Ann. Immunol. (Inst. Pasteur) 1984, 135 C, 39-44
QUANTITATIVE OF I D I O T Y P E - B E A R I N G
ASSAYS IgE ANTIBODIES
by T. Inada and A. Nisonoff
Bosenstiel Research Center, Department o] Biology, Brandeis Universitg, Waltham, Massachusetts 02254 (USA)
SUMMARY The present report describes a method allowing quantitation of antiarsonate IgE antibodies and the estimation of the fraction of such antibodies which expresses the recurring CRI~ idiotypic determinant. This method permits an analysis of the regulation of this idiotype within IgE-type antibodies. KEY-WORDS: IgE, Idiotype; Arsonate, Radioimmunoassay.
INTRODUCTION There have been few studies relating to the presence of major intrastrain cross-reactive idiotypes (CRI) on antibodies of the IgE class, and very little is known about the regulation of CRI-bearing IgE. This is attributable in part to the very low concentration of IgE in serum and the consequent difficulty of carrying out quantitative serological measurements of CRI. The presence of CRI on IgE was demonstrated in the anti-phosphorylcholine system by Kishimoto et al.  and in the antiGAT system (glutamic acid, alanine, tyrosine copolymer)iby Dessein el al.  and Lowy et al. . Our own studies, done in collaboration with the laboratory of Z. Ovary and using monoclonal anti-CRI , demonstrated the presence of an idiotype, designated CRI A, on IgE antibodies directed to the p-azophenylarsonate (Ar) hapten in A/J mice. Our initial attempts to identify CRIA on IgE anti-Ar, carried out several years earlier by the method of reverse passive cutaneous anaphylaxis ((RPCA); S. Kojima, Z. Ovary and A. Nisonoff, unpublished results) were inconsisManuserit re~u le 7 septembre 1983. Supported by grant AI-12895 from the National Institutes of Health,
T. INADA AND A. N I S O N O F F
tent because of non-specific permeability of venules induced by rabbit anti-IgE sera. Methods used for identifying CRI + IgE antibodies include RPCA [1, 2, 4], the adherence of IgE antibodies to immobilized anti-Id  and the in vitro degranulation of mast cells from immunized mice by tile action of anti-Id antibodies . This preliminary study reports the development of sensitive radioimmunoassays for the quantitation of IgE anti-Ar antibodies and for the estimation of the fraction of such antibodies which expresses CRIb. The preparation of a hybridoma producing IgE anti-Ar (CRIb) of A / J derivation is also described.
M A T E R I A L S AND METHODS Affinity-purified rabbit anli- I gE. This reagent was obtained through the generosity of Dr Amar S. Tung, Merck, Sharpe and Dohme Research Labor~itories. It was labelled with 1~5I using the chloramine-T method. The specific activity immediately after labelling was 48 ~Ci/~g.
Radioimmunoassay /or IgE anti-Ar antibodies. The wells of polyvinylchloride microtitre plates were coated with a BSA-Ar conjugate (1 mg/ml) and saturated with 3% horse serum . Fifty ~1 of the serum to be tested was added, together with 20 ~1 of 1% normal A / J serum, and allowed to stand for 4 h at room temperature. After washing, 100 ~1 of a solution containing 8 ng of purified, labelled rabbit anti-IgE was added and allowed to stand overnight at room temperature. The wells were then washed with tap water and with 0.5 M NaC1.
Radioimmunoassay /or CRI + IgE. The percentage of IgE ~nti-Ar antibodies which express CRIA was determined by incubating anti-CRIA antiserum (anti-Id vs. the CRI + monoclonal antibody, R16.7) with the unknown serum for 30 to 60 min at 370 C before carrying out the assay for IgE anti-Ar described above. Evidence that anti-Id(R16.7) prevents the binding of CRI + antibodies to BSA-Ar is presented below.
Preparation o/an IgE anli-Ar hybridoma. Fusions were carried out with the spleen cells of A / J mice whose sera contained IgE anti-Ar antibodies, as shown by PCA reactions. To induce the antibodies, A / J mice were inoculated i. p. on day 0 with 5 ~g K L H - A r and 2-4 mg
Ar BSA CRI GAT
= = = =
p-azophenylarsonate. bovine serum albumin. cross-reacting idiotype. glutamic acid, alanine, tyrosine (copolymer). KLH = keyhole limpet haemocyanin.
PCA = passive cutaneous anaphylaxis. PEG ~ polyethylene glycol. RPCA ~ reverse passive cutaneous anaphylaxis. SDS ~ sodium dodecyl sulphate.
