- Email: [email protected]
Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test
Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test G. J. Gleich,
M.D.,
J. B. Larson,
M.D.,
R. T. Jones,
B.S., and
H. Baer,
Ph.D.
Rochester, iMin?l., and Rockville, Md.
The potencies of 11 tmmeroial extracts of short ragweed pollen were cc&yzed by skin test end point titration. and compared to potency as measwred in vitro: (I) by the radioallergosorbent test (RAST), (2’) by AgE concentrations, and (3j by protein nitrogen determinations (PNU). RAST potency was determined by the capacity of the extract to inhibit the binding of IgE antibody to solid-phase allergen in the first step of the RAST, and it was expressed as the quantity of extract needed for 50 p cent in,hibition of binding. Potencies determined by skin testing in. 7 patients zere strongly related among the various patients in spite of a l,OOO-fold difference in the patients’ sensitivity to the extracts. Similarly, potency as measured by RAST inhibition showed about n l,OOO-fold difference among the extracts, and the RAST potencies were strongly related to potency as measzlred by skin testing. Puri$ed antigens derived from short ragweed inhibited binding of IgE antibodies, but th,e slopes of the inhibition owrves were significantly less than those produced by crude or partially purified short ragweed. AgE concentration and PNU concentrations also were correlated with skin test potency, but the range of potencies was less than that found by RAST inhibition. Measwenaent of the potency of estraots by BAST inhibition should prove useful as a general procedure for the standardization of allergy extracts.
At the present time the potency of allergy extracts is usually listed as the number of protein nitrogen units (PNU) per milliliter of extract* or simply in terms of weight per volume of solute to solvent used to prepare the extract. In the case of ragweed pollens and grass pollens, purified antigens that appear to account for the bulk of the allergenic properties of these materials may be used as measuresof the potency of these extracts.?-* In particular, the studies of Baer and his associates2demonstrate that the in vivo reactivity of short ragweed extracts correlates extremely well with the antigen E (AgE) content of the extracts,% and for short ragweed extract AgE could be used as a measure of t,he potency of the extract. Since the introduction of the RAST for the measurement of IgE antibodies by Wide, Bennich, and Johansson,” this test has been utilized both for the diagnosis of allergy to various antigens, 6 for the measurement of changes in IgE From the Mayo Clinic and Mayo Foundation, Departments of Medicine, Immunology, and Microbiology, The Allergic Diseases Research Laboratory, Rochester, and the Bureau of Biologic%, Food and Drug Administration, Rockvillc. Supported in part by a grant from the Food and Drug Administration, Bureau of Biologies, NIH-DBS-72-2087. Received for publication July 16, 1973. Reprint requests to: Dr. Gerald J. Gleieh, Allergic Diseases Research Laboratory, Mayo Clinic and Mayo Foundation, Rochester, Minn. 55901. Vol.
53, No. 3, pp. 158-169
VOLUME NUMBER
53 3
Potency of allergy
extracts
159
antibodies to specific antigens,T-9 and, finally, for the measurement of the potency of allergy extracts.10-15 For the measurement of the potency of allergy extracts by RAST, two general approaches have been used: (1) extracts have been coupled to microcrystalline cellulose and the quantity of extract needed to achieve a maximum in the RAST determined, and (2) extracts have been tested to determine their inhibitory capacity in the first step of the RAST. In this study we have measured the potency of short ragweed extracts by determining their capacity to inhibit the binding of IgE antibodies to ragweed antigens in the first step of the RAST and have compared these measurements of potency to those obtained by skin test end point titrations, to AgE concentrations, and to PNU concentrations. MATERIALS AND METHODS Allergens and allergenic extracts Extracts of short ragweed (Ambrosia elation) were purchased from 11 different companies in the United States. All but two of these were nonglycerinated. The manufacturers’ claimed potency in terms of weight per volume or PNU measurement of potency for these extracts is shown in Table I. AgE was obtained from the National Institute of Allergy and Infectious Disease through the courtesy of Dr. Charles Kirkpatrick. Antigen K (AgK) and antigen Ra3 (Ra3) were purchased from Worthington Biochemicals, Freehold, New Jersey. Lyophilized short ragweed extract was a gift from Mr. George Center, Center Laboratories, Port Washington, New York (Lot 00802FD). Partially purified ragweed extract was prepared by extraction of defatted pollen with distilled water followed by gel filtration of this extract on a column of Sephadex G-25. The void volume peak was concentrated using an Amicon ultrafiltration device and a PM-10 membrane and then passed through the G-25 column a second time. After reconcentration and dialysis against 2 mM NH,HCO,, the solution was dried for 36 hours at 68” C. The dry weight of extract was 26 mg. per milliliter. In Fig. 3 this partially purified extract is labeled BEG-RW.
Skin testing To assay the in vivo potency of the crude short ragweed pollen extracts, we determined that dilution of the extract that would yield a standard 6 x 6 mm. (2+) wheal-and-flare reaction 15 minutes after the intradermal injection of extract sufficient to produce an initial 3 x 3 mm. wheal. The extracts were initially diluted so as to contain approximately 5,000 PNU per milliliter using pH 7.4 phosphate-buffered saline containing 0.5 per cent human serum albumin. This buffer was both isotonic and iso-osmolar when compared to human serum. If only the weight per volume potency was supplied by the manufacturer, we estimated the initial dilution using the relationship between weight per volume and PNU suggested by Sheldon, Lovell, and MathewsIs Fivefold dilutions of the extracts were prepared, and 7 patients with varying degrees of sensitivity to short ragweed were tested to determine the dilution producing a 6 x 6 wheal. The dilutions were prepared and used within 4 weeks of preparation. Between testing periods the extracts were kept at 4” C. but were brought up to room temperature during the actual testing procedure. In cases in which an exact 6 x 6 wheal was not obtained, the end point was extrapolated to a value between the dilutions that bracketed the hypothetic 6 x 6 wheal. The potency of the extract was expressed in terms of skin test units, i.e., the number of dilutions necessary to yield that concentration producing a 6 x 6 wheal. For example, the extracts were initially diluted to approximately 5,000 PNU per milliliter and subsequently diluted serially 1 to 5. If the fifth of these dilutions, containing approximately 1.6 PNU per milliliter, yielded a 2+ reaction, then the potency of the resultant extract would be 55 times the initial dilution of the original crude extract obtained from the manufacturer. In this way the number of skin test allergenic units were determined for each of the 11 extracts. In actual practice a given extract was tested at three skin sites on the patient’s forearm with dilutions
160
Gleich
TABLE
I.
et al.
Potency
of
Short ragweed extract
1 l t
20 3A 40 5x ti0 7w
extracts
of
Designated potency
4/26/74 l/3/74 4/25/74
l-10
10/24/73 7/l/73 l/k)74
represent
A# &g/ml.)
1076
4,546 317.5
48,000 20,600
110 111
8,333 4,762
l-10
to
1,724 2oo.f! i 8.0 13.9
11,364 2,500 41167
ifi
153
as
the
number
the
various
of
extracts
5Ocjr, RAST in the
inhibition
units
per
milliliter.
figures.
differing 25-fold in concentration in order to determine the dilution of extract that yielded an approximate 6 x 6 response. The particular dilutions used in a patient were determined from preliminary estimates of his sensitivity based on skin tests done at the time of the original diagnosis of his ragweed hay fever. Usually one of these preliminary skin tests on the forearm gave a reaction close to 6 x 6; if not, appropriate dilutions above or below those originally selected were placed on the forearm skin in order to determine which dilution approximated a 6 x 6 reaction. The final tests to defme the end point were performed in duplicate on the upper arm. Three dilutions were tested: (1) the dilution yielding about a 6 x 6 wheal, (2) a dilution fivefold more concentrated, and (3) a dilution fivefold less concentrated. To test ttte 11 extracts it was necessary to carry out these tests over a 2 day period.
