was tested for IgE by radioimmunoassay.

RAST. As shown in Fig. 1, neither increasing the quantity of solid-phase antigen from 0.5 mg. per tube to 5 mg. per tube in Step 1 of the RAST nor increasing the quantity of anti-IgE from 2.6 to 26 ng. in Step 2 of the RAST resulted in linear relationships between the volume of allergic serum and the per cent of counts bound. In both instances the per cent of counts bound approached a maximum when as little as 10 ~1 of allergic serum was added, although with volumes of serum up to 0.5 ~1 (see Fig. 1, B) , the binding curve approximated a straight line. These findings indicated that even with large quantities of first and second step reagents, binding approached a maximum with quantities of allergic serum above 2 ~1. Because excess antibody to IgE is clearly present in Step 2 of the RAST, we wondered whether the quantity of solid-phase ragweed antigen in Step 1 was sufficient to combine with all of the IgE antibodies. To explore this possibility we tested the supernatant from the first step of the RAST in a second RAST procedure to determine whether residual antibodies reactive with solidphase ragweed antigen remained in the supernatant. In addition, solid-phase particles were tested for the quantity of IgE bound using double-antibody radioimmunoassay. The results from a typical assay are shown in Table II. First, even with 10 ~1 of allergic serum, the maximum binding region was approached and increasing the quantity of allergic serum from 10 to 50 ~1 increased the quantity of anti-IgE bound by only a few per cent. Second, residual IgE antibody remained in the supernatant and more activity was present in supernatants from tubes containing less solid-phase antigen. Finally, the quantities of IgE bound to the solid-phase antigen increased as the quantities of solid-phase antigen and amount of allergic serum increased. These results indicate that considerable IgE antibody remains in supernatants after Step 1 of the RAST. In

350

Gleich

and

III.

Quantitation

TABLE

Serum FCS Normal Allergic

FCS Normal Allergic

*In

tested

(~1)

J. ALLERGY

Jones

of

IgE antibodies

Solid-phase ragweed antigen

2.0 2.0 0.05 0.1 0.5 1.0 2.0

0.5 mg. !

2.0 2.0 0.05 0. I 0.5 1.o 2.0

3.0

I

I

by

CLIN.

IMMUNOL. MAY 1975

RAST* Per cent of counts

By serum

bound

By supematfint

ha.)

rnQwe3d

0.30 0.28 2.32 2.07 9.51 13.52 18.52

0.35 0.26 0.45 0.55 1.57 2.40 4.02

1.3 1.3 1.7 1.3 1.8 i::

0.66 0.67 2.55 5.43 I 1.06 15.19 20.59

0.26 0.33 0.57 0.48 0.83 1.24 2.18

1.3 I.3 I.5 1.9 2.5 2.8 4.7

this experiment partially purified ragweed extra& was coupled to activated mierocrystalline celluIose using a ratio of 130 mg. of ragweed antigen per 500 mg. activated ecllulose; 70 mg. of the ragweed protein (absorbance at 280 nm.) bound to cellulose.

TASiE

IV. Use of ragweed

Sepharose

as a solid-phase

antigen

in the RAST* IgE bound

Serum KS Normal Allergic

FCS Normal Allergic

tested

By aerum

)

0.5 crude ragweed

1.34 1.66 25.68

1.34 0.95 2.02

0.85 I.15 2.27

:i 50

extract

27.05 28.36 33.15

2.12 2.20 2.58

2.85 3.29 3.88

0.5 partially purified

1.70 2.48 26.48

1.11 0.86 2.24

0.91 0.99 2.77

extract ragweed

27.92 31.29 33.64

2.46 2.98 3.66

3.00 2.95 4.41

2: 5

to

~~)

e-

50 50 5

:: 50 *In

U)

Solid-phese antfgm btB.)

this experiment either lyophilized crude ragweed extract or partially pUti8d extrillctz was coupled to Sepharose 2B; 100 mg. lyophilized extract or 250 mg. partially puliffed extract was coupled to 25-ml. aliquots of CNBr-aotivated Seplmrose 28. The supernataut was tested for residual IgE antibody using a crude ragweed extract coupled to cellulose. The Sepharoae 2B prepared with partially purified ragweed extract contained 388 pg per milligram, assuming 20 mg. of dry weight per milliliter of packed Sepharose 2B.

