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A rast assay for monitoring specific IgE antibodies in the mouse
Journal o f Immunological Methods, 47 (1981) 309--320
309
Elsevier/North-Holland Biomedical Press
A RAST ASSAY FO R M O N I T O R I N G SPECIFIC IgE ANTIBODIES IN THE MOUSE
M.A. KINGS and A.L. DE WECK Institute o f Clinical Immunology, Berne, Switzerland
(Received 5 March 1981, accepted 23 July 1981) Mouse monoclonal anti-ovalbumin IgE was isolated from serum of BALB/c mice implanted with the IgE secreting hybridoma (14-205). Purified IgE was used to immunize rabbits and for the preparation of an antiserum specific for mouse IgE. The latter was used to develop a RAST assay to replace the PCA technique in monitoring antigen-specific IgE responses in the mouse.
INTRODUCTION RAST (Radioallergosorbent Test) is a diagnostic test based u p o n the d e t ectio n o f allergen specific antibodies of the IgE t y p e by 12SI-labelled antiIgE on Sephadex particles (Wide et al., 1967) or paper discs (Ceska et al., 1972), to which allergens have been chemically coupled. Subsequently this technique has been adapted f or ot he r a n t i b o d y classes bot h in man and animals. Of particular interest are studies in rats and mice, which are bot h used as models o f IgE mediated allergic diseases. Fo r a long time, passive cutaneous anaphylaxis (PCA) has been the only means of detecting h o m o c y t o t r o p i c antibodies o f the IgE class. Radioimmunoassays (RIAs) for the rat system have been developed (Pauwels et al., 1977a; Karlsson et al., 1979), because . o f the availability of rat m y e l o m a IgE. However, owing to the unavailability o f mouse IgE myelomas, 24 or 48 h PCA tests carried o u t in the rat or mouse have remained until n o w the only means of assaying mouse IgE. The advent o f mo n o c l onal mouse IgEs p r o d u c e d by the h y b r i d o m a technique (BSttcher and H~immerling, 1978) and directed against such antigens as the d i n i t r o p h e n y l h a pt e n (DNP) or ovalbumin (OVA) has made it possible to obtain larger a m o u n t s o f mouse IgE and has allowed RIAs to be developed for total and specific mouse IgE (Eshhar et al., 1980; Kelly et al., 1980; Liu et al., 1980). The RAST m e t h o d described here uses antigen-coated paper discs and [ 12 s I ] r ab b it anti-mouse IgE for estimation o f mouse specific antibodies directed against benzylpenicilloyl-ovalbumin (BPO-OVA).
0022-1759/81/0000---O000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
310 MATERIALS AND METHODS Activation and preparation of antigen coated discs Ovalbumin was purchased from Sigma Chemicals, U.S.A. BPO22-OVA (ovalbumin carrying 22 benzylpenicilloyl groups) was prepared and analyzed as described elsewhere (Nakagawa et al., 1980). Filter paper discs, diameter 5.0 mm, t y p e 589/3 (Schleicher Schuell, Feldbach, F.R.G.) were activated and subsequently coated with the allergens BPO22OVA or OVA by the method of Lee and Heiner (1978). Immunization o f mice for monitoring IgE responses Female BALB/c mice, 6--8 weeks old (G.I. Bomholtgaard, Ry, Denmark), were given a primary injection (i.p.) of 1 pg BPOv.5-OVA in 1 mg AI(OH)3, and booster injections by the same route. The mice were bled (0.2 ml) from the orbital sinus, and the serum samples tested consisted of a pooled serum from 5 mice. Each serum pool was titrated by passive cutaneous anaphylaxis (PCA) in 250--300 g Wistar rats from our own colony. The sensitization period was 48 h, and the end-point expressed as the reciprocal of antiserum dilution yielding a reaction 5 mm in diameter. Isolation and purification o f mouse IgE Ovalbumin-specific monoclonal IgE antibodies (Kindly donated by Dr. K. Turner) were obtained from serum of BALB/c mice (PCA titer 1/160,000) in which the IgE-secreting hybridoma (14-205; BSttcher and H~immerling, 1978) was propagated in vivo. This serum was diluted 1 : 1 in 0.5 M TrisHC1-NaC1 buffer, pH 8.0, and applied directly to an immunoabsorbent column (10 cm X 1 cm) of 10 mg ovalbumin coupled to every 1 g of CNBractivated Sepharose (Pharmacia, Uppsala, Sweden). The IgE eluted with 0.2 M glycine-HC1 buffer, pH 2.4, was immediately adjusted to pH 8.2 with 1 M Tris (Merck, Darmstadt, F.R.G.) and concentrated by vacuum dialysis. The IgE fraction was tested for purity by conventional immunoelectrophoresis (IEP) and Ouchterlony techniques, using various commercial antimouse immunoglobulin antisera (Nordic, Immunological laboratories, The Netherlands). IgG1 was found to be a major contaminant (approx. 30%) of the IgE fraction. However, in view of the low concentration of IgE contained in the serum, further absorption was avoided and the IgE fraction was injected as such into an outbred rabbit for antiserum production. Subsequent purification of IgE was achieved by coupling rabbit anti-IgE antibodies (precipitated with 33% saturated ammonium sulfate) to CNBractivated Sepharose. The 0.2 M glycine-HC1 eluted IgE fraction reacted in immunodiffusion with rabbit anti-mouse IgE and anti-mouse IgE (supplied by Drs. Lee and Sehon), b u t n o t with anti-mouse IgG, IgM or IgA antisera (Nordic). A precipitin line with anti-normal mouse serum, assumed to be due to the presence of L chains in the IgE fraction was corroborated with antimouse kappa or lambda antisera (Miles, Stoke Poges, U.K.). The purified IgE
311 was shown to contain kappa light chains. The pure isolated IgE was positive in 48 h mouse PCA with a titer of between 1/10,000, and 1/50,000 on challenge with ovalbumin and was negative after the same IgE fraction was heatinactivated at 56 ° for 4 h.
Purification and labelling o f rabbit anti-mouse IgE Outbred rabbits were injected with 100 pg mouse IgE in 0.5 ml complete Freund's adjuvant (Difco, Detroit, MI) subcutaneously at sites adjacent to the inguinal, axillary and cervical lymph nodes at m o n t h l y intervals, and bled at regular intervals. The antiserum was rendered monospecific by absorption with y o u n g BALB/c mouse serum polymerised with glutaraldehyde according to the m e t h o d of Ternynck and Avrameas (1976). In immunodiffusion, the absorbed antiserum yielded a precipitin line of identity with purified mouse IgE and two native sera from mouse carrying IgE antiovalbumin hybridomas. N o reaction line was visible with pure mouse IgG, derived from the MOPC 21 t u m o r line (Bionetics, Stans, Switzerland) with mouse IgG (Nordic) or normal mouse serum. In addition, this antiserum was tested in reverse PCA (rPCA) in the rat, to ascertain whether it could bind to and subsequently release histamine from IgE-bearing skin mast cells. This was in fact observed and the endpoint dilution yielding a 5 mm diameter blue spot was between 1/10,000 and 1/100,000. In the absence of sufficient amounts of pure mouse IgE to make an immunosorbent column, advantage was taken of the cross-reactivity between mouse and rat IgE, and the rabbit anti-mouse IgE antiserum was applied to a rat IgE IR 162 immunoabsorbent column. (Rat IgE IR2 and IR162 were kindly donated by Dr. H. Bazin.) The anti-mouse IgE antibodies were eluted by 0.2 M glycine-HC1, pH 2.4, into a vessel containing 1 M Tris, pH 8.2, and concentrated by vacuum dialysis. They were further dialyzed against 0.5 M phosphate buffer, pH 7.5, and the protein concentration estimated by its extinction coefficient at 280 nm (EI~ cm = 13.6 for rat IgE). The purified anti-IgE antibodies were labelled with 12sI by the chloramine T m e t h o d described by Greenwood et al. (1963). Briefly, 70--100 pg of specific a n t i b o d y (previously dialyzed against 0.5 M phosphate buffer, pH 7.5) in a total volume of 100 gl was added to 10 pl (1 mCi) 125I (Radiochemical Centre, Amersham, U.K.) and 10 pl of 1 mg/ml chloramine T (Merck, Darmstadt, F.R.G.) in 0.1 M phosphate buffer, pH 7.5, and mixed gently for 5 min. The reaction was stopped by the addition of 10 pl 2.5 mg/ ml sodium metabisulfite (Merck, Darmstadt, F.R.G.) in 0.1 M phosphate buffer, pH 7.5, mixed for 2 min before the addition of 100 pl of I mg/ml sodium iodide (Merck, Darmstadt, F.R.G.) in 0.1 M phosphate buffer, pH 7.5. Furthermore, 100 pl of incubation buffer was added comprising the following: 0.5 M phosphate buffer, pH 7.5, 0.3% h u m a n serum albumin (Fluka, Buchs, Switzerland), 0.5% Tween 20, 0.02% sodium azide (Merck, Darmstadt, F.R.G.) and 20% fetal calf serum (Gibco, Basel, Switzerland). The whole of the reaction mixture was applied directly to a 10 cm × 1 cm
312 Sephadex G 50 column (Pharmacia, Sweden), previously saturated with 1 ml 20% ovalbumin (Sigma Chemicals, U.S.A.) and equilibrated with 0.1 M phosphate buffer, pH 7.5. Elution was carried out in 10-drop fractions and each fraction counted in the gamma counter. From the profile obtained, the labelled protein was localized in a single sharp peak and the fractions pooled and stored at +4°C until ready for use. The specific activity of the labelled protein was between 0.85 and 2.0 p~i/pg.