Measurement
of the potency
RAST
Potency in vitro 50% RAST inhibition w. per ml.1
36,300 16,900
l-20
used
AgE,
PNU)
u/7/75
tSymbols
PNU,
l-20
4/26/74 defined
by
vitro
l-20 1-33 5% 40.000 PNU ’ l-10
11 A
is
in
IMMUNOL. MARCH 1974
44 6 0 0 205
10 0 vitro units.
measured
CLIN.
18,500 8,250 9.350 4;560 48,100 19.000 35;200
l/11/74 7/12/73
in
as
PNU/ml.
5%
(27,080
*Potency inhibition
ragweed
EXpiratiOll date
ii/+75
:t
short
1. ALLERGY
of ragweed
extracts
by RAST inhibition
The RAST procedure was carried out as described previously9 with the following modifications : (1) for the solid-phase ragweed antigen, lyophilizod short ragweed obtained from Center Laboratories was covalently linked to microcrystalline cellulose at a rat,io of 10 mg. of lyophilized powder per 500 mg. of cellulose; (2) in the second stage of’ the KAST 20 ng. of anti-IgE labeled with 1311 were used rather than 2 ng.; and (3: aliquots of a serum pool prepared by mixing equal volumes of serum from 5 subjects highly allergic to short ragweed pollen were used in the first step of the RAST to assure that most antigens recognized by TgE antibodies would he detected in the RAST inhibition procedure. None of these patients had ever been treated by hyposensitization. 111 the actual performance of the test, first, inhibitor in RAST diluent was added in 100 ~1 of a 1:5 dilution of pooled allergic serum in a volume up to 400 pl. Second, RAST diluent was added, and the volume was adjusted to 500 ~1 when necessary with additional RAST diluent. Finally, within a few minutes of the above additions 500 pl of the solid-phase ragweed antigen at a concentration of 1 mg. per milliliter was added, and the tubes were capped and rotated overnight at room temperature. Following washing, 0.65 ml. of RAST diluent was added, followed by 20 ng. of anti-IgE in pH 7.5 phosphate albumin in a volume of 1OQ ~1. The RAST diluent consisted of pH 7.5 0.1 M KH,PO,-K,HPO,, 470 ml.; fetal calf serum (Grand Island Biologicals, Grand Island, New York), 20 ml.; 10 per cant NaN,, 5 ml. ; and tween 20, 5 ml. When reacted with 0.5 mg. solid-phase ragweed antigen, the 100 pl of the 1:5 dilution of the allergic serum pool yielded a maximum of 19 per cent to 22 per cent binding, while controls, either normal human serum or fetal calf serum, yielded a maximum of 1.2 per cent
For
VOLUME NUMBER
inhibition,
53 3
and
Potency
in vivo
by
skin
of allergy
161
extracts
testing*
(skin
Potency in viva test units x lo61
D. S.
L. c.
J. K.
J. M.
J. B.
W. R.
J. P.
5.39
37.8 3.17 17.2 34.4 48.8 68.4 16.8 377 52.7
163 222 1”:9 244. 104 1,050 1,070 30.5
0.121 0.078 0.078 0.036 9.76 0.183 1.02 9.77 0.169
0.0614 0.0133 0.0566 0.0032 0.390 0.109 0.236 0.265 0.0975
139.1 8.73 16.3 2.06 67.4 34.8 77.5 601 35.3
5.48 1.80 1.41 1.17 6.64 3.32 16.8 229 3.95
19.5 67.4
137 244
1.29 1.95
0.0645 0.226
19.5 122
2.29 1.33 0.15 5.55 2.73 20.4 244 2.48 3.91 9.77 example,
if
5 ~1 yielded
508%
inhibition,
then
one ml.
of the
extract
contains
4.82 12.0 1000 L-5
=
200 RAST
binding and usually less than 1 per cent binding. In the performance of the RAST inhibition test, varying quantities of extracts were added to determine that amount necessary to produce a 50 per cent inhibition in the binding of IgE antibody to the solid-phase antigen, In all cases the negative control with normal human or fetal calf serum, whichever was less, was subtracted from all of the tests.
Radial immunodiffusion nitrogen
for measurement
The concentration of AgE in values were extrapolated from a and anti-AgE. The number of phosphotungstic acid and analysis
Statistical
tests
Results covariance.is evaluations.
were evaluated A programmable
by
of AgE
and
measurement
of protein
each extract was measured by radial immunodiffusion,ir standard curve obtained by the use of U. 8. reference PNU per milliliter was determined by precipitation of the dissolved precipitate.
tests of rank Hewlett-Packard
and AgE with
correlation coefiicients and by analysis of calculator was used for the statistical
9810A
RESULTS
The results of the determinations of the biologic potency of the allergy extracts in vivo by skin test end point titration are shown in Table I. It is remarkable that even though these extracts were supposedly close to the same potency in terms of the manufacturers’ estimate (either weight per volume or PNU), nonetheless their potency by skin test titration varied by almost l,OOOfold. To determine whether the skin test procedure yielded reliable estimates of the potency of the extracts among the patients, we determined the correlations between the potency in various patients, and the results are shown in Table II. All of the rank correlation coefficients in Table II are positive and indicate that the skin test procedure yielded remarkably good agreement between the measurements of potencies of the extracts in different patients. Most of the rank correlation coefficients in Table II are significant at p < 0.0005 and all are
162
Gleich
J. ALLERGY
et al.
CLIN.
IMMUNOL. MARCH 1974
s
87
r,= + 0.69
P< 0.07
B
s!
I--
6
6
9
potency,
logqounits
7
Skin
test
FIG. 1. Comparison of potency of short ragweed lop, Patients D. S. and ‘J. P. BoHom, Patients symbols denoting individual extracts.)
W.
extracts by skin test end point titrations: R. and J. M. [See legend to Fig. 2 for
significant at p < 0.01. In F’ig. 1 the results of skin test end point titrations in 2 patients are compared. Comparison of potencies tested in I). S. and J. 1’. showed the best agreement (I‘, L- + 0.99), while comparison of the potencies of the various extracts between W. R. and J. M. (P, = + 0.69) showed the worst agreement. However, even in the latter case the potency measurements were strongly related. The potencies of the 11 short ragweed extracts were also measured by RAST inhibition, and the results of these studies are shown in Table I and Fig. 2. As shown in Fig. 2, addition of increasing amounts of short ragweed extract produced increasing inhibition of the binding of IgE antibodies to the solidphase antigens. When these inhibition curves are plotted on semilog paper, they approximate linear relationships in all cases; all the correlation coefficients save one were greater than + 0.99, and that one was + 0.97. Particularly in the region between 30 per cent and 70 per cent inhibition the semilog plots were reasonably linear, and we selected the volume of allergen extract producing 50 per cent inhibition as the end point in these titrations. The results in Big. 2 indicate that the 11 extracts differed by over l,OOO-fold in their potency. Furthermore,
VOLUME NUMBER 100
53 3
Potency
163
extracts
I
7
FIG. 2. Measurement of the potency of extracts of short Potency is determined by the number of microliters needed tion in the RAST. The extracts listed in Table I were tested: 6, 0; 7, ; 8, +; 9,+; 10, o; 11, A.
ragweed by RAST inhibition. to produce 50 per cent reduc1, 0; 2, 0; 3, A; 4, 0; 5, X;
n
TABLE extracts
of allergy
II.
Relationship by skin testing
between the in 7 patients*
measurements
of the
potency
of
short
ragweed
Patients Patients
J. P.
1. c.