Table III the results of a similar experiment in which smaller volumes of serum were tested are shown. With 0.5 mg. solid-phase antigen, residual IgE antibody was clearly present in the supernatant when 0.5 ~1 allergk serum was tested; with 3.0 mg solid-phase antigen, 1.0 ~1 was required before residual antibody was found. In neither case was there a linear relationship between serum tested and counts bound. In previous studies we had measured the quantities of bovine serum albumin bound to solid-phase particles and we had found that larger quantities of antigen were bound to Sepharose particles than to cellulose or Sephadex.’ To determine whether Sepharose might be a more efficient solid-phase immunosorbent than

VOLUME NUMBER

55 5

Radioallergosorbent

TABLE V. Reaction

Serum

tested

(pl)

FCS

50

Normal

50

Allergic

5

FCS

:“o 50 50

Normal

50

Allergic

5

of allergic

Solid-phase ragwaad

5

1; :i

various Per cent

solid-phase of counts

ragweed bound

IgE bound to solid-phase antigen (rig.1

By serum

By supernatant

0.72

0.31

2.0

0.26

1.5

22.10

5.38

4.4

Cellulose

25.02 26.17 28.40 0.39

11.52 8.53 16.19 0.25

2; 14.1 1.3

Phlorogkinol + DVS

0.51

0.38

0.9

22.55

3.86

2.6

Cellulose + CNBr

26.87 30.41 29.36 0.35 0.87 23.72

10.15 5.69 15.88 0.29 0.35 8.23

::: 7.8 1.05 0.94 9.61

24.43 26.96 26.75 0.68 0.45 26.46 28.71

12.26 8.83 17.58 0.40 0.30 6.28 8.10

16.83 11.54 25.99 0.90 0.72 2.79 3.58

31.31 29.50

11.84 18.39

!-Z:Z

Cellulose + DVS

351

antigens*

0.52

:t 50 FCS Normal Allergic

with

II

Cellulose + Phloroglucinol + CNBr

:i 50 FCS Normal Allergic

serum

test.

“0.5 mg. of solid-phase antigen was tested in each experiment. The various solid-phase ragweeds were prepared by activation of cellulose with the different reagents as described in “Materials and Methods”; 500 mg. of each activated cellulose was reacted with 130 mg. of partially purified ragweed extra&r The cellulose-phloroglucinol-CNBr contained 101 cg ragweed protein per milligram; the cellulose-phloroglucinol-DVS, 107 cg per milligram; the and the cellulose-DVS, 111 c(g per milligram. cellulose-CNBr, 140 fig per milligram; Rupernatants were tested with the solid-phase ragweed described in the footnote to Table II.

microcrystalline cellulose, we coupled partially purified ragweed antigen or crude ragweed extract to Sepharose 2B and tested ragweed-Sepharose in the RAST. The results are shown in Table IV. The per cent of counts bound in the RAST was virtually at a maximum when 10 to 20 ~1 allergic serum was added, but, in contrast to the results with microcrystalline cellulose, the supernatants from the RAST procedure contained little residual IgE antibody. Also in contrast to the results with microcrystalline cellulose, the quantities of IgE protein on the Sepharose particles detectable by double-antibody radioimmunoassay were considerably less. Because the Sepharose ragweed antigens absorbed out virtually all of the IgE antibodies reactive with the ragweed antigens, we expected the quantity of IgE antibodies on the solid-phase particles at the end of the first stage of the RAST to be greater than in the earlier experiment. The failure to find an increased quantity of IgE on the Sepharose 2B ragweed suggests that the IgE bound was sterically hindered and unable to compete with radiolabeled IgE for the anti-IgE antibody combining sites in the radioimmu-

352

Gleich

and

TABLE VI. Testing

J. ALLERGY

Jones

of allergic

and

normal

serums

by

RAST and Per cent

Patient

Serum

taste4

R. B. R. B. R. B. R. D. R. D. R. D. J. Y. J. Y. J. Y. M. H. M. It.

w. w. w. w.

(fill

By serum

analysis

of counts

CLIN.