R A S T assay for mouse specific IgE antibodies To each polystyrene tube (5 ml, 75 mm × 12 mm, type 55-476, Starstedt, F.R.G.), was added an antigen-coated disc and 50 pl of a 1/50 dilution of mouse serum to be assayed. Control tubes contained 50 pl of 1/50 normal mouse serum or incubation buffer respectively. The tubes were covered and allowed to stand at room temperature for 3 h. They were then washed 3 times (3 ml volumes of 0.9% NaC1 containing 0.2% Tween 20) with intervals of 10 min between washings. After the final wash, 50 pl of rabbit [12sI]anti-mouse IgE (approx. 30,000 cpm after dilution in incubation buffer) was added, the tubes covered and allowed to stand at room temperature for 17 h. The tubes were then washed 3 times as above and then counted in a gamma counter (LKB 12/60). Procedure for assessing the specificity of [125i]anti.ig E through direct binding studies with antigen coated micro tubes (modified from Catt and Tregear, 1976) Micro-tubes (type M 179A, Dynatech, Switzerland) were coated with 100 pl of 10 pg/ml of the following antigens in 0.1 M Na2CO3 pH 9.6: mouse IgE, IgG, IgGl and bovine serum albumin (BSA -- Fluka Buchs, Switzerland) as background control. All were assayed in quadruplicate. After incubating for 2 h at 37°C, the tubes were washed 3 times with 0.9% NaC1 containing 0.05% Tween 20. The tubes were then saturated by complete filling with 3% BSA in 0.1 M Na2CO3, pH 9.6 and incubated for 30 min at 37°C. After washing 3 times as described above, 100 pl of rabbit 12sI anti-mouse IgE (approx. 40,000 c p m ) in phosphate buffered saline 0.01 M pH 7.38 was added and allowed to stand at room temperature for 17 h. Washing was repeated as above and the tubes were counted. RESULTS
Specificity o f [12SI]anti-mouse IgE antibodies Direct binding studies were carried out using antigen-coated tubes as described above. Briefly, purified mouse IgE, IgG and IgG1 were coated on individual tubes to which rabbit anti-mouse [12sI]IgE was added. The results are shown in Fig. 1. Maximum binding was obtained with IgE while binding of the labelled anti-IgE to other antibody classes was markedly lower and became negligible after deduction of the background level obtained with a 1/100 dilution of normal mouse serum (NMS).
313 R A S T inhibition s t u d i e s Discs c o a t e d with m o u s e IgE were i n c u b a t e d f o r 17 h in a series o f t u b e s with 50 pl o f a serially d i l u t e d m o u s e serum c o n t a i n i n g IgE a n t i b o d i e s against B P O - O V A (48 h PCA titers against B P O - O V A : 1 / 4 0 0 and against OVA: 1 / 8 0 0 ) , t o g e t h e r with 50 pl o f r a b b i t [~2SI]anti-mouse IgE (approx. 3 0 , 0 0 0 c p m ) . A f t e r washing and c o u n t i n g , the r e d u c t i o n in binding (% i n h i b i t i o n ) as c o m p a r e d to m a x i m u m binding to a IgE-coated disc in the absence o f c o m p e t i n g s e r u m was calculated a f t e r p r i o r d e d u c t i o n o f c o n t r o l , a c c o r d i n g t o the f o r m u l a
binding in absence binding in p r e s e n c e of i n h i b i t o r of i n h i b i t o r % inhibition = X 100 binding in absence o f i n h i b i t o r T h e results are s h o w n in Fig. 2 and d e m o n s t r a t e a d o s e - d e p e n d e n t inhibition, which was n o t t h e case w i t h n o r m a l m o u s e s e r u m (results n o t shown). E f f e c t o f s e r u m dilution on the R A S T assay Fig. 3 shows the e f f e c t o f serial d i l u t i o n o f m o u s e reaginic serum binding to B P O - O V A - c o a t e d discs• Sera A, B, C, D and E possess PCA titers o f 1 / 6 4 0 0 , 1 2 8 0 , 640, 1 6 0 a n d 30 respectively• Dilutions less t h a n 1 / 2 5 s h o w e d variable decrease in binding, p r o b a b l y d u e to i n h i b i t i o n b y n o n - r e l a t e d serum p r o t e i n s (results n o t shown). H o w e v e r , dilutions greater t h a n 1 / 2 5 s h o w e d a progressive decrease in binding. In c o n s e q u e n c e , a d i l u t i o n o f 1 / 5 0 was c h o s e n as t h e o p t i m a l d i l u t i o n f o r assaying all u n k n o w n sera and was f o u n d to be the m o s t s a t i s f a c t o r y t o assay sera having PCA titers ranging f r o m 1 / 1 0 I0" 98" 7. 6cpm X I0 3 5 4 3
, . ° •
°
:.:.: :.:, %° • , %•., %•.,
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IIgE IgG IgG I BSA I:100 NMS
1/256~128 1/64 1/32 1/16 I/'8 I/4 .serum dilution
~2
I
Fig. 1. Binding of [12SI]anti-mouse IgE to microtubes coated with various mouse immunoglobulins, BSA and normal mouse serum as indicated. For conditions of the experiment, see Materials and Methods. Fig. 2. RAST inhibition. Competitive inhibition of binding of [12SI]anti-mouse IgE to mouse IgE-coated discs by serial dilutions of mouse reaginic serum with high PCA titer.
314
2-5
net cpm xlO 3
C
2oo
serum
,oo
>5o
dilution
Fig. 3. E f f e c t o f s e r u m d i l u t i o n o n t h e R A S T assay ( [ 1 2 S I ] a n t i - m o u s e IgE c o n s t a n t ) . Sera A, B, C, D a n d E r e p r e s e n t d i l u t i o n s o f m o u s e s e r u m d i r e c t e d against t h e a n t i g e n B P O - O V A , h a v i n g P C A titers o f 1 / 6 4 0 0 , 1 2 8 0 , 640, 160, 30 respectively.
to 1/6400. However, since binding is low for sera with titers of less than 1/100, sera suspected of having low IgE levels were tested at 1/25 and 1/50 dilutions. Each serum was assayed at least in duplicate, and positive control sera possessing high, medium and low PCA titers were tested each time from pooled aliquots in order to check the degree of binding in each series of assays.