W. R.
D. S.
J. B.
J. K.
J. M. J. K. J. B.
0.85 0.87 0.85 0.99 0.90 0.86
0.83 0.74 0.87 0.90 0.75 -
0.69 0.70 0.73 0.89
0.86 0.89 0.85 -
0.85 0.72 -
0.73
-
-
-
Ei. L. c.
---
*Eleven short ragweed extracts were tested in each patient, and the extract potencies between patients were compared by the Spearman rank correlation test. Numbers are correlation coefficients, r,. All are positive. For n = 11 and a one-tailed test, rs values greater than + 0.52 are significant at p < 0.05; r, values greater than + 0.69 are significant at p < 0.01; and r, values greater than + 0.85 are significant at p < 0.0005.
it is evident by simple inspection that in all instances, save one, the inhibition curves are parallel one to another. Comparison of the slopes of the 11 inhibition curves by analysis of covariance did not reveal a difference (F10,33 = 1.30; p > 0.25). The results shown in Fig. 2 represent the pooled data from three individual experiments. In these experiments the potency of individual extracts in the group with 50 per cent inhibition points between 0.1 and 0.6 ~1 of short ragweed extract varied slightly among the three experiments. However, in no case did these variations result in a change in rank order of more than one position. In Fig. 3 inhibition curves for crude ragweed extract, partially purified ragweed extract, and antigens purified from ragweed pollen are shown. Here the results are expressed as per cent inhibition given by the inhibitor in terms of micrograms, because all of the inhibitors used in this experiment were quantified in weight units. The crude ragweed extract was obtained as a lyophilized preparation from Center Laboratories. The more highly purified extract
164
Gleich
FIG. 3. Measurement denotes the partially lyophilized preparation
TABLE III. testing
and
J. ALLERGY
et al.
Comparison by RAST,
of the potency of ragweed antigens by purified antigen prepared in our laboratory. from Center Laboratories. of the AgE, and
potency PNU*
of
short
ragweed
extracts
Rank Paiient
E J:B: J.P. 5-"$ w. xi. *All correlation
RAST
AgE
0.93
0.88 0.84 0.85 0.82 0.81 0.76 0.60
0.79 0.78 0.77 0.74 0.66 0.56 coefficients
correlation
are positive.
For
significance
levels,
CLIN.
IMMUNOL. MARCH 1974
RAST inhibition. ARGRW Crude RW denotes the
as determined
coeiTl&ntr,
by
skin
rs PNU
0.94 0.80 0.85 0.78 0.79 0.66 0.59 see caption to Table II.
produced in our own laboratory was the most potent inhibitor, presumably because it was free of low molecular contaminating materials. The lyophilized crude ragweed extract, and the purified extract prepared in our own laboratory had slopes that did not differ (F,, !) =. 0.72 ; p > 0.25). In contrast, antigens purified from ragweed pollen, particularly antigens K and Ra3, were considerably less potent as inhibitors in the RAST, and the slopes of the inhibition curves produced by antigens K and Ra3 were significantly le,ssthan those given by t,he crude anti partially purified ragweed extract. AgE was clearly a more potent inhibitor than antigens K and Ra3, and on a weight basis compared favorably with the crude ragweed preparation. However, it is notable that the inhibition curve produced by &SE has a lower slope than that given by the crude ragweed extract (F,, y = 37.i; p < 0.001) and by the partially purified ragweed extract ( F1, ,0 -= 27.2; p < 0.001). The experiment with AgE was repeated twice, and in each case the sloptl of the inhibition curve was less than with crude or partially purified extracts. Finally, the same result was seen when serum from a single subject was used as the IgE a.ntibody. Comparison of the estimates of potency of the 11 short ragweed extracts b.y skin test end point titration and by RAST inhibition revealed that these were
VOLUME NUMBER
53 3
Potency
r,= + 0.93 P
4
extracts
165
x o .
.
+
0
.
t
of allergy
l
r, =+0.56 PKO.05
A 11
6
I
q
I
7 Skin
test
8 potency,
FIG. 4. Comparison of potency of short ragweed point titrations: Top, Patient J. M. Bottom, Patient denoting individual extracts.)
I
9
loglO units extracts by RAST and by skin test end W. R. [See legend to Fig. 2 for symbols
remarkably well correlated. These results are shown in Table III and Fig. 4. In Table III the relationship between the potency of short ragweed extracts as determined by RAST inhibition and by skin test end point titration is listed. A one-sided test of significance was applied to these coefficients, and all were significant at p < 0.05 and 5 of the 7 were significant at p < 0.005. In Fig. 4 the best and the worst of these correlations are shown. The AgE and PNU contents of the extracts are listed in Table I, and in Table III the rank correlation coefficients for the relationship between potency as measured by skin test and as measured by AgE and PNU content are shown. Remarkably, both of these compared favorably to the correlations found between potency by RAST and potency by skin testing. All of the correlation coefficients in Table III are positive, and most are significant at p < 0.01. Finally, all of the in vitro measurements of potency were themselves significantly correlated, and in Fig. 5 the relationship between RAST potency and AgE concentration is shown.
166
Gleich
J. ALLERGY
et al.
.2 %
,f$ 4.0 .e 4
0 rs = +0.93 P < 0.001
+
.$ OS! g
.
CLIN.
IMMUNOL. MARCH 197A
x .
0
3.0-
Log,0[AgEl FIG. 5. Relationship concentration. (See
between legend to
potency measurements by Fig. 2 for symbols denoting
RAST inhibition and individual extracts.]
by
AgE
DISCUSSION
These experiments support the studies of Ceska, Eriksson, and Varga10-13 and Foucard and his co-workers,14,I5 indicating that the R.AST procedure may be utilized to measure the potency of pollen extracts. In most of their studies extracts were coupled to cellulose, and the capacity of these solid-phase antigens to bind IgE antibodies was measured. In one report they utilized an inhibition procedure similar to that employed herc.15 The principle of this assay is shown in Fig. 6. In essenceit is based on a competition between solid- and fluid-phase antigens for IgE antibody receptor sites in the first step of the RAST. As the quantity of soluble antigen is increased, fewer IgE antibodies complex with the solid-phase antigen, and the per cent binding of anti-IgE in the second step of the RAST is reduced. By careful titration the quantity of soluble antigen needed to achieve a 50 per cent inhibition of binding can be determined. Moreover, the inhibition responses approximate straight lines and have measurable slope and intercept, thus permitting comparison not only of the quantity of antigen needed for 50 per cent inhibition but also the antigenic relatedness of the various substances.1” We believe that the modifications of the RAST inhibition procedure utilized in this study offer significant advantages for measurement of the potency of allergy extracts. First, the 50 per cent inhibition points were readily determined and allowed precise measurement of the potency of the various extracts. It is remarkable that the range of potency of the extracts, approximately l,OOO-fold, was the same when determined by RAST inhibition and by skin test end point. titration. Second, one can utilize a standardized solid-phase antigen and a standardized pool of IgE antibodies to ragweed antigens. We have found that the solid-phase antigens are remarkably stable when stored as suspensions at do C., so that a preparation of an active allergen could be used for several years.