IMMUNOL. MAY 1975

of supernatants’ bound By supmetant

8.13 13.49 16.19 3.36 5.83

:;

24.06 23.77

0.45 0.46 1.64 0.82 3.76 8.75 12.15

::

0.37 0.29

0.26 0.31

Normal : *In

this experimentthe solid-phase ragweedcel%ulo~e usedto test the serummd supernstants is the sameas describedin the footnote to Table IV.

noassay. Control experiments in which Sepharose 2B not coupled to ragweed antigen was tested revealed that virtually all of the IgE antibody was present in the supernatants from the first step of the RAST, indicating that little nonspecific trapping of IgE antibody occurred. Finally, the counts bound by the negative controls were considerably higher with the Sephame-ragweed than in prior experiments with cellulose-ragweed. The results of the preceding experiments indicated that under ordinary conditions insuBeient quantities of ragweed antigenie determinants are present on microcrystalline cellulose to remove all of the IgIll antibodies from the volumes of allergic serum ordinarily tested by RAST. As an alternative explanation, steric hindrance might prevent binding of IgE antibodies to ragweed antigens. To determine whether the latter possibility was likely, we coupled partially purified ragweed antigen to microcrystalline cellulose by 4 different methods: (1) the usual procedure employing cyanogen bromide, (2) a reaction procedure utilizing divinylsulfone, (3) a reaction procedure in which microcrystalline ceilulose is initially substituted with phlorogfueinol and epichlorohydrin and activated with eyanogen bromide, and (4) the same reaction procedure activated with divinylsulfone. All of these polymers were tested in EAST to compare their efllcieney as immunosorbents. The results of this experiment are shown in Table V. If IgE antibodies were prevented from binding to ragweed antigens because of steric hindrance, one would expect that the epichlorohydrin-phloroglucinol solid-phase antigens possessinga “spacer” would he more effective as immunosorbents. Although there was a tendency for these latter solid-phase antigens to be somewhat more efficient in removing IgE antibodies from serum compared to cellufo~~ wi%31out “spacers,” this tendency was not marked and discouraged belief that steric

VOLUME NUMBER

Radioallergosorbent

55 5

Serum,

log,,

test.

II

353

$1

FIG. 2. Semilog plots of RAST results. Comparison of the reactivity of 8 individual allergic serums and a serum pool with solid-phase ragweed is shown. The correlation coefftcients for all regression lines were to.98 or greater. The reactivity of the serum pool is indicated by the plus signs (+), and it was composed of equal volumes of the 5 most reactive serums, X, 0, A, 0, and

0.

hindrance was the principal cause of the inability of the solid-phase antigens to remove IgE antibodies. Rather it seems likely that insufficient ragweed antigen is present on cellulose to enable it to remove IgE antibody from the usual quantities of allergic serum tested in RAST. Further evidence to support this contention is presented in Table VI. Here serums from 6 ragweed-sensitive patients were tested by RAST and the supernatants analyzed for residual IgE antibod) activity. Significant residual IgE antibody to ragweed was detected in supernatants in which the initial R,AST values were as low as 3 per cent. Clearly, residual IgE antibody remains in the supernatant with initial binding levels of 5 per cent or greater. The results presented indicate that IgE antibody is in excess in the first step of RAST. Therefore, RAST must be standardized in relationship to a reference, and we investigated various ways to analyze these results. The reactivity of 8 serums from ragweed-allergic subjects was tested and the results are shown in Figs. 2 and 3. Inspection of the semilog regression lines in Fig. 2 suggests that there are differences in slope among the 9 samples (8 individual serums and a serum pool), and this was confirmed by analysis of covariance [F (8,29) = 9.24; p < 0.0005] . The slope of the regression line produced by the serum pool did not differ significantly from the lines yielded by the 5 serums composing it. However, the slopes of lines produced by the 3 less reactive serums were significantly different from the serum pool [F (3,18) = 3’7.7; p < 0.0005]. The slopes of the log-log regression lines in Fig. 3 are quite similar on simple inspection, even though statistical analysis revealed that they differed [F (8,29) = 4.88; p < O&05]. In particular, the differences in slopes between the serums

354

Gleich

and

J. ALLERGY

Jones

CLIN.