QUANTITATION OF IDIOTYPE-BEARING IgE
of alum, and were boosted 28 days later with the same mixture of antigen and adjuvant. Two weeks later, 4-5 • 107 spleen cells were transferred into A/J recipients which had received 600 R on the same day. Shortly after the adoptive transfer, the recipients were immunized with the same mixture of KLH-Ar and alum that had been used for the donors. They were bled 7 days later and PEA titres of the sera were determined. Mice whose serum contained IgE anti-Ar were sacrificed for the preparation of hybridomas 3 to 4 days after the bleeding. Hybridomas were prepared by the method of KShler and Milstein  using the non-secreting Sp2/0 line  and polyethylene glycol (PEG 1,000). Individual wells were screened for the presence of anti-Ar antibodies by radioimmunoassay. Out of more than 500 wells containing viable cells, approximately 120 contained anti-Ar antibodies. All these anti-Ar-positive wells were tested for the presence of IgE anti-Ar by PEA in rats. (This preceded our development of a suitable radioimmunoassay). Three of the wells contained IgE antibodies. Only one hybridoma, designated L3, survived the subsequent cloning procedures. L3 monoclonal antibodies were obtained by. the production of ascites in CAF1 mice. The concentrations of antibody m ascites were, however, low (average, 30 ~g/ml). The IgE antibodies were affinity-purified on Sepharose 4B to which BGG-Ar was conjugated. The antibody was eluted with 0.5 M p-aminophenylarsonate at neutral pH. A total of 650 ~g was recovered. Analysis by the Ouchterlony method yielded lines of precipitation with a rabbit anti-r reagent or rabbit anti-mouse Fab, but not with antisera to mouse y1, "~2b, ya2, or ~ chains. Electrophoresis of protein L3 in a 10% polyacrylamide gel containing sodium dodecyl sulphate (SDS) and reducing agent yielded 2 bands of molecular weights 84,000• and 24,000• respectively. The heavy chains of another IgE monoclonal antibody (T2) and of an IgG1 monoclonal antibody (R16.7) exhibited molecular weights of 83,000 and 49,000, respectively.
R E S U L T S AND DISCUSSION
Specificity or rabbit anti-IgE. The specificity of the radiolabelled rabbit anti-IgE is shown by the data in figure 1. Positive results were obtained in the r a d i o i m m u n o a s s a y when protein L3 was tested, but no significant binding was observed when radiolabelled monoclonal a n t i b o d y R16.7 (IgGlk) or A / J serum anti-Ar antibodies were used. The serum antibodies were generated by immunizing with K L H - A R in Freund's a d j u v a n t , a procedure t h a t yields a very low ratio of IgE to IgG antibodies (as shown by PCA reactions). When this ratio is extremely low, the results obtained for IgE are artifactually decreased (data not shown). The results obtained with protein L3 indicate t h a t the assay can detect less t h a n 0.1 ng of IgE anti-Ar in a volume of 50 ill.
Concentrations of IgE anti-Ar in immune sera. To induce IgE anti-Ar antibodies, the general method of immunization described above for the production of IgE-produeing hybridomas was used. This involves the use of alum as a d j u v a n t and an adoptive transfer after i m m u n i z a t i o n of the donor.
A. N I S O N O F F
PCA; :i :::! PCA 50
:: :.:: :
,? ~ •
:=:: i:::::::::.: ii:::::: :.: :: .:..
R16.7 i:i~:~ii:iii:il ,~:.::,::
,6o "s.obo ",~',ooo
I0 no Anti-At ANTIBODY
FIG. 1. - - R e s u l t s of r a d i o i m m u n o a s s a y s , u s i n g z~5I-labelled specifically purified r a b b i t a n t i m o u s e I g E as t h e d e v e l o p i n g r e a g e n t . S a m p l e v o l u m e s were 50 ~l each. As i n d i c a t e d in t h e figure, t h e l i m i t of s e n s i t i v i t y of t h e PCA r e a c t i o n w i t h L3 a n t i b o d y ( m o n o c l o n a l I g E a n t i - A r ) is ~ 2 ng.