VOLUME NUMBER
53 3
Potency
Solid
FIG. 6. antigens
of allergy
extracts
167
phase Ag
Diagrammatic representation to IgE antibodies.
of
competitive
binding
Also, one can store the IgE antibodies for considerable
of
soluble
and
solid-phase
periods of time either at -70’ C. or after lyophilization. Finally, by using an aliquot of a designated standard, one could express potencies in terms of this material, thus allowing ready comparison of results from different laboratories. Third, titration of extracts allows one to plot an inhibition curve and, in the case of short ragweed, the results indicate that purified antigens, such as AgE, AgK, and Ra3, were significantly less potent inhibitors of the reaction between IgE antibodies and short ragweed antigens than crude extracts or partially purified extracts. This even held true for AgE, the most potent antigen derived from ragweed pollen, in keeping with the findings of Reisman and associateP using in vivo neutralization of reagins. Thus one can obtain qualitative data indicating whether or not a given extract possesses all of the antigenic determinants recognized by the IgE antibodies. In the case of the 11 extracts we tested, the slopes did not differ, suggesting that all of the extracts possessed a full complement of antigenic determinants characteristic of short ragweed, even though they differed in potency by l,OOO-fold. The simplicity of the RAST inhibition procedure would seem to recommend it for routine quantitation of the potency of short ragweed extracts. Whether these same reagents can be used for the determination of the potency of extracts related to short ragweed or whether one needs individualized reagents for those pollens remains to be determined, although preliminary results suggest that there is extensive cross-reactivity among extracts related to short ragweed. Furthermore, one needs to determine whether this system is applicable to the measurement of the potency of other allergy extracts, specifically those of tree, grass, animal dander, and mold antigens, namely those most significant antigens in allergy practice. Although specific studies must be done to validate the principle in each of these cases, nonetheless it would seem likely, because of the generality of the method, that this procedure will prove useful for the measurement of the potency of extracts of these other allergens. Furthermore, the and his co-workers14l I5 results of Ceska and his associates10-13 and Foucard provide further support for this belief. These results confirm the prior studies of Baer and his associates2 that the
168
Gleich
et al.
J. ALLERGY
CLIN.
IMMUNOL. MARCH 1974
AgE content of extracts and their in viva potency, as measured by skin test, are positively and significantly correlated. However, one difference between this study and the prior study concerns the correlation of potency on the basis of PNU. Here we found significant correlations between skin t.est pot,ency and the PNU content. Furthermore, as shown in Table III, the three in vitro measurements of potency, RAST 50 per cent inhibition units, AgE content, and PNTi, yielded comparable rank correlation coefficients with the potency as determined by, skin testing. Also, the potencies of the 11 extracts as measured in vitro by the three procedures were internally correlated; RAST potency was related to AgE: and PNU potencies, rs = -+ 0.93, and rs =- +- 0.93, respectively, and the AgE potency was related to PNIT, rs = + 0.93. All of these rank order correlation coefficients are significant at p < 0.005 (one-tailed test). Why PNU determina,tions were a good measure of potency in this study and an unsatisfactory method in the previous study is not clear. In any event, the potencies by PR’Ir determinations varied about tenfold, whereas the in vivo potency by skin testing and the in vitro potency by RAST inhibition both showed a l,OOO-fold difference. REFERENCES 1 Stull, A., Cooke, R. A., and Tennant, J.: The allergen content of pollen extracts. Its determination and its deterioration, J. ALLERGY 4: 455j 1933. 2 Baer, H., Godfrey, H., Maloney, C. J., Norman, P. S., and Liohtenstein, L. M.: The potency and antigen E content of commercially prepared ragweed extracts, J. ALLERGY 45: 347, 1970.
and characterization of allergens from the 3 .Johnson, P., and Marsh, D. G.: The isolation pollen of rye grass (LoZizLm perenne), Eur. Polymer J. 1: 63, 1965. 4 Baer, H., Maloney, C. J., Norman, P. S., and Marsh, D. G.: The potency and rye Group I antigen content of commercially prepared grass pollen extracts, J. ALLERGY &IN. IMMUNOL. 51: 116, 1973. (Abst.) 5 Wide, L., Bennich, H., and Johansson, 8. G. 0.: Diagnosis of allergy by an in vitro test for allergen antibodies, Lancet 2: 1106, 1967. 6 Berg, T., Bennich, H., and Johansson, S. G. 0.: In vitro diagnosis of atopic allergy. 1. A comparison between provocation tests and the radioallergosorbent test, Int. Arch. Allergy Appl. Immunol. 40: 770, 1971. 7 Berg, T., and Johansson, 8. 0. 0.: In vitro diagnosis of atopic allergy. IV. Seasonal variations of IgE antibodies in children allergic to pollens. A study of untreated children and of children treated with inhalation of disodium cromoglycate, Int. Arch. Allergy Appl. Immunol. 41: 45,2, 1971. 8 Lichtenstein, L. M., Ishizaka, K., Norman, P. S., Sobotka, A. K., and Hill, 3. M.: IgE antihody measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy, J. Clin. Invest. 52: 472, 1973. 9 Yunginger, J. W., and Gleich, G. J.: Seasonal changes in IgE antibodies and their relationshin to IgG antibodies during immunotherapy for ragweed hay fever, J. Clin. Invest. 52: 1268, 1973. 10 Ceska, M., Eriksson, R., and Varga, J. M.: Radioimmunosorbent assay of allergens, J. ALLERGY CLIN. IMMUNOL. 49: 1, 1972. 11 Ceska, M., Eriksson, R., and Varga, J. M.: Characterization of allergen extracts by polyacrylamide gel isoelectrofoeusing and radioimmunosorbent allergen assay. I. Distribution of allergenic components in birch pollen extracts, Int. Arch. Allergy Appl. Immunol. 42: 430, 1972. 12 Varga, J. M., and Ceska, M.: Characterization of allergen extracts by polyacrylamide gel isoelectrofoeusing and radioimmunosorbent allergen assay. II. Dog and cat allergens, Int. Arch. Allergy Appl. Immunol. 42: 43,8, 1972. 13 Varga, J. M., and Ceska, M.: Characterization of allergen extracts by gel isoeleetrofocusing and radioimmunosorbent allergen assay. Allergens in timothy pollen, J. ALLERQY CLIN. IMMUNOL. 49: 274, 1972.
VOLUME NUMBER
53 3
Potency
of allergy
extracts
169
14 Foueard, T., Bennich, II., and Johansson, 8. G. 0.: Studies on the stability of diluted allergen extracts using the radioallergosorbdnt test (RAST), Clin. Allergy 3: 91, 19’73. 16 Foucard, T., Johansson, S. G. O., Bennich, H., and Berg, T.: In vitro estimation of allergens by a radioimmune antiglobulin technique using human IgE antibodies, Int. Arch. Allergy Appl. Immunol. 43: 360, 1972. 16 Sheldon, J. M., Lovell, R. G., and Mathews, K. P.: A manual of clinical allergy, Philadelphia, 1967, W. B. Saunders Company, p. 516. 17 Mancini, G., Carbonara, A. O., and Heremans, J. F.: Immunochemical quantitation of antigens by single radial immunodiffusion, Immunochemistry 2: 235, 1965. 18 Dixon, W. J., and Massey, F. J., Jr.: Introduction to statistical analysis, New York, 1957, McGraw-Hill Book Company, Inc., pp. 209 and 294. 19 Yunginger, J. W., and Gleich, G. J.: Measurement of ragweed antigen E by double antibody radioimmunoassay,J. ALLERQY CLIN.IMMUNOL.~O: 326,1972. 20 Reisman, R. E., Bonstein, H. S., Rose, N. R., and Arbesman, C. E.: Studies of purified fractions of ragweed pollen, J. ALLERQY 35: 227, 1964.
Select the Foundation Question
ONE best of America 3.
answer for the Self-Assessment
In the treatment of effect of theophylline
following Program:
question
patients with asthma, compounds is
from
the
most
the
Allergy
important
(A) stimulation of the medullary respiratory center (B) inhibition of immunologic release of histamine from leukocytes (C) pulmonary arteriolar dilation (D) relaxation of bronchiolar smooth muscle (E) mucolytic activity through reduction of mucopolysaccharide and protein content of sputum The correct this Journal.
answer
and
bibliographic
references
will
be found
on page
188
of
M.D.,
J. B. Larson,
M.D.,
R. T. Jones,
B.S., and
H. Baer,
Ph.D.