IMMUNOL MAY 1975

- - -.i----...a0.

0.3

0.7

1.0

1.;

1.7

Serum, log,,, pl FIG. 3. Log-log same symbols.

plots of RAST The correlation

results. The data shown in Fig. 2 are repiatted coefkients for all regression lines were +0.98

using the or greater.

with low levels of binding and those with high levels are clearly less than in Fig. 2. Finally, we tested the reactivity of serums from subjects allergic to ragweed or grass pollen with either a ragweed or grass solid-phase antigen, respectively, and we compared the slopesof the log-log plots. The results are shown in Fig. 4. The slopes of the regression lines were all quite similar on inspection, although statistical comparison of the results from all the eight samples revealed a difference [F (‘7,26) = 2.83; p < 0.051. The slopes of the regression lines from the ragweed-sensitive serums did not differ among themselves, nor did the slopes of the lines from the grass-sensitive serums. Comparison of the slopes of the regression lines of ragweed-sensitive serums to the slope of the grass pool did not reveal a difYerence, nor did the converse comparison. Also, the slopes of the regression lines yielded by the grass and ragweed pools did not differ.

In the only published analysis of the ability of the RAST to quantitate IgE antibodies, Johansson and co-workers found the radioactivity bound to s&dphase antigens (and they used a variety of antigens) was related to the etmcentration of IgE antibodies provided that both the solid-phase antigen and the labeled anti-IgE were present in excess.3 They also found that sig&~id dose-response curves were obtained when volume of serum tested and anti-f&% bound

VOLUME NUMBER

55 5

Radioallergosorbent

0.3 ' 0

0.3

0.7

Serum,

1 1.0

/ 1.3

I 1.7

test.

II

355

I 2.0

log,,,pl

FIG. 4. Comparison of log-log plots with two antigenic systems. In this experiment ragweed serums were tested with a solid-phase ragweed, and grass serums were tested against a solid-phase grass antigen. The solid lines indicate the dose-response curves with ragweed antigens and the dashed lines indicate the results with grass antigens. The ragweed serum pool was the some as that used previously (see legend for Fig. 2) and is represented by the open circles. The grass serum pool was composed of equal volumes of serum from 5 grass-sensitive patients and is represented by the solid triangles. The remaining 6 regression lines illustrate results with serum from individual patients sensitive either to ragweed or to grass. The solid-phase ragweed was prepared by reaction of the partially purified ragweed extract with CNBr-activated cellulose and contained 101 pg ragweed protein per gram cellulose. The solid-phase grass antigen is described in “Materials and Methods.”

were plotted on a log-log scale. Dose-response curves with a variety of solid-phase antigens and serums from sensitive patients were parallel. They concluded that all of the antigenie systems they had investigated could be standardized in terms of a single reference serum. Our results with solid-phase ragweed antigens differ from those of Johansson, Bennich, and Berg3 in some respects and agree in others. In contrast to their study we find that the volumes of serum usually employed in RAST provide an excess of IgE antibodies in the first step of the RAST and that even small volumes of serum from several subjects contain an excess of IgE antibody. In agreement with their study, we find that the log-log plots of results tend to yield regression lines with comparable slopes. The finding that IgE antibodies are in excess in the first step of the RAST has several implications. Because of the excess of IgE antibody, there will be competition among these antibodies for the various antigenic determinants on the particle, and IgE antibodies of high afinity will be more likely to bind to antigen than those of low afinity. Thus RAST results are a function of both the quantity and the &r&y of the IgE antibodies in the test serum. One could have an allergic serum with a low absolute concentration of IgE antibodies of high affinity, and this serum could yield greater binding in RAST than another serum

356

Gleich

and

J. ALLERGY

Jones

CLIN.