I00. rs : 0 91 P = <001
tSF~ :LI
net cpm' ~lo3
SE~3g ~SEN~O
SEM~tO
OL I0
100
PCA h1~OeOs 0
lO,O00
Fig. 4. C o r r e l a t i o n b e t w e e n OVA-specific R A S T a n d PCA titers in 10 d i f f e r e n t sera. Each p o i n t r e p r e s e n t s t h e m e a n o f 5 R A S T e s t i m a t i o n s in t h e same assay p e r individual serum. Bars i n d i c a t e S.E.M.
IOO
315
rs:081 p : (001
RAST net I cpm xlo 3
8 •
l
OI I0
I00
I000 PCA titres
I0,000
Fig. 5. C o r r e l a t i o n b e t w e e n B P O - O V A - s p e c i f i c R A S T a n d PCA titers. C o r r e l a t i o n coeffic i e n t c a l c u l a t e d a c c o r d i n g t o S p e a r m a n R a n k (rs) test.
"2 3200 0
~oi 32.00
A
Q_
net
x 103 ~-
o ~L
0
800
200
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0
I 2 weeks
cpm xto3
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net
m
cpm ~,~
a. 8 0 0
-~_
B
o
d
3
Y weeks
"4
o 6400 t C
"3ne t cpm -2 XlO3
1 ~ 4oo
IOO1 0
I 2 weeks
3
Fig. 6. Direct p l o t t i n g o f P C A titers a n d R A S T . A i n d i c a t e s t h e PCA profile (o-o-o-o) a n d t h e R A S T assays of t h e same sera e s t i m a t e d o n d i f f e r e n t days (A-~-~, A-A-A, m-m-m). B s h o w s t h e R A S T a n d P C A profile o f sera h a v i n g PCA titers o f less t h a n 1 / 3 2 0 , w h e n assayed in t h e same b a t c h as t h e curve (m-m-m) in A. C s h o w s 2 O V A R A S T profiles assayed a t d i f f e r e n t t i m e s (A_~.A, -.m-m) w i t h t h e i r r e s p e c t i v e PCA profile (o-o-o).
316
TABLE 1 Heat-incubation impairs the ability of mouse IgE to bind specifically to antigen coated discs. Antigen coated discs were incubated for 3 h with native serum (i), or heatinactivated serum (ii), followed by washing 3 times, and in one instance (iii) incubated with native serum for an additional 3 h. Disc coated with
Incubation
Bound cpm (net)
BPO-OVA BPO-OVA BPO-OVA OVA OVA OVA
(i) Native serum only a (ii) Heat-inactivated b serum only (iii) Heat-inactivated followed by native serum (i) Native serum only a (ii) Heat-inactivated serum only b (iii) Heat-inactivated serum followed by native serum
2 758 668 2 791 1 297 274 1 392
a Native serum used at dilutions 1/100 and 1/200. Initial PCA titer of 1/2000 and 1/200 for BPO-OVA and OVA respectively. b Native serum diluted 1/100 and 1/200 then heat-inactivated at 56°C for 3 h.
Experiments to demonstrate correlation between R A S T titers
values and PCA
I n d i v i d u a l sera having PCA titers ranging f r o m 1 / 2 0 t o 1 / 6 4 0 0 for the antigens B P O - O V A a n d O V A respectively, as e s t i m a t e d in 48-h rat P C A tests ( m e a n o f 2 d u p l i c a t e estimates in t w o animals) were c o m p a r e d for b o u n d c p m in t h e R A S T assay b y S p e a r m a n r a n k c o r r e l a t i o n test (rs). F o r the antigen O V A (Fig. 4), the rs was 0 . 9 0 7 , P < 0.01. Each p o i n t represents the m e a n o f 5 R A S T e s t i m a t i o n s per each individual s e r u m in t h e same assay, with t h e bars i n d i c a t i n g s t a n d a r d error o f the m e a n (S.E.M.). F o r the antigen B P O - O V A (Fig. 5), a similar significant c o r r e l a t i o n b e t w e e n P C A and R A S T was f o u n d (r s -- 0 . 8 1 1 , P ~ 0.01). Fig. 6 A - - C shows t h e direct p l o t o f PCA titers and R A S T n e t c p m values d u r i n g the c o u r s e o f an a d o p t i v e transfer e x p e r i m e n t . It is clear t h a t a l t h o u g h the degree o f binding assessed as a b s o l u t e n e t c p m b o u n d changes with intervals o f 1 w e e k b e t w e e n assays as a f u n c t i o n o f i s o t o p e d e c a y (Fig. 6A, C), the R A S T profile always follows t h a t o f t h e P C A titer.
E f f e c t o f heat inactivation on R A S T binding A s e r u m having a PCA titer o f 1 / 2 0 0 0 and 1 / 2 0 0 f o r B P O - O V A and O V A respectively was d i l u t e d 1 / 1 0 0 and 1 / 2 0 0 in the R A S T i n c u b a t i o n b u f f e r and h e a t - i n a c t i v a t e d at 56°C for 3 h. A native serum c o n t r o l was also diluted 1 / 1 0 0 a n d 1 / 2 0 0 a n d b o t h c o n t r o l - a n d h e a t - i n a c t i v a t e d sera were assayed b y R A S T for binding to specific a n t i g e n - c o a t e d discs. Fig. 7 shows t h a t heati n a c t i v a t i o n a p p a r e n t l y i m p a i r e d the R A S T assay. A f u r t h e r e x p e r i m e n t was carried o u t to ascertain w h e t h e r (a) the h e a t - i n a c t i v a t e d IgE a n t i b o d i e s were possibly n o t b i n d i n g t o the allergen-coated disc, or (b) w h e t h e r IgE was
317 20. 1.6. net I-2 cpm x 105
"8 b
"4
d
I0--0250 I00 2-00 I0"--0250 I0--0Z-O0 serum
dilution
Fig. 7. Effect of heat-inactivation on mouse serum having an initial PCA titer of 1/2000 and 1/200 for BPO-OVA and OVA respectively, assessed from subsequent binding in specific RAST. a: Binding of native serum to BPO-OVA discs at dilutions of 1/100 and 1/200; b : Binding of heat-inactivated serum to BPO-OVA discs at dilutions of 1/100 and 1/200; c : Binding of native serum to OVA discs at dilutions of 1/100 and 1/200; d : Binding of heat-inactivated serum to OVA discs at dilutions of 1/100 and 1/200.
b i n d i n g t o t h e disc b u t was u n a b l e t o r e a c t w i t h t h e 12SI-labelled a n t i b o d y . A n t i g e n ~ c o a t e d discs were p r e i n c u b a t e d for 3 h with i n a c t i v a t e d serum, w a s h e d 3 t i m e s a n d r e i n c u b a t e d w i t h n a t i v e s e r u m f o r a n a d d i t i o n a l 3 h. I n c o r r e s p o n d i n g c o n t r o l s , discs w e r e i n c u b a t e d e i t h e r w i t h n a t i v e o r w i t h h e a t i n a c t i v a t e d s e r u m . As s h o w n i n T a b l e 1, p r e i n c u b a t i o n w i t h h e a t - i n a c t i v a t e d s e r u m d o e s n o t i m p a i r t h e a b i l i t y o f t h e a n t i g e n - c o a t e d disc t o b i n d s u b s e -
[ ] PCA t i t r e IIIIIS J L ". BALB/C ~i~i~i~iCBA
/
"•
1280~
6
~
'-8 ' _ net
520'
7
~P
"-
80
I
I~
o
17
i!~iiil
"e
cpm
' x=°s
..4
!iiiiiiijiiiiiiit"
a. 20' i:l
iijiiil !iiiiiJ 0
I weeks
2
0
I weeks
2
0
2 weeks
Fig. 8. Comparison of PCA and RAST values of an anti-BPO-OVA ongoing IgE response in different mouse strains. IF] represents PCA titer and [[[[[, i" i and i~ii:,represent RAST values in SJL, BALB/c and CBA respectively.