Rochester, iMin?l., and Rockville, Md.
The potencies of 11 tmmeroial extracts of short ragweed pollen were cc&yzed by skin test end point titration. and compared to potency as measwred in vitro: (I) by the radioallergosorbent test (RAST), (2’) by AgE concentrations, and (3j by protein nitrogen determinations (PNU). RAST potency was determined by the capacity of the extract to inhibit the binding of IgE antibody to solid-phase allergen in the first step of the RAST, and it was expressed as the quantity of extract needed for 50 p cent in,hibition of binding. Potencies determined by skin testing in. 7 patients zere strongly related among the various patients in spite of a l,OOO-fold difference in the patients’ sensitivity to the extracts. Similarly, potency as measured by RAST inhibition showed about n l,OOO-fold difference among the extracts, and the RAST potencies were strongly related to potency as measzlred by skin testing. Puri$ed antigens derived from short ragweed inhibited binding of IgE antibodies, but th,e slopes of the inhibition owrves were significantly less than those produced by crude or partially purified short ragweed. AgE concentration and PNU concentrations also were correlated with skin test potency, but the range of potencies was less than that found by RAST inhibition. Measwenaent of the potency of estraots by BAST inhibition should prove useful as a general procedure for the standardization of allergy extracts.
At the present time the potency of allergy extracts is usually listed as the number of protein nitrogen units (PNU) per milliliter of extract* or simply in terms of weight per volume of solute to solvent used to prepare the extract. In the case of ragweed pollens and grass pollens, purified antigens that appear to account for the bulk of the allergenic properties of these materials may be used as measuresof the potency of these extracts.?-* In particular, the studies of Baer and his associates2demonstrate that the in vivo reactivity of short ragweed extracts correlates extremely well with the antigen E (AgE) content of the extracts,% and for short ragweed extract AgE could be used as a measure of t,he potency of the extract. Since the introduction of the RAST for the measurement of IgE antibodies by Wide, Bennich, and Johansson,” this test has been utilized both for the diagnosis of allergy to various antigens, 6 for the measurement of changes in IgE From the Mayo Clinic and Mayo Foundation, Departments of Medicine, Immunology, and Microbiology, The Allergic Diseases Research Laboratory, Rochester, and the Bureau of Biologic%, Food and Drug Administration, Rockvillc. Supported in part by a grant from the Food and Drug Administration, Bureau of Biologies, NIH-DBS-72-2087. Received for publication July 16, 1973. Reprint requests to: Dr. Gerald J. Gleieh, Allergic Diseases Research Laboratory, Mayo Clinic and Mayo Foundation, Rochester, Minn. 55901. Vol.
53, No. 3, pp. 158-169
VOLUME NUMBER
53 3
Potency of allergy
extracts
159
antibodies to specific antigens,T-9 and, finally, for the measurement of the potency of allergy extracts.10-15 For the measurement of the potency of allergy extracts by RAST, two general approaches have been used: (1) extracts have been coupled to microcrystalline cellulose and the quantity of extract needed to achieve a maximum in the RAST determined, and (2) extracts have been tested to determine their inhibitory capacity in the first step of the RAST. In this study we have measured the potency of short ragweed extracts by determining their capacity to inhibit the binding of IgE antibodies to ragweed antigens in the first step of the RAST and have compared these measurements of potency to those obtained by skin test end point titrations, to AgE concentrations, and to PNU concentrations. MATERIALS AND METHODS Allergens and allergenic extracts Extracts of short ragweed (Ambrosia elation) were purchased from 11 different companies in the United States. All but two of these were nonglycerinated. The manufacturers’ claimed potency in terms of weight per volume or PNU measurement of potency for these extracts is shown in Table I. AgE was obtained from the National Institute of Allergy and Infectious Disease through the courtesy of Dr. Charles Kirkpatrick. Antigen K (AgK) and antigen Ra3 (Ra3) were purchased from Worthington Biochemicals, Freehold, New Jersey. Lyophilized short ragweed extract was a gift from Mr. George Center, Center Laboratories, Port Washington, New York (Lot 00802FD). Partially purified ragweed extract was prepared by extraction of defatted pollen with distilled water followed by gel filtration of this extract on a column of Sephadex G-25. The void volume peak was concentrated using an Amicon ultrafiltration device and a PM-10 membrane and then passed through the G-25 column a second time. After reconcentration and dialysis against 2 mM NH,HCO,, the solution was dried for 36 hours at 68” C. The dry weight of extract was 26 mg. per milliliter. In Fig. 3 this partially purified extract is labeled BEG-RW.
Skin testing To assay the in vivo potency of the crude short ragweed pollen extracts, we determined that dilution of the extract that would yield a standard 6 x 6 mm. (2+) wheal-and-flare reaction 15 minutes after the intradermal injection of extract sufficient to produce an initial 3 x 3 mm. wheal. The extracts were initially diluted so as to contain approximately 5,000 PNU per milliliter using pH 7.4 phosphate-buffered saline containing 0.5 per cent human serum albumin. This buffer was both isotonic and iso-osmolar when compared to human serum. If only the weight per volume potency was supplied by the manufacturer, we estimated the initial dilution using the relationship between weight per volume and PNU suggested by Sheldon, Lovell, and MathewsIs Fivefold dilutions of the extracts were prepared, and 7 patients with varying degrees of sensitivity to short ragweed were tested to determine the dilution producing a 6 x 6 wheal. The dilutions were prepared and used within 4 weeks of preparation. Between testing periods the extracts were kept at 4” C. but were brought up to room temperature during the actual testing procedure. In cases in which an exact 6 x 6 wheal was not obtained, the end point was extrapolated to a value between the dilutions that bracketed the hypothetic 6 x 6 wheal. The potency of the extract was expressed in terms of skin test units, i.e., the number of dilutions necessary to yield that concentration producing a 6 x 6 wheal. For example, the extracts were initially diluted to approximately 5,000 PNU per milliliter and subsequently diluted serially 1 to 5. If the fifth of these dilutions, containing approximately 1.6 PNU per milliliter, yielded a 2+ reaction, then the potency of the resultant extract would be 55 times the initial dilution of the original crude extract obtained from the manufacturer. In this way the number of skin test allergenic units were determined for each of the 11 extracts. In actual practice a given extract was tested at three skin sites on the patient’s forearm with dilutions
160
Gleich
TABLE
I.
et al.
Potency
of
Short ragweed extract
1 l t
20 3A 40 5x ti0 7w
extracts
of
Designated potency
4/26/74 l/3/74 4/25/74
l-10
10/24/73 7/l/73 l/k)74
represent
A# &g/ml.)
1076
4,546 317.5
48,000 20,600
110 111
8,333 4,762
l-10
to
1,724 2oo.f! i 8.0 13.9
11,364 2,500 41167
ifi
153
as
the
number
the
various
of
extracts
5Ocjr, RAST in the
inhibition
units
per
milliliter.
figures.
differing 25-fold in concentration in order to determine the dilution of extract that yielded an approximate 6 x 6 response. The particular dilutions used in a patient were determined from preliminary estimates of his sensitivity based on skin tests done at the time of the original diagnosis of his ragweed hay fever. Usually one of these preliminary skin tests on the forearm gave a reaction close to 6 x 6; if not, appropriate dilutions above or below those originally selected were placed on the forearm skin in order to determine which dilution approximated a 6 x 6 reaction. The final tests to defme the end point were performed in duplicate on the upper arm. Three dilutions were tested: (1) the dilution yielding about a 6 x 6 wheal, (2) a dilution fivefold more concentrated, and (3) a dilution fivefold less concentrated. To test ttte 11 extracts it was necessary to carry out these tests over a 2 day period.