Results obtained with a number of individual mice are shown in table I. After the adoptive transfer of 5 x 106, 25 x l0 s or 50 x 10 e splenic lymphocytes, recipients were i m m e d i a t e l y immunized with 10 ~g K L H - A r and 2 mg alum; t h e y were bled 11 days later. In the mice which received 25 x 106 or 50 x 106 cells, the concentration of IgE anti-Ar antibodies varied from 2.7 to 6.6 ~g/ml (protein L3 was used as s t a n d a r d
TABLE I. - - Assays for total IgE and CRI + anti-At antibodies
in individual A/J sera.
l-1 2-1 2-2 2-3 3-1 3-2 3-3 3-4 3-5
5 x l0 s 25 X 10 a ,, ,, 50 X l 0 s ,, ,, ,, ,,
~g/.ml 6.4 288 194 248 243 369 414 297 216
Inhibition of I g E b y anti-Id(R16.7)
Inhibition by rabbit anti-ovalbumin
ND 33 20 9 2 5 15 24 23
ND 0 (*) 0 0 0 0 0 0 0
~g/ml <1 3.8 3.4 6.6 2.7 5.7 4.0 5.0 3.2
(*) S l i g h t e n h a n c e m e n t of b i n d i n g of 125I was c a u s e d in each case b y t h e r a b b i t a n t i - o v a l b u min . ND=not determined.
QUANTITATION OF IDIOTYPE-BEARING IgE
in these assays). The ratio of total anti-Ar to IgE anti-Ar varied from 38/1 to 104/1.
Presence of CRI. in IgE anti-Ar antibodies. Also shown in table I are data on the inhibition of binding of IgE anti-Ar to BSA-Ar-eoated wells by anti-Id(R16.7) (anti-CRI.). Five of the 8 sera that had significant titres of IgE anti-Ar expressed CRIA in a substantial proportion of the IgE antibodies (15 to 33%). These results confirm and quantify the presence of CRI. in IgE anti-Ar of A / J mice.
Controls for the assay of CRIA in IgE anti-Ar. The specificity of the inhibition of binding by anti-Id(R16.7) was demonstrated by its failure to inhibit the binding to BSA-Ar-coated wells of R49.20, a CRI2 A / J monoclonal antibody with anti-Ar activity, or of anti-Ar antibodies in the sera of each of two idiotypically suppressed mice. When a pool of non-suppressed serum was tested, 42% inhibition of binding of anti-Ar antibodies was observed. In all of these experiments, the developing reagent was 12q-labelled, affinity-purifiedr abbit antimouse Fab. These preliminary results demonstrate the feasibility of carrying out quantitative assays for IgE anti-Ar antibodies and for the percentage of such antibodies which express CRI A.
RI~SUMI~ DOSAGE DES ANTICORPS IGE PORTEURS D'IDIOTYPES
Nous d6crivons une m6thode p e r m e t t a n t de titrer les anticorps IgE anti-arsonate et d'6valuer la fraction de ces anticorps qui exprime le d6terminant idiotypique r6eurrent CRI A. Cette m6thode permet l'analyse de la r6gulation de cet idiotype au sein des anticorps de type IgE. MOTS-CLI~S : IgE, I d i o t y p e ; Arsonate, Dosage radio-immunologique.
REFERENCES  DESSEIN, A., Ju, S.-T., DORF, E., BENACERRAF, B. & GERMAIN, R. N., IgE response to synthetic polypeptide antigens. - - II. Idiotypic analysis of the response to L-glutamic acid 5~176 (GAT). J. Immunol., 1980, 124, 71-76.  HIRANO, T., KOJIMA, S., HIRAYAMA, N., NELLES, M. J., INADA, Z., NISONOFF, A. & OVARY, Z., Presence of an intrastrain cross-reactive idiotype on A/J antibodies of the IgE class specific for the p-azophenylarsonate group. J. Immunol., 1983, 130, 1300-1302.
T. INADA AND A. NISONOFF
 LowY, I., PROUVOSE-DANoN, A., ABADIE, A. & THEZE, J., Fine specificity
and idiotype analysis of the IgE response to the synthetic terpolymer L-glutamic-acid~~176 1~ (GAT) and its dinitrophenyl conjugate (DNP-GAT). Mol. Immunol., 1980, 17, 1033-1038. KISHIMOTO, T., SHIGEMOTO,S., WATANABE, T. & YAMAMURA,Y., Demonstration of phosphorylcholine-specific IgE B cells in CBA/N mice. J. Immunot., 1979, 123, 1039-1043. KLINMAN, i . R., PICKARD, A. R., SIGAL, N. H., GEARHART, P. J., METCALF, E. S. & PIEnCE, S. K., Assessing B-cell diversification by antigenreceptor and precursor cell analysis. Ann. Immunol. (Inst. Pasteur), 1976, 127 C, 489-502. K()HLER, G. & MILSTEIN, C., Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Europ. J. Immunol., 1976, 6, 511-519. SHULMAN,M., WILDE, C. D. & K(iHLER, G., A better cell line for making hybridomas secreting specific antibodies. Nature (Lond.), 1978, 276, 269-270.