IMMUNOL. MAY 1975

with an equal concentration of TgE antibody of low affinity. The presence of antibodies of other immunoglobulin classes, such as TgC, could interfere with the binding of IgE antibodies to solid-phase antigens in RAST especially if the quantity of solid-phase antigen was limiting. Such interfcrencc could bc of particular importance in studies of hyposensitization becansc IgO antibody to ragweed could result in spuriously low values for IgE antibody. In a prior study of hyposensitization we did not detect interference of IgG antibodyl”* IL in the R.AST, and this lack of interference may have been due t,o the relatively high concentration of antigen we coupled to the solid-phase particles. The finding that semilog dose-response curves were not parallel indicates that one cannot standardize RAST results in this manner.” In our studies of IgE antibodies by RAST, we have expressed the results as counts bound divided by counts added x 100 and have analyzed the results by nonparametric statistical methods.x However, because of the need to compare results from different experiments, it would be desirable to express RAST results in terms of a reference serum or pool of serums. In keeping with the findings of cJohansson and his associates,” our results suggest that RAST results can be standardized using a pool of allergic serums and plotting the results on a log-log scale. Further, it appears that the IIAST could be standardized with a single antigenic system and the results expressed in terms of the reactivity of that system. However, until reference standards are available, RAST results from different laboratories cannot. be compared. Clearly, tests such as that used by Zeiss and associates,‘2 which measures total IgE antibody, and new tests to determine the affinity of IgE antibody are needed for studies of changes in the absolute quantities and affinities of IgE antibodies. We thank

Dr. John

Yunginger

for

his critical

review

of this

manuscript.

RENRENCES 1 Wide, L., Ben&h, H., and Johansson, S. G. 0.: Diagnosis of allergy by an in-vitro test for allergen antibodies, Lancet 2: 1105, 1967. 2 Gleich, G. J., and Jones, R. T.: Measurement of IgE antibodies by the radioallergosorbent test. I. Technical considerations in the performance of the test, J. ALLERGY CLIE. TMYIXOL. 66: 334, 1975. 3 Johansson, S. G. O., Bennich, H., and Berg, T.: In vitro diagnosis of atopic allergy. TII. Quantitative estimation of circulating IgE antibodies by the radioallergosorbent test, Int. Arch. Allergy Appl. Immunol. 41: 443, 1971. 4 Lichtenstein, I,. M., Ishizaka, K., Norman, P. S., Sobotka, A. K., and Hill, B. M.: .IgE antibody measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy, J. Clin. Invest. 62: 472, 1973. 5 Porath, a., and Sundberg, L.: High capacity chemisorbents for protein immobilization, Nature [New Biol.] 288: 261, 1972. 6 Sundberg, L. : Personal communication. 7 Yunginger, J. W., and G&h, G. J.: Comparison of the protein-binding capacities of cyanogen bromide-activated polysaccharides, J. ALLERQY CLrw. IMMXJNOL. 60: 109, 1972. 8 Gleieh, G. J., Averbeck, A. K., and Swedlund, H. A.: Measurement of IgE in normal and allergic serum by radioimmunoassay, J. Lab. Clin. Med. 77: $90, 1971. 9 Midgley, A. R., Jr., Niswender, G. D., and R.&n, R. W.: Principles for the assessment of the reliability of radioimmunoassay methods (precision, accuracy, sensitivity, speci&?ity) , a?~ Karolinska Symposia on research methods in reproductive endocrinology; First Symposium:Immunoassay of gonadotrophins, Acta Endoerinol. Suppl. 142: 163, 1969.

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10 Yunginger, J. W., and Gleich, G. J.: Seasonal changes in IgE antibodies and their relationship to IgG antibodies during immunotherapy for ragweed hay fever, J. Clin. Invest. 52: 1268, 1973. 11 Gleich, G. J., and Yunginger, J. W.: Seasonal changes in JgE antibodies in patients with ragweed hay fever: Analysis of the effects of hyposensitization. Proceedings of the Eighth International Congress of Allergology, Oct. 14-20, 1973, Tokyo, Japan, Amsterdam, 1974, Excerpta Medica Foundation, pp. 44-53. 12 Zeiss, C. R., Pruzansky, J. J., Patterson, R., and Roberts, M.: A solid-phase radioimmunoassay for the quantitation of human reaginic antibody against ragweed antigen E, J. Tmmunol. 110: 414, 1973. 13 Greenwood, F. C., Hunter, W. M., and Glover, J. S.: The preparation of iarI-labelled human growth hormone of high specific radioactivity, Biochem. J. 89: 114, 1963.