Measurement
of the potency
RAST
Potency in vitro 50% RAST inhibition w. per ml.1
36,300 16,900
l-20
used
AgE,
PNU)
u/7/75
tSymbols
PNU,
l-20
4/26/74 defined
by
vitro
l-20 1-33 5% 40.000 PNU ’ l-10
11 A
is
in
IMMUNOL. MARCH 1974
44 6 0 0 205
10 0 vitro units.
measured
CLIN.
18,500 8,250 9.350 4;560 48,100 19.000 35;200
l/11/74 7/12/73
in
as
PNU/ml.
5%
(27,080
*Potency inhibition
ragweed
EXpiratiOll date
ii/+75
:t
short
1. ALLERGY
of ragweed
extracts
by RAST inhibition
The RAST procedure was carried out as described previously9 with the following modifications : (1) for the solid-phase ragweed antigen, lyophilizod short ragweed obtained from Center Laboratories was covalently linked to microcrystalline cellulose at a rat,io of 10 mg. of lyophilized powder per 500 mg. of cellulose; (2) in the second stage of’ the KAST 20 ng. of anti-IgE labeled with 1311 were used rather than 2 ng.; and (3: aliquots of a serum pool prepared by mixing equal volumes of serum from 5 subjects highly allergic to short ragweed pollen were used in the first step of the RAST to assure that most antigens recognized by TgE antibodies would he detected in the RAST inhibition procedure. None of these patients had ever been treated by hyposensitization. 111 the actual performance of the test, first, inhibitor in RAST diluent was added in 100 ~1 of a 1:5 dilution of pooled allergic serum in a volume up to 400 pl. Second, RAST diluent was added, and the volume was adjusted to 500 ~1 when necessary with additional RAST diluent. Finally, within a few minutes of the above additions 500 pl of the solid-phase ragweed antigen at a concentration of 1 mg. per milliliter was added, and the tubes were capped and rotated overnight at room temperature. Following washing, 0.65 ml. of RAST diluent was added, followed by 20 ng. of anti-IgE in pH 7.5 phosphate albumin in a volume of 1OQ ~1. The RAST diluent consisted of pH 7.5 0.1 M KH,PO,-K,HPO,, 470 ml.; fetal calf serum (Grand Island Biologicals, Grand Island, New York), 20 ml.; 10 per cant NaN,, 5 ml. ; and tween 20, 5 ml. When reacted with 0.5 mg. solid-phase ragweed antigen, the 100 pl of the 1:5 dilution of the allergic serum pool yielded a maximum of 19 per cent to 22 per cent binding, while controls, either normal human serum or fetal calf serum, yielded a maximum of 1.2 per cent
For
VOLUME NUMBER
inhibition,
53 3
and
Potency
in vivo
by
skin
of allergy
161
extracts
testing*
(skin
Potency in viva test units x lo61
D. S.
L. c.
J. K.
J. M.
J. B.
W. R.
J. P.
5.39
37.8 3.17 17.2 34.4 48.8 68.4 16.8 377 52.7
163 222 1”:9 244. 104 1,050 1,070 30.5
0.121 0.078 0.078 0.036 9.76 0.183 1.02 9.77 0.169
0.0614 0.0133 0.0566 0.0032 0.390 0.109 0.236 0.265 0.0975
139.1 8.73 16.3 2.06 67.4 34.8 77.5 601 35.3
5.48 1.80 1.41 1.17 6.64 3.32 16.8 229 3.95
19.5 67.4
137 244
1.29 1.95
0.0645 0.226
19.5 122
2.29 1.33 0.15 5.55 2.73 20.4 244 2.48 3.91 9.77 example,
if
5 ~1 yielded
508%
inhibition,
then
one ml.
of the
extract
contains
4.82 12.0 1000 L-5
=
200 RAST
binding and usually less than 1 per cent binding. In the performance of the RAST inhibition test, varying quantities of extracts were added to determine that amount necessary to produce a 50 per cent inhibition in the binding of IgE antibody to the solid-phase antigen, In all cases the negative control with normal human or fetal calf serum, whichever was less, was subtracted from all of the tests.
Radial immunodiffusion nitrogen
for measurement
The concentration of AgE in values were extrapolated from a and anti-AgE. The number of phosphotungstic acid and analysis
Statistical
tests
Results covariance.is evaluations.
were evaluated A programmable
by
of AgE
and
measurement
of protein
each extract was measured by radial immunodiffusion,ir standard curve obtained by the use of U. 8. reference PNU per milliliter was determined by precipitation of the dissolved precipitate.
tests of rank Hewlett-Packard
and AgE with
correlation coefiicients and by analysis of calculator was used for the statistical
9810A
RESULTS
The results of the determinations of the biologic potency of the allergy extracts in vivo by skin test end point titration are shown in Table I. It is remarkable that even though these extracts were supposedly close to the same potency in terms of the manufacturers’ estimate (either weight per volume or PNU), nonetheless their potency by skin test titration varied by almost l,OOOfold. To determine whether the skin test procedure yielded reliable estimates of the potency of the extracts among the patients, we determined the correlations between the potency in various patients, and the results are shown in Table II. All of the rank correlation coefficients in Table II are positive and indicate that the skin test procedure yielded remarkably good agreement between the measurements of potencies of the extracts in different patients. Most of the rank correlation coefficients in Table II are significant at p < 0.0005 and all are
162
Gleich
J. ALLERGY
et al.
CLIN.
IMMUNOL. MARCH 1974
s
87
r,= + 0.69
P< 0.07
B
s!
I--
6
6
9
potency,
logqounits
7
Skin
test
FIG. 1. Comparison of potency of short ragweed lop, Patients D. S. and ‘J. P. BoHom, Patients symbols denoting individual extracts.)
W.
extracts by skin test end point titrations: R. and J. M. [See legend to Fig. 2 for
significant at p < 0.01. In F’ig. 1 the results of skin test end point titrations in 2 patients are compared. Comparison of potencies tested in I). S. and J. 1’. showed the best agreement (I‘, L- + 0.99), while comparison of the potencies of the various extracts between W. R. and J. M. (P, = + 0.69) showed the worst agreement. However, even in the latter case the potency measurements were strongly related. The potencies of the 11 short ragweed extracts were also measured by RAST inhibition, and the results of these studies are shown in Table I and Fig. 2. As shown in Fig. 2, addition of increasing amounts of short ragweed extract produced increasing inhibition of the binding of IgE antibodies to the solidphase antigens. When these inhibition curves are plotted on semilog paper, they approximate linear relationships in all cases; all the correlation coefficients save one were greater than + 0.99, and that one was + 0.97. Particularly in the region between 30 per cent and 70 per cent inhibition the semilog plots were reasonably linear, and we selected the volume of allergen extract producing 50 per cent inhibition as the end point in these titrations. The results in Big. 2 indicate that the 11 extracts differed by over l,OOO-fold in their potency. Furthermore,
VOLUME NUMBER 100
53 3
Potency
163
extracts
I
7
FIG. 2. Measurement of the potency of extracts of short Potency is determined by the number of microliters needed tion in the RAST. The extracts listed in Table I were tested: 6, 0; 7, ; 8, +; 9,+; 10, o; 11, A.
ragweed by RAST inhibition. to produce 50 per cent reduc1, 0; 2, 0; 3, A; 4, 0; 5, X;
n
TABLE extracts
of allergy
II.
Relationship by skin testing
between the in 7 patients*
measurements
of the
potency
of
short
ragweed
Patients Patients
J. P.
1. c.