318
1280
e'° PCA "*-~RAST
-/~"-~ _
~320
•
f 1"6
12
4~
eO
•~ U
20 0 I
5
8
12
16
21
28
33
50
64
days after anti-idiotype trealmenl
HPCA
640
HRAST
~ &
160
'-
40
.I 6 .12 nel
0.8 cpm XlO 3 04
(.)
a.
I0-
0 days afler anti-tdiotype treatment
Fig. 9. C o m p a r i s o n b e t w e e n PCA titers a n d R A S T values in t w o g r o u p s o f mice pres e n t i n g a n o n g o i n g IgE r e s p o n s e against OVA. A n i m a l s f r o m g r o u p A are t r e a t e d w i t h a n t i - i d i o t y p e a n t i s e r u m ( f o r e x p e r i m e n t a l details, see Blaser et al., 1 9 8 1 ) , while a n i m a l s o f g r o u p B are c o n t r o l s t r e a t e d w i t h n o r m a l m o u s e serum.
quently native IgE, suggesting that heat-inactivation indeed impairs the binding capacity of IgE. R A S T assays p e r f o r m e d on sera f r o m various m o u s e strains
As shown in Fig. 8, the anti-mouse IgE 125I-labelled antibody is also able to react with IgE-containing sera of the SJL and CBA strains, and not only with the BALB/c IgE which is the origin of the monoclonal IgE hybridoma used for the preparation of the rabbit anti-IgE antiserum. The RAST assay was also used to monitor IgE responses of BALB/c mice treated with OVA anti-idiotype serum (Blaser et al., 1981). As can be seen from Fig. 9A and B, the RAST and PCA profiles follow parallel courses although the RAST assay appears to reflect variations n o t detected in PCA. DISCUSSION
The RAST technique is now well established in the human system for measuring antigen-specific IgE antibodies in serum of allergic individuals.
319 The same principle has been used to estimate specific rat IgE antibodies (Pauwels et al., 1977b). Very recently and while the work presented here was underway, RAST assays have been described in the mouse system (Liu et al., 1980; Karlsson et al., 1980; Kelly et al., 1980). From the results presented here, the RAST assay described is specific for mouse IgE and permits the monitoring of antigen-specific IgE responses in mouse serum. In particular, the IgE-immunosorbent purified anti-IgE serum appears not to react with mouse IgG~, the other h o m o c y t o t r o p i c antibody in mice. It is possible that the rabbit anti-IgE antiserum, having been raised by immunization with a monoclonal anti-ovalbumin antibody of BALB/c origin, would contain anti-idiotypic antibodies (against the anti-ovalbumin idiotype) or anti-BALB/c allotypes restricted to IgE. Such an IgE-associated mouse allotype has recently been described (Sanchez et al., 1980) and anti-idiotypic antibodies have frequently complicated the preparation of anti-rat IgE antibodies. Our anti-IgE is n o t restricted to the recognition of BALB/c strain IgE and is not anti-idiotypic in nature, since it binds to IgE specific for antigens (e.g. BPO) other than ovalbumin. Consequently, it can be used in a broader way to detect mouse IgE. The purification of the anti-IgE serum on a rat IgE m y e l o m a immunosorbent column is probably responsible for the specificity against a c o m m o n IgE isotypic determinant of the eluted antibody. The significant correlation between PCA titers and RAST values implies t h a t the same antibody is measured, and is in agreement with results of Evans et al. (1972) who showed a similar significant correlation between h u m a n Prausnitz-Kfistner and the RAST test. Therefore the RAST assay could replace the cumbersome PCA technique for estimating mouse IgE antibody. PCA shows a marked variability between recipient animals and exhibits at best a l-fold dilution error in titers. The RAST assay undoubtedly gives greater precision than PCA and the lowest dilutions of serum that can be assayed are between 1/20 and 1/40. No a t t e m p t has y e t been made to standardize the assay with regard to defined RAST units, since only the evolution of the IgE profile was of interest in our current studies on IgE regulation; all sera were assayed in one batch at the end of an experiment. Standard sera from pools having known PCA titers of 1/2000, 1/640 and 1/20, respectively, are used concurrently and facilitate estimating the presence of IgE a n t i b o d y in an u n k n o w n serum while providing a useful check on the degree of binding in every assay. The added advantage of this technique is t h a t only 10 or 20 pl of undiluted serum is required to make a working dilution for the RAST assay and a variety of antigens can be assayed. As shown in this paper, we also looked at the effect of heat-inactivation on the subsequent binding of IgE antibodies. According to Prouvost-Danon (1976), the degree of thermolability of mouse IgE at 56°C depends on the dilution, the pH and the medium in which the serum was diluted. Although we did not heat-inactivate at the optimal conditions outlined in the above
320 paper, the results s h o w n in Fig. 7 indicate a significant decrease in binding. This c o u l d be d u e to a loss o f the h e a t - i n a c t i v a t e d s e r u m ' s ability to bind to t h e a n t i g e n - c o a t e d disc or its ability t o bind t o [~2sI]anti-IgE, o r b o t h . Results p r e s e n t e d in Table 1 a p p e a r t o indicate t h a t the f o r m e r is the case. In s o m e e x p e r i m e n t s n o t s h o w n here, we o b t a i n e d indications t h a t serum samples t h a w e d and f r o z e n several times m a y s h o w a m a r k e d decrease in their I g E - b i n d i n g c a p a c i t y , suggesting t h a t preservation o f m o u s e serum samples f o r IgE R A S T assays deserves particular a t t e n t i o n . ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y the Swiss N a t i o n a l Science F o u n d a t i o n , G r a n t No. 3 . 9 4 4 . 7 8 , and the a u t h o r s w o u l d like t o t h a n k Miss M. S c h l u c h t e r f o r her assistance and Drs. W~ilti and C o n r o y for helpful discussions. REFERENCES Blaser, K., T. Nakagawa and A.L. de Week, 1981, J. Immunol. 126, 1180. BSttcher, I. and G. Hiimrnerling, 1978, Nature (Lond.) 275,761. Catt, U. and G.W. Tregear, 1976, Science 158, 1570. Ceska, M., R. Eriksson and J.M. Varga, 1972, J. Allergy clin. Immunol. 4 9, 1. Eshhar, Z., M. Ofarim and T. Waks, 1980, J. Immunol. 124, 2775. Evans, R., R.E. Reisman, J.I. Wypych and C.E. Arbesman, 1972, J. Allergy clin. Immunol. 49, 285. Greenwood, F.C., W.M. Hunter and J.S. Glover, 1963, Biochem. J. 89, 114. Karlsson, T., J.R. Ellerson, I. Dahlbom and H. Bennich, 1979, Scand. J. Immunol. 9,217. Karlsson, T., I. BSttcher, M. Albring and H. Bennich, 1980, Workshop on Immunoassays, Paris, 4th International Congress of Immunology, Abstract No. 19.6.14. Kelly, A., G.M. Lang, P.G. Bundesen, V. Holford-Strevens, I. BSttcher and A.W. Sehon, 1980, J. Immunol. Methods 39,317. Lee, W. and D. Heiner, 1978, J. Immunol. Methods, 20,185. Liu, F., J. Bohn, E. Ferry, H. Yamanroto, C. Molinardo, L. Sherman, N. Klinman and D. Katz, 1980, J. Immunol. 124, 2728. Nakagawa, T., K. Blaser and A.L. de Week, 1980, Int. Arch. appt. Allergy 63, 212. Pauwels, R., H. Bazin, B. Platteau and M. Van der Straeten, 1977a, J. Immunol. Methods 18, 133. Pauwels, R., H. Bazin, B. Platteau and M. Van der Straeten, 1977b, Ann. Immunol. 128C, 675. Prouvost-Danon, A., R. Binaghi and A. Abadie, 1976, Immunochem. 14, 81. Sanchez, M., K. Yoshihiro, K. Okumura and T. Tada, 1980, 4th Immunology Congress, Paris, Abstract No. 2.3.13. Ternynck, T. and S. Avrameas, 1976, Scand. J. Immunol. Suppl. 3, 29. Wide, L., H. Bennich and S.G.O. Johansson, 1967, Lancet ii, 1105.