W. R.
D. S.
J. B.
J. K.
J. M. J. K. J. B.
0.85 0.87 0.85 0.99 0.90 0.86
0.83 0.74 0.87 0.90 0.75 -
0.69 0.70 0.73 0.89
0.86 0.89 0.85 -
0.85 0.72 -
0.73
-
-
-
Ei. L. c.
---
*Eleven short ragweed extracts were tested in each patient, and the extract potencies between patients were compared by the Spearman rank correlation test. Numbers are correlation coefficients, r,. All are positive. For n = 11 and a one-tailed test, rs values greater than + 0.52 are significant at p < 0.05; r, values greater than + 0.69 are significant at p < 0.01; and r, values greater than + 0.85 are significant at p < 0.0005.
it is evident by simple inspection that in all instances, save one, the inhibition curves are parallel one to another. Comparison of the slopes of the 11 inhibition curves by analysis of covariance did not reveal a difference (F10,33 = 1.30; p > 0.25). The results shown in Fig. 2 represent the pooled data from three individual experiments. In these experiments the potency of individual extracts in the group with 50 per cent inhibition points between 0.1 and 0.6 ~1 of short ragweed extract varied slightly among the three experiments. However, in no case did these variations result in a change in rank order of more than one position. In Fig. 3 inhibition curves for crude ragweed extract, partially purified ragweed extract, and antigens purified from ragweed pollen are shown. Here the results are expressed as per cent inhibition given by the inhibitor in terms of micrograms, because all of the inhibitors used in this experiment were quantified in weight units. The crude ragweed extract was obtained as a lyophilized preparation from Center Laboratories. The more highly purified extract
164
Gleich
FIG. 3. Measurement denotes the partially lyophilized preparation
TABLE III. testing
and
J. ALLERGY
et al.
Comparison by RAST,
of the potency of ragweed antigens by purified antigen prepared in our laboratory. from Center Laboratories. of the AgE, and
potency PNU*
of
short
ragweed
extracts
Rank Paiient
E J:B: J.P. 5-"$ w. xi. *All correlation
RAST
AgE
0.93
0.88 0.84 0.85 0.82 0.81 0.76 0.60
0.79 0.78 0.77 0.74 0.66 0.56 coefficients
correlation
are positive.
For
significance
levels,
CLIN.
IMMUNOL. MARCH 1974
RAST inhibition. ARGRW Crude RW denotes the
as determined
coeiTl&ntr,
by
skin
rs PNU
0.94 0.80 0.85 0.78 0.79 0.66 0.59 see caption to Table II.
produced in our own laboratory was the most potent inhibitor, presumably because it was free of low molecular contaminating materials. The lyophilized crude ragweed extract, and the purified extract prepared in our own laboratory had slopes that did not differ (F,, !) =. 0.72 ; p > 0.25). In contrast, antigens purified from ragweed pollen, particularly antigens K and Ra3, were considerably less potent as inhibitors in the RAST, and the slopes of the inhibition curves produced by antigens K and Ra3 were significantly le,ssthan those given by t,he crude anti partially purified ragweed extract. AgE was clearly a more potent inhibitor than antigens K and Ra3, and on a weight basis compared favorably with the crude ragweed preparation. However, it is notable that the inhibition curve produced by &SE has a lower slope than that given by the crude ragweed extract (F,, y = 37.i; p < 0.001) and by the partially purified ragweed extract ( F1, ,0 -= 27.2; p < 0.001). The experiment with AgE was repeated twice, and in each case the sloptl of the inhibition curve was less than with crude or partially purified extracts. Finally, the same result was seen when serum from a single subject was used as the IgE a.ntibody. Comparison of the estimates of potency of the 11 short ragweed extracts b.y skin test end point titration and by RAST inhibition revealed that these were
VOLUME NUMBER
53 3
Potency
r,= + 0.93 P
4
extracts
165
x o .
.
+
0
.
t
of allergy
l
r, =+0.56 PKO.05
A 11
6
I
q
I
7 Skin
test
8 potency,
FIG. 4. Comparison of potency of short ragweed point titrations: Top, Patient J. M. Bottom, Patient denoting individual extracts.)
I
9
loglO units extracts by RAST and by skin test end W. R. [See legend to Fig. 2 for symbols
remarkably well correlated. These results are shown in Table III and Fig. 4. In Table III the relationship between the potency of short ragweed extracts as determined by RAST inhibition and by skin test end point titration is listed. A one-sided test of significance was applied to these coefficients, and all were significant at p < 0.05 and 5 of the 7 were significant at p < 0.005. In Fig. 4 the best and the worst of these correlations are shown. The AgE and PNU contents of the extracts are listed in Table I, and in Table III the rank correlation coefficients for the relationship between potency as measured by skin test and as measured by AgE and PNU content are shown. Remarkably, both of these compared favorably to the correlations found between potency by RAST and potency by skin testing. All of the correlation coefficients in Table III are positive, and most are significant at p < 0.01. Finally, all of the in vitro measurements of potency were themselves significantly correlated, and in Fig. 5 the relationship between RAST potency and AgE concentration is shown.
166
Gleich
J. ALLERGY
et al.
.2 %
,f$ 4.0 .e 4
0 rs = +0.93 P < 0.001
+
.$ OS! g
.
CLIN.
IMMUNOL. MARCH 197A
x .
0
3.0-
Log,0[AgEl FIG. 5. Relationship concentration. (See
between legend to
potency measurements by Fig. 2 for symbols denoting
RAST inhibition and individual extracts.]
by
AgE
DISCUSSION
These experiments support the studies of Ceska, Eriksson, and Varga10-13 and Foucard and his co-workers,14,I5 indicating that the R.AST procedure may be utilized to measure the potency of pollen extracts. In most of their studies extracts were coupled to cellulose, and the capacity of these solid-phase antigens to bind IgE antibodies was measured. In one report they utilized an inhibition procedure similar to that employed herc.15 The principle of this assay is shown in Fig. 6. In essenceit is based on a competition between solid- and fluid-phase antigens for IgE antibody receptor sites in the first step of the RAST. As the quantity of soluble antigen is increased, fewer IgE antibodies complex with the solid-phase antigen, and the per cent binding of anti-IgE in the second step of the RAST is reduced. By careful titration the quantity of soluble antigen needed to achieve a 50 per cent inhibition of binding can be determined. Moreover, the inhibition responses approximate straight lines and have measurable slope and intercept, thus permitting comparison not only of the quantity of antigen needed for 50 per cent inhibition but also the antigenic relatedness of the various substances.1” We believe that the modifications of the RAST inhibition procedure utilized in this study offer significant advantages for measurement of the potency of allergy extracts. First, the 50 per cent inhibition points were readily determined and allowed precise measurement of the potency of the various extracts. It is remarkable that the range of potency of the extracts, approximately l,OOO-fold, was the same when determined by RAST inhibition and by skin test end point. titration. Second, one can utilize a standardized solid-phase antigen and a standardized pool of IgE antibodies to ragweed antigens. We have found that the solid-phase antigens are remarkably stable when stored as suspensions at do C., so that a preparation of an active allergen could be used for several years.