309
Elsevier/North-Holland Biomedical Press
A RAST ASSAY FO R M O N I T O R I N G SPECIFIC IgE ANTIBODIES IN THE MOUSE
M.A. KINGS and A.L. DE WECK Institute o f Clinical Immunology, Berne, Switzerland
(Received 5 March 1981, accepted 23 July 1981) Mouse monoclonal anti-ovalbumin IgE was isolated from serum of BALB/c mice implanted with the IgE secreting hybridoma (14-205). Purified IgE was used to immunize rabbits and for the preparation of an antiserum specific for mouse IgE. The latter was used to develop a RAST assay to replace the PCA technique in monitoring antigen-specific IgE responses in the mouse.
INTRODUCTION RAST (Radioallergosorbent Test) is a diagnostic test based u p o n the d e t ectio n o f allergen specific antibodies of the IgE t y p e by 12SI-labelled antiIgE on Sephadex particles (Wide et al., 1967) or paper discs (Ceska et al., 1972), to which allergens have been chemically coupled. Subsequently this technique has been adapted f or ot he r a n t i b o d y classes bot h in man and animals. Of particular interest are studies in rats and mice, which are bot h used as models o f IgE mediated allergic diseases. Fo r a long time, passive cutaneous anaphylaxis (PCA) has been the only means of detecting h o m o c y t o t r o p i c antibodies o f the IgE class. Radioimmunoassays (RIAs) for the rat system have been developed (Pauwels et al., 1977a; Karlsson et al., 1979), because . o f the availability of rat m y e l o m a IgE. However, owing to the unavailability o f mouse IgE myelomas, 24 or 48 h PCA tests carried o u t in the rat or mouse have remained until n o w the only means of assaying mouse IgE. The advent o f mo n o c l onal mouse IgEs p r o d u c e d by the h y b r i d o m a technique (BSttcher and H~immerling, 1978) and directed against such antigens as the d i n i t r o p h e n y l h a pt e n (DNP) or ovalbumin (OVA) has made it possible to obtain larger a m o u n t s o f mouse IgE and has allowed RIAs to be developed for total and specific mouse IgE (Eshhar et al., 1980; Kelly et al., 1980; Liu et al., 1980). The RAST m e t h o d described here uses antigen-coated paper discs and [ 12 s I ] r ab b it anti-mouse IgE for estimation o f mouse specific antibodies directed against benzylpenicilloyl-ovalbumin (BPO-OVA).
0022-1759/81/0000---O000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
310 MATERIALS AND METHODS Activation and preparation of antigen coated discs Ovalbumin was purchased from Sigma Chemicals, U.S.A. BPO22-OVA (ovalbumin carrying 22 benzylpenicilloyl groups) was prepared and analyzed as described elsewhere (Nakagawa et al., 1980). Filter paper discs, diameter 5.0 mm, t y p e 589/3 (Schleicher Schuell, Feldbach, F.R.G.) were activated and subsequently coated with the allergens BPO22OVA or OVA by the method of Lee and Heiner (1978). Immunization o f mice for monitoring IgE responses Female BALB/c mice, 6--8 weeks old (G.I. Bomholtgaard, Ry, Denmark), were given a primary injection (i.p.) of 1 pg BPOv.5-OVA in 1 mg AI(OH)3, and booster injections by the same route. The mice were bled (0.2 ml) from the orbital sinus, and the serum samples tested consisted of a pooled serum from 5 mice. Each serum pool was titrated by passive cutaneous anaphylaxis (PCA) in 250--300 g Wistar rats from our own colony. The sensitization period was 48 h, and the end-point expressed as the reciprocal of antiserum dilution yielding a reaction 5 mm in diameter. Isolation and purification o f mouse IgE Ovalbumin-specific monoclonal IgE antibodies (Kindly donated by Dr. K. Turner) were obtained from serum of BALB/c mice (PCA titer 1/160,000) in which the IgE-secreting hybridoma (14-205; BSttcher and H~immerling, 1978) was propagated in vivo. This serum was diluted 1 : 1 in 0.5 M TrisHC1-NaC1 buffer, pH 8.0, and applied directly to an immunoabsorbent column (10 cm X 1 cm) of 10 mg ovalbumin coupled to every 1 g of CNBractivated Sepharose (Pharmacia, Uppsala, Sweden). The IgE eluted with 0.2 M glycine-HC1 buffer, pH 2.4, was immediately adjusted to pH 8.2 with 1 M Tris (Merck, Darmstadt, F.R.G.) and concentrated by vacuum dialysis. The IgE fraction was tested for purity by conventional immunoelectrophoresis (IEP) and Ouchterlony techniques, using various commercial antimouse immunoglobulin antisera (Nordic, Immunological laboratories, The Netherlands). IgG1 was found to be a major contaminant (approx. 30%) of the IgE fraction. However, in view of the low concentration of IgE contained in the serum, further absorption was avoided and the IgE fraction was injected as such into an outbred rabbit for antiserum production. Subsequent purification of IgE was achieved by coupling rabbit anti-IgE antibodies (precipitated with 33% saturated ammonium sulfate) to CNBractivated Sepharose. The 0.2 M glycine-HC1 eluted IgE fraction reacted in immunodiffusion with rabbit anti-mouse IgE and anti-mouse IgE (supplied by Drs. Lee and Sehon), b u t n o t with anti-mouse IgG, IgM or IgA antisera (Nordic). A precipitin line with anti-normal mouse serum, assumed to be due to the presence of L chains in the IgE fraction was corroborated with antimouse kappa or lambda antisera (Miles, Stoke Poges, U.K.). The purified IgE
311 was shown to contain kappa light chains. The pure isolated IgE was positive in 48 h mouse PCA with a titer of between 1/10,000, and 1/50,000 on challenge with ovalbumin and was negative after the same IgE fraction was heatinactivated at 56 ° for 4 h.
Purification and labelling o f rabbit anti-mouse IgE Outbred rabbits were injected with 100 pg mouse IgE in 0.5 ml complete Freund's adjuvant (Difco, Detroit, MI) subcutaneously at sites adjacent to the inguinal, axillary and cervical lymph nodes at m o n t h l y intervals, and bled at regular intervals. The antiserum was rendered monospecific by absorption with y o u n g BALB/c mouse serum polymerised with glutaraldehyde according to the m e t h o d of Ternynck and Avrameas (1976). In immunodiffusion, the absorbed antiserum yielded a precipitin line of identity with purified mouse IgE and two native sera from mouse carrying IgE antiovalbumin hybridomas. N o reaction line was visible with pure mouse IgG, derived from the MOPC 21 t u m o r line (Bionetics, Stans, Switzerland) with mouse IgG (Nordic) or normal mouse serum. In addition, this antiserum was tested in reverse PCA (rPCA) in the rat, to ascertain whether it could bind to and subsequently release histamine from IgE-bearing skin mast cells. This was in fact observed and the endpoint dilution yielding a 5 mm diameter blue spot was between 1/10,000 and 1/100,000. In the absence of sufficient amounts of pure mouse IgE to make an immunosorbent column, advantage was taken of the cross-reactivity between mouse and rat IgE, and the rabbit anti-mouse IgE antiserum was applied to a rat IgE IR 162 immunoabsorbent column. (Rat IgE IR2 and IR162 were kindly donated by Dr. H. Bazin.) The anti-mouse IgE antibodies were eluted by 0.2 M glycine-HC1, pH 2.4, into a vessel containing 1 M Tris, pH 8.2, and concentrated by vacuum dialysis. They were further dialyzed against 0.5 M phosphate buffer, pH 7.5, and the protein concentration estimated by its extinction coefficient at 280 nm (EI~ cm = 13.6 for rat IgE). The purified anti-IgE antibodies were labelled with 12sI by the chloramine T m e t h o d described by Greenwood et al. (1963). Briefly, 70--100 pg of specific a n t i b o d y (previously dialyzed against 0.5 M phosphate buffer, pH 7.5) in a total volume of 100 gl was added to 10 pl (1 mCi) 125I (Radiochemical Centre, Amersham, U.K.) and 10 pl of 1 mg/ml chloramine T (Merck, Darmstadt, F.R.G.) in 0.1 M phosphate buffer, pH 7.5, and mixed gently for 5 min. The reaction was stopped by the addition of 10 pl 2.5 mg/ ml sodium metabisulfite (Merck, Darmstadt, F.R.G.) in 0.1 M phosphate buffer, pH 7.5, mixed for 2 min before the addition of 100 pl of I mg/ml sodium iodide (Merck, Darmstadt, F.R.G.) in 0.1 M phosphate buffer, pH 7.5. Furthermore, 100 pl of incubation buffer was added comprising the following: 0.5 M phosphate buffer, pH 7.5, 0.3% h u m a n serum albumin (Fluka, Buchs, Switzerland), 0.5% Tween 20, 0.02% sodium azide (Merck, Darmstadt, F.R.G.) and 20% fetal calf serum (Gibco, Basel, Switzerland). The whole of the reaction mixture was applied directly to a 10 cm × 1 cm
312 Sephadex G 50 column (Pharmacia, Sweden), previously saturated with 1 ml 20% ovalbumin (Sigma Chemicals, U.S.A.) and equilibrated with 0.1 M phosphate buffer, pH 7.5. Elution was carried out in 10-drop fractions and each fraction counted in the gamma counter. From the profile obtained, the labelled protein was localized in a single sharp peak and the fractions pooled and stored at +4°C until ready for use. The specific activity of the labelled protein was between 0.85 and 2.0 p~i/pg.
R A S T assay for mouse specific IgE antibodies To each polystyrene tube (5 ml, 75 mm × 12 mm, type 55-476, Starstedt, F.R.G.), was added an antigen-coated disc and 50 pl of a 1/50 dilution of mouse serum to be assayed. Control tubes contained 50 pl of 1/50 normal mouse serum or incubation buffer respectively. The tubes were covered and allowed to stand at room temperature for 3 h. They were then washed 3 times (3 ml volumes of 0.9% NaC1 containing 0.2% Tween 20) with intervals of 10 min between washings. After the final wash, 50 pl of rabbit [12sI]anti-mouse IgE (approx. 30,000 cpm after dilution in incubation buffer) was added, the tubes covered and allowed to stand at room temperature for 17 h. The tubes were then washed 3 times as above and then counted in a gamma counter (LKB 12/60). Procedure for assessing the specificity of [125i]anti.ig E through direct binding studies with antigen coated micro tubes (modified from Catt and Tregear, 1976) Micro-tubes (type M 179A, Dynatech, Switzerland) were coated with 100 pl of 10 pg/ml of the following antigens in 0.1 M Na2CO3 pH 9.6: mouse IgE, IgG, IgGl and bovine serum albumin (BSA -- Fluka Buchs, Switzerland) as background control. All were assayed in quadruplicate. After incubating for 2 h at 37°C, the tubes were washed 3 times with 0.9% NaC1 containing 0.05% Tween 20. The tubes were then saturated by complete filling with 3% BSA in 0.1 M Na2CO3, pH 9.6 and incubated for 30 min at 37°C. After washing 3 times as described above, 100 pl of rabbit 12sI anti-mouse IgE (approx. 40,000 c p m ) in phosphate buffered saline 0.01 M pH 7.38 was added and allowed to stand at room temperature for 17 h. Washing was repeated as above and the tubes were counted. RESULTS
Specificity o f [12SI]anti-mouse IgE antibodies Direct binding studies were carried out using antigen-coated tubes as described above. Briefly, purified mouse IgE, IgG and IgG1 were coated on individual tubes to which rabbit anti-mouse [12sI]IgE was added. The results are shown in Fig. 1. Maximum binding was obtained with IgE while binding of the labelled anti-IgE to other antibody classes was markedly lower and became negligible after deduction of the background level obtained with a 1/100 dilution of normal mouse serum (NMS).
313 R A S T inhibition s t u d i e s Discs c o a t e d with m o u s e IgE were i n c u b a t e d f o r 17 h in a series o f t u b e s with 50 pl o f a serially d i l u t e d m o u s e serum c o n t a i n i n g IgE a n t i b o d i e s against B P O - O V A (48 h PCA titers against B P O - O V A : 1 / 4 0 0 and against OVA: 1 / 8 0 0 ) , t o g e t h e r with 50 pl o f r a b b i t [~2SI]anti-mouse IgE (approx. 3 0 , 0 0 0 c p m ) . A f t e r washing and c o u n t i n g , the r e d u c t i o n in binding (% i n h i b i t i o n ) as c o m p a r e d to m a x i m u m binding to a IgE-coated disc in the absence o f c o m p e t i n g s e r u m was calculated a f t e r p r i o r d e d u c t i o n o f c o n t r o l , a c c o r d i n g t o the f o r m u l a
binding in absence binding in p r e s e n c e of i n h i b i t o r of i n h i b i t o r % inhibition = X 100 binding in absence o f i n h i b i t o r T h e results are s h o w n in Fig. 2 and d e m o n s t r a t e a d o s e - d e p e n d e n t inhibition, which was n o t t h e case w i t h n o r m a l m o u s e s e r u m (results n o t shown). E f f e c t o f s e r u m dilution on the R A S T assay Fig. 3 shows the e f f e c t o f serial d i l u t i o n o f m o u s e reaginic serum binding to B P O - O V A - c o a t e d discs• Sera A, B, C, D and E possess PCA titers o f 1 / 6 4 0 0 , 1 2 8 0 , 640, 1 6 0 a n d 30 respectively• Dilutions less t h a n 1 / 2 5 s h o w e d variable decrease in binding, p r o b a b l y d u e to i n h i b i t i o n b y n o n - r e l a t e d serum p r o t e i n s (results n o t shown). H o w e v e r , dilutions greater t h a n 1 / 2 5 s h o w e d a progressive decrease in binding. In c o n s e q u e n c e , a d i l u t i o n o f 1 / 5 0 was c h o s e n as t h e o p t i m a l d i l u t i o n f o r assaying all u n k n o w n sera and was f o u n d to be the m o s t s a t i s f a c t o r y t o assay sera having PCA titers ranging f r o m 1 / 1 0 I0" 98" 7. 6cpm X I0 3 5 4 3
, . ° •
°
:.:.: :.:, %° • , %•., %•.,
:.:.: , %• • , . , • ,
•..,
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:~ 6 0
• • ,,%,
.__ (.-
,:.:
_E 4 0
2
IIgE IgG IgG I BSA I:100 NMS
1/256~128 1/64 1/32 1/16 I/'8 I/4 .serum dilution
~2
I
Fig. 1. Binding of [12SI]anti-mouse IgE to microtubes coated with various mouse immunoglobulins, BSA and normal mouse serum as indicated. For conditions of the experiment, see Materials and Methods. Fig. 2. RAST inhibition. Competitive inhibition of binding of [12SI]anti-mouse IgE to mouse IgE-coated discs by serial dilutions of mouse reaginic serum with high PCA titer.
314
2-5
net cpm xlO 3
C
2oo
serum
,oo
>5o
dilution
Fig. 3. E f f e c t o f s e r u m d i l u t i o n o n t h e R A S T assay ( [ 1 2 S I ] a n t i - m o u s e IgE c o n s t a n t ) . Sera A, B, C, D a n d E r e p r e s e n t d i l u t i o n s o f m o u s e s e r u m d i r e c t e d against t h e a n t i g e n B P O - O V A , h a v i n g P C A titers o f 1 / 6 4 0 0 , 1 2 8 0 , 640, 160, 30 respectively.
to 1/6400. However, since binding is low for sera with titers of less than 1/100, sera suspected of having low IgE levels were tested at 1/25 and 1/50 dilutions. Each serum was assayed at least in duplicate, and positive control sera possessing high, medium and low PCA titers were tested each time from pooled aliquots in order to check the degree of binding in each series of assays.