VOLUME NUMBER
53 3
Potency
Solid
FIG. 6. antigens
of allergy
extracts
167
phase Ag
Diagrammatic representation to IgE antibodies.
of
competitive
binding
Also, one can store the IgE antibodies for considerable
of
soluble
and
solid-phase
periods of time either at -70’ C. or after lyophilization. Finally, by using an aliquot of a designated standard, one could express potencies in terms of this material, thus allowing ready comparison of results from different laboratories. Third, titration of extracts allows one to plot an inhibition curve and, in the case of short ragweed, the results indicate that purified antigens, such as AgE, AgK, and Ra3, were significantly less potent inhibitors of the reaction between IgE antibodies and short ragweed antigens than crude extracts or partially purified extracts. This even held true for AgE, the most potent antigen derived from ragweed pollen, in keeping with the findings of Reisman and associateP using in vivo neutralization of reagins. Thus one can obtain qualitative data indicating whether or not a given extract possesses all of the antigenic determinants recognized by the IgE antibodies. In the case of the 11 extracts we tested, the slopes did not differ, suggesting that all of the extracts possessed a full complement of antigenic determinants characteristic of short ragweed, even though they differed in potency by l,OOO-fold. The simplicity of the RAST inhibition procedure would seem to recommend it for routine quantitation of the potency of short ragweed extracts. Whether these same reagents can be used for the determination of the potency of extracts related to short ragweed or whether one needs individualized reagents for those pollens remains to be determined, although preliminary results suggest that there is extensive cross-reactivity among extracts related to short ragweed. Furthermore, one needs to determine whether this system is applicable to the measurement of the potency of other allergy extracts, specifically those of tree, grass, animal dander, and mold antigens, namely those most significant antigens in allergy practice. Although specific studies must be done to validate the principle in each of these cases, nonetheless it would seem likely, because of the generality of the method, that this procedure will prove useful for the measurement of the potency of extracts of these other allergens. Furthermore, the and his co-workers14l I5 results of Ceska and his associates10-13 and Foucard provide further support for this belief. These results confirm the prior studies of Baer and his associates2 that the
168
Gleich
et al.
J. ALLERGY
CLIN.
IMMUNOL. MARCH 1974
AgE content of extracts and their in viva potency, as measured by skin test, are positively and significantly correlated. However, one difference between this study and the prior study concerns the correlation of potency on the basis of PNU. Here we found significant correlations between skin t.est pot,ency and the PNU content. Furthermore, as shown in Table III, the three in vitro measurements of potency, RAST 50 per cent inhibition units, AgE content, and PNTi, yielded comparable rank correlation coefficients with the potency as determined by, skin testing. Also, the potencies of the 11 extracts as measured in vitro by the three procedures were internally correlated; RAST potency was related to AgE: and PNU potencies, rs = -+ 0.93, and rs =- +- 0.93, respectively, and the AgE potency was related to PNIT, rs = + 0.93. All of these rank order correlation coefficients are significant at p < 0.005 (one-tailed test). Why PNU determina,tions were a good measure of potency in this study and an unsatisfactory method in the previous study is not clear. In any event, the potencies by PR’Ir determinations varied about tenfold, whereas the in vivo potency by skin testing and the in vitro potency by RAST inhibition both showed a l,OOO-fold difference. REFERENCES 1 Stull, A., Cooke, R. A., and Tennant, J.: The allergen content of pollen extracts. Its determination and its deterioration, J. ALLERGY 4: 455j 1933. 2 Baer, H., Godfrey, H., Maloney, C. J., Norman, P. S., and Liohtenstein, L. M.: The potency and antigen E content of commercially prepared ragweed extracts, J. ALLERGY 45: 347, 1970.
and characterization of allergens from the 3 .Johnson, P., and Marsh, D. G.: The isolation pollen of rye grass (LoZizLm perenne), Eur. Polymer J. 1: 63, 1965. 4 Baer, H., Maloney, C. J., Norman, P. S., and Marsh, D. G.: The potency and rye Group I antigen content of commercially prepared grass pollen extracts, J. ALLERGY &IN. IMMUNOL. 51: 116, 1973. (Abst.) 5 Wide, L., Bennich, H., and Johansson, 8. G. 0.: Diagnosis of allergy by an in vitro test for allergen antibodies, Lancet 2: 1106, 1967. 6 Berg, T., Bennich, H., and Johansson, S. G. 0.: In vitro diagnosis of atopic allergy. 1. A comparison between provocation tests and the radioallergosorbent test, Int. Arch. Allergy Appl. Immunol. 40: 770, 1971. 7 Berg, T., and Johansson, 8. 0. 0.: In vitro diagnosis of atopic allergy. IV. Seasonal variations of IgE antibodies in children allergic to pollens. A study of untreated children and of children treated with inhalation of disodium cromoglycate, Int. Arch. Allergy Appl. Immunol. 41: 45,2, 1971. 8 Lichtenstein, L. M., Ishizaka, K., Norman, P. S., Sobotka, A. K., and Hill, 3. M.: IgE antihody measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy, J. Clin. Invest. 52: 472, 1973. 9 Yunginger, J. W., and Gleich, G. J.: Seasonal changes in IgE antibodies and their relationshin to IgG antibodies during immunotherapy for ragweed hay fever, J. Clin. Invest. 52: 1268, 1973. 10 Ceska, M., Eriksson, R., and Varga, J. M.: Radioimmunosorbent assay of allergens, J. ALLERGY CLIN. IMMUNOL. 49: 1, 1972. 11 Ceska, M., Eriksson, R., and Varga, J. M.: Characterization of allergen extracts by polyacrylamide gel isoelectrofoeusing and radioimmunosorbent allergen assay. I. Distribution of allergenic components in birch pollen extracts, Int. Arch. Allergy Appl. Immunol. 42: 430, 1972. 12 Varga, J. M., and Ceska, M.: Characterization of allergen extracts by polyacrylamide gel isoelectrofoeusing and radioimmunosorbent allergen assay. II. Dog and cat allergens, Int. Arch. Allergy Appl. Immunol. 42: 43,8, 1972. 13 Varga, J. M., and Ceska, M.: Characterization of allergen extracts by gel isoeleetrofocusing and radioimmunosorbent allergen assay. Allergens in timothy pollen, J. ALLERQY CLIN. IMMUNOL. 49: 274, 1972.
VOLUME NUMBER
53 3
Potency
of allergy
extracts
169
14 Foueard, T., Bennich, II., and Johansson, 8. G. 0.: Studies on the stability of diluted allergen extracts using the radioallergosorbdnt test (RAST), Clin. Allergy 3: 91, 19’73. 16 Foucard, T., Johansson, S. G. O., Bennich, H., and Berg, T.: In vitro estimation of allergens by a radioimmune antiglobulin technique using human IgE antibodies, Int. Arch. Allergy Appl. Immunol. 43: 360, 1972. 16 Sheldon, J. M., Lovell, R. G., and Mathews, K. P.: A manual of clinical allergy, Philadelphia, 1967, W. B. Saunders Company, p. 516. 17 Mancini, G., Carbonara, A. O., and Heremans, J. F.: Immunochemical quantitation of antigens by single radial immunodiffusion, Immunochemistry 2: 235, 1965. 18 Dixon, W. J., and Massey, F. J., Jr.: Introduction to statistical analysis, New York, 1957, McGraw-Hill Book Company, Inc., pp. 209 and 294. 19 Yunginger, J. W., and Gleich, G. J.: Measurement of ragweed antigen E by double antibody radioimmunoassay,J. ALLERQY CLIN.IMMUNOL.~O: 326,1972. 20 Reisman, R. E., Bonstein, H. S., Rose, N. R., and Arbesman, C. E.: Studies of purified fractions of ragweed pollen, J. ALLERQY 35: 227, 1964.
Select the Foundation Question
ONE best of America 3.
answer for the Self-Assessment
In the treatment of effect of theophylline
following Program:
question
patients with asthma, compounds is
from
the
most
the
Allergy
important
(A) stimulation of the medullary respiratory center (B) inhibition of immunologic release of histamine from leukocytes (C) pulmonary arteriolar dilation (D) relaxation of bronchiolar smooth muscle (E) mucolytic activity through reduction of mucopolysaccharide and protein content of sputum The correct this Journal.
answer
and
bibliographic
references
will
be found
on page
188
of