I00. rs : 0 91 P = <001
tSF~ :LI
net cpm' ~lo3
SE~3g ~SEN~O
SEM~tO
OL I0
100
PCA h1~OeOs 0
lO,O00
Fig. 4. C o r r e l a t i o n b e t w e e n OVA-specific R A S T a n d PCA titers in 10 d i f f e r e n t sera. Each p o i n t r e p r e s e n t s t h e m e a n o f 5 R A S T e s t i m a t i o n s in t h e same assay p e r individual serum. Bars i n d i c a t e S.E.M.
IOO
315
rs:081 p : (001
RAST net I cpm xlo 3
8 •
l
OI I0
I00
I000 PCA titres
I0,000
Fig. 5. C o r r e l a t i o n b e t w e e n B P O - O V A - s p e c i f i c R A S T a n d PCA titers. C o r r e l a t i o n coeffic i e n t c a l c u l a t e d a c c o r d i n g t o S p e a r m a n R a n k (rs) test.
"2 3200 0
~oi 32.00
A
Q_
net
x 103 ~-
o ~L
0
800
200
0
0
I 2 weeks
cpm xto3
.~
200
0
net
m
cpm ~,~
a. 8 0 0
-~_
B
o
d
3
Y weeks
"4
o 6400 t C
"3ne t cpm -2 XlO3
1 ~ 4oo
IOO1 0
I 2 weeks
3
Fig. 6. Direct p l o t t i n g o f P C A titers a n d R A S T . A i n d i c a t e s t h e PCA profile (o-o-o-o) a n d t h e R A S T assays of t h e same sera e s t i m a t e d o n d i f f e r e n t days (A-~-~, A-A-A, m-m-m). B s h o w s t h e R A S T a n d P C A profile o f sera h a v i n g PCA titers o f less t h a n 1 / 3 2 0 , w h e n assayed in t h e same b a t c h as t h e curve (m-m-m) in A. C s h o w s 2 O V A R A S T profiles assayed a t d i f f e r e n t t i m e s (A_~.A, -.m-m) w i t h t h e i r r e s p e c t i v e PCA profile (o-o-o).
316
TABLE 1 Heat-incubation impairs the ability of mouse IgE to bind specifically to antigen coated discs. Antigen coated discs were incubated for 3 h with native serum (i), or heatinactivated serum (ii), followed by washing 3 times, and in one instance (iii) incubated with native serum for an additional 3 h. Disc coated with
Incubation
Bound cpm (net)
BPO-OVA BPO-OVA BPO-OVA OVA OVA OVA
(i) Native serum only a (ii) Heat-inactivated b serum only (iii) Heat-inactivated followed by native serum (i) Native serum only a (ii) Heat-inactivated serum only b (iii) Heat-inactivated serum followed by native serum
2 758 668 2 791 1 297 274 1 392
a Native serum used at dilutions 1/100 and 1/200. Initial PCA titer of 1/2000 and 1/200 for BPO-OVA and OVA respectively. b Native serum diluted 1/100 and 1/200 then heat-inactivated at 56°C for 3 h.
Experiments to demonstrate correlation between R A S T titers
values and PCA
I n d i v i d u a l sera having PCA titers ranging f r o m 1 / 2 0 t o 1 / 6 4 0 0 for the antigens B P O - O V A a n d O V A respectively, as e s t i m a t e d in 48-h rat P C A tests ( m e a n o f 2 d u p l i c a t e estimates in t w o animals) were c o m p a r e d for b o u n d c p m in t h e R A S T assay b y S p e a r m a n r a n k c o r r e l a t i o n test (rs). F o r the antigen O V A (Fig. 4), the rs was 0 . 9 0 7 , P < 0.01. Each p o i n t represents the m e a n o f 5 R A S T e s t i m a t i o n s per each individual s e r u m in t h e same assay, with t h e bars i n d i c a t i n g s t a n d a r d error o f the m e a n (S.E.M.). F o r the antigen B P O - O V A (Fig. 5), a similar significant c o r r e l a t i o n b e t w e e n P C A and R A S T was f o u n d (r s -- 0 . 8 1 1 , P ~ 0.01). Fig. 6 A - - C shows t h e direct p l o t o f PCA titers and R A S T n e t c p m values d u r i n g the c o u r s e o f an a d o p t i v e transfer e x p e r i m e n t . It is clear t h a t a l t h o u g h the degree o f binding assessed as a b s o l u t e n e t c p m b o u n d changes with intervals o f 1 w e e k b e t w e e n assays as a f u n c t i o n o f i s o t o p e d e c a y (Fig. 6A, C), the R A S T profile always follows t h a t o f t h e P C A titer.
E f f e c t o f heat inactivation on R A S T binding A s e r u m having a PCA titer o f 1 / 2 0 0 0 and 1 / 2 0 0 f o r B P O - O V A and O V A respectively was d i l u t e d 1 / 1 0 0 and 1 / 2 0 0 in the R A S T i n c u b a t i o n b u f f e r and h e a t - i n a c t i v a t e d at 56°C for 3 h. A native serum c o n t r o l was also diluted 1 / 1 0 0 a n d 1 / 2 0 0 a n d b o t h c o n t r o l - a n d h e a t - i n a c t i v a t e d sera were assayed b y R A S T for binding to specific a n t i g e n - c o a t e d discs. Fig. 7 shows t h a t heati n a c t i v a t i o n a p p a r e n t l y i m p a i r e d the R A S T assay. A f u r t h e r e x p e r i m e n t was carried o u t to ascertain w h e t h e r (a) the h e a t - i n a c t i v a t e d IgE a n t i b o d i e s were possibly n o t b i n d i n g t o the allergen-coated disc, or (b) w h e t h e r IgE was
317 20. 1.6. net I-2 cpm x 105
"8 b
"4
d
I0--0250 I00 2-00 I0"--0250 I0--0Z-O0 serum
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Fig. 7. Effect of heat-inactivation on mouse serum having an initial PCA titer of 1/2000 and 1/200 for BPO-OVA and OVA respectively, assessed from subsequent binding in specific RAST. a: Binding of native serum to BPO-OVA discs at dilutions of 1/100 and 1/200; b : Binding of heat-inactivated serum to BPO-OVA discs at dilutions of 1/100 and 1/200; c : Binding of native serum to OVA discs at dilutions of 1/100 and 1/200; d : Binding of heat-inactivated serum to OVA discs at dilutions of 1/100 and 1/200.
b i n d i n g t o t h e disc b u t was u n a b l e t o r e a c t w i t h t h e 12SI-labelled a n t i b o d y . A n t i g e n ~ c o a t e d discs were p r e i n c u b a t e d for 3 h with i n a c t i v a t e d serum, w a s h e d 3 t i m e s a n d r e i n c u b a t e d w i t h n a t i v e s e r u m f o r a n a d d i t i o n a l 3 h. I n c o r r e s p o n d i n g c o n t r o l s , discs w e r e i n c u b a t e d e i t h e r w i t h n a t i v e o r w i t h h e a t i n a c t i v a t e d s e r u m . As s h o w n i n T a b l e 1, p r e i n c u b a t i o n w i t h h e a t - i n a c t i v a t e d s e r u m d o e s n o t i m p a i r t h e a b i l i t y o f t h e a n t i g e n - c o a t e d disc t o b i n d s u b s e -
[ ] PCA t i t r e IIIIIS J L ". BALB/C ~i~i~i~iCBA
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Fig. 8. Comparison of PCA and RAST values of an anti-BPO-OVA ongoing IgE response in different mouse strains. IF] represents PCA titer and [[[[[, i" i and i~ii:,represent RAST values in SJL, BALB/c and CBA respectively.
318
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Fig. 9. C o m p a r i s o n b e t w e e n PCA titers a n d R A S T values in t w o g r o u p s o f mice pres e n t i n g a n o n g o i n g IgE r e s p o n s e against OVA. A n i m a l s f r o m g r o u p A are t r e a t e d w i t h a n t i - i d i o t y p e a n t i s e r u m ( f o r e x p e r i m e n t a l details, see Blaser et al., 1 9 8 1 ) , while a n i m a l s o f g r o u p B are c o n t r o l s t r e a t e d w i t h n o r m a l m o u s e serum.
quently native IgE, suggesting that heat-inactivation indeed impairs the binding capacity of IgE. R A S T assays p e r f o r m e d on sera f r o m various m o u s e strains
As shown in Fig. 8, the anti-mouse IgE 125I-labelled antibody is also able to react with IgE-containing sera of the SJL and CBA strains, and not only with the BALB/c IgE which is the origin of the monoclonal IgE hybridoma used for the preparation of the rabbit anti-IgE antiserum. The RAST assay was also used to monitor IgE responses of BALB/c mice treated with OVA anti-idiotype serum (Blaser et al., 1981). As can be seen from Fig. 9A and B, the RAST and PCA profiles follow parallel courses although the RAST assay appears to reflect variations n o t detected in PCA. DISCUSSION
The RAST technique is now well established in the human system for measuring antigen-specific IgE antibodies in serum of allergic individuals.
319 The same principle has been used to estimate specific rat IgE antibodies (Pauwels et al., 1977b). Very recently and while the work presented here was underway, RAST assays have been described in the mouse system (Liu et al., 1980; Karlsson et al., 1980; Kelly et al., 1980). From the results presented here, the RAST assay described is specific for mouse IgE and permits the monitoring of antigen-specific IgE responses in mouse serum. In particular, the IgE-immunosorbent purified anti-IgE serum appears not to react with mouse IgG~, the other h o m o c y t o t r o p i c antibody in mice. It is possible that the rabbit anti-IgE antiserum, having been raised by immunization with a monoclonal anti-ovalbumin antibody of BALB/c origin, would contain anti-idiotypic antibodies (against the anti-ovalbumin idiotype) or anti-BALB/c allotypes restricted to IgE. Such an IgE-associated mouse allotype has recently been described (Sanchez et al., 1980) and anti-idiotypic antibodies have frequently complicated the preparation of anti-rat IgE antibodies. Our anti-IgE is n o t restricted to the recognition of BALB/c strain IgE and is not anti-idiotypic in nature, since it binds to IgE specific for antigens (e.g. BPO) other than ovalbumin. Consequently, it can be used in a broader way to detect mouse IgE. The purification of the anti-IgE serum on a rat IgE m y e l o m a immunosorbent column is probably responsible for the specificity against a c o m m o n IgE isotypic determinant of the eluted antibody. The significant correlation between PCA titers and RAST values implies t h a t the same antibody is measured, and is in agreement with results of Evans et al. (1972) who showed a similar significant correlation between h u m a n Prausnitz-Kfistner and the RAST test. Therefore the RAST assay could replace the cumbersome PCA technique for estimating mouse IgE antibody. PCA shows a marked variability between recipient animals and exhibits at best a l-fold dilution error in titers. The RAST assay undoubtedly gives greater precision than PCA and the lowest dilutions of serum that can be assayed are between 1/20 and 1/40. No a t t e m p t has y e t been made to standardize the assay with regard to defined RAST units, since only the evolution of the IgE profile was of interest in our current studies on IgE regulation; all sera were assayed in one batch at the end of an experiment. Standard sera from pools having known PCA titers of 1/2000, 1/640 and 1/20, respectively, are used concurrently and facilitate estimating the presence of IgE a n t i b o d y in an u n k n o w n serum while providing a useful check on the degree of binding in every assay. The added advantage of this technique is t h a t only 10 or 20 pl of undiluted serum is required to make a working dilution for the RAST assay and a variety of antigens can be assayed. As shown in this paper, we also looked at the effect of heat-inactivation on the subsequent binding of IgE antibodies. According to Prouvost-Danon (1976), the degree of thermolability of mouse IgE at 56°C depends on the dilution, the pH and the medium in which the serum was diluted. Although we did not heat-inactivate at the optimal conditions outlined in the above
320 paper, the results s h o w n in Fig. 7 indicate a significant decrease in binding. This c o u l d be d u e to a loss o f the h e a t - i n a c t i v a t e d s e r u m ' s ability to bind to t h e a n t i g e n - c o a t e d disc or its ability t o bind t o [~2sI]anti-IgE, o r b o t h . Results p r e s e n t e d in Table 1 a p p e a r t o indicate t h a t the f o r m e r is the case. In s o m e e x p e r i m e n t s n o t s h o w n here, we o b t a i n e d indications t h a t serum samples t h a w e d and f r o z e n several times m a y s h o w a m a r k e d decrease in their I g E - b i n d i n g c a p a c i t y , suggesting t h a t preservation o f m o u s e serum samples f o r IgE R A S T assays deserves particular a t t e n t i o n . ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y the Swiss N a t i o n a l Science F o u n d a t i o n , G r a n t No. 3 . 9 4 4 . 7 8 , and the a u t h o r s w o u l d like t o t h a n k Miss M. S c h l u c h t e r f o r her assistance and Drs. W~ilti and C o n r o y for helpful discussions. REFERENCES Blaser, K., T. Nakagawa and A.L. de Week, 1981, J. Immunol. 126, 1180. BSttcher, I. and G. Hiimrnerling, 1978, Nature (Lond.) 275,761. Catt, U. and G.W. Tregear, 1976, Science 158, 1570. Ceska, M., R. Eriksson and J.M. Varga, 1972, J. Allergy clin. Immunol. 4 9, 1. Eshhar, Z., M. Ofarim and T. Waks, 1980, J. Immunol. 124, 2775. Evans, R., R.E. Reisman, J.I. Wypych and C.E. Arbesman, 1972, J. Allergy clin. Immunol. 49, 285. Greenwood, F.C., W.M. Hunter and J.S. Glover, 1963, Biochem. J. 89, 114. Karlsson, T., J.R. Ellerson, I. Dahlbom and H. Bennich, 1979, Scand. J. Immunol. 9,217. Karlsson, T., I. BSttcher, M. Albring and H. Bennich, 1980, Workshop on Immunoassays, Paris, 4th International Congress of Immunology, Abstract No. 19.6.14. Kelly, A., G.M. Lang, P.G. Bundesen, V. Holford-Strevens, I. BSttcher and A.W. Sehon, 1980, J. Immunol. Methods 39,317. Lee, W. and D. Heiner, 1978, J. Immunol. Methods, 20,185. Liu, F., J. Bohn, E. Ferry, H. Yamanroto, C. Molinardo, L. Sherman, N. Klinman and D. Katz, 1980, J. Immunol. 124, 2728. Nakagawa, T., K. Blaser and A.L. de Week, 1980, Int. Arch. appt. Allergy 63, 212. Pauwels, R., H. Bazin, B. Platteau and M. Van der Straeten, 1977a, J. Immunol. Methods 18, 133. Pauwels, R., H. Bazin, B. Platteau and M. Van der Straeten, 1977b, Ann. Immunol. 128C, 675. Prouvost-Danon, A., R. Binaghi and A. Abadie, 1976, Immunochem. 14, 81. Sanchez, M., K. Yoshihiro, K. Okumura and T. Tada, 1980, 4th Immunology Congress, Paris, Abstract No. 2.3.13. Ternynck, T. and S. Avrameas, 1976, Scand. J. Immunol. Suppl. 3, 29. Wide, L., H. Bennich and S.G.O. Johansson, 1967, Lancet ii, 1105.