Molecular Pergamon

Immunology, Vol. 16, pp. 597-607 Press Ltd. 1979. Printed in Great

Britain

STANDARDIZATION OF RADIOIMMUNOASSAY: DIFFERENCES IN REACTIVITY OF RAT IgEs WITH ANTIBODIES TO A MONOCLONAL IgE KEN A. KELLY’.

ALEC

H. SEHONl

and HERVI? BAZINZ

‘Department of Immunology, Faculty of Medicine, University of Manitoba. Winnipeg, Manitoba, Canada, R3E OW3 and *Faculty of Medicine, Experimental Immunology Unit University of Louvain, Bruxelles, Belgium (Firsr

recrired

1 June 1978; in rrrkd

form 29 Nocemher

1978)

AbstractPExamination of antisera raised against a rat monoclonal IgE,,, revealed that antibodies to its myelotypic determinants had a much higher affinity for IgE,,, than antibodies to its class-specific determinants. Moreover, in competiti~~einhibition radio~mmunoassays designed for the quantitation of total IgE in rat sera, using ilnmunosp~cifically purified antibodies to the class-specific IgE determinants, it was found that purified IgE standards isolated from other myeloma protein sources were not asefficient as IgE,,, in inhibiting the binding of ‘251PIgE,62: these results suggest the existence of subclasses of IgE, In these assays, it was found imperative, therefore, to use antibodies against class-specific determinants, i.e. anti--1gE antibodies devoid of anti-myelotypic antibodies, in conjunction with lz5 I-labelled purified IgE other than IgEl,, which had been used as the immunizing antigen.

Normal levels of human IgE in circulation arevery low (100-300 ng/ml) and in patients with atopic disorders elevation of IgE concentration by a factor of 3-10 has been observed in a number of allergic disorders (Johansson et al., 1972). Hence, for the quantitation of both total IgE and IgE antibodies to specific allergens, solid phase radioimmunoassays (RIAs), which possess the unique features of extremely high sensitivity, specificity and accuracy have been successfully employed. In the radioimmunosorbent test, designated as RIST, anti-IgE antibodies are coupled to Sepharose or cellulose particles and L251-labelled IgE and IgE present in the test sample compete for the antibody combining sites (Bennich & Johansson, 1971). Another competitive solid phase RIA is the double antibody solid phase (DASP) assay for human (Gleich et ul., 1970) and rat,IgE (tshizaka et ui.. 1976). A noncompetitive RIA is the paper disc radioimmunosorbent test, referred to as PRIST,which consists of adding first the sample containing IgE to a paper disc to which anti-IgE antibodies have been coupled; the IgE bound to this reverse immunosorbent is quantitated in terms of its ability to combine further with ‘251-anti-IgE antibodies. This technique has been applied to the quantitation of human IgE (Ceska & Lundkvist, 1972) and, while this manuscript was in preparation, a report appeared describing the quantitation of total rat IgE levels in sera using PRIST _ -*In this context, the term homologous refers to the particular monoclonal IgE produced by a given immunocytoma which is used as the immunizing antigen for the production of antibodies; by analogy. the term heterologous refers to IgE produced by another immunocytoma or present in sera of normal rats.

(Pauwels et af., 1977~). Human IgE antibodies to specific allergens have been quantitated by the radioallergosorbent test, referred to as RAST (Wide et cd., 1967). In this test, the serum of a patient with a specific allergy is added to an allergosorbent. prepared by covalently coupling the specific allergen to a solid phase. The IgE antibodies bound to the insolubilized allergen are then quantitated in terms of their ability to bind ‘251-anti-lgE antibodies. A modification of the RAST has been developed for the quantitation of human IgE antibodies in absolute amounts (Schellenberg & Adkinson, 1975). While the present study was in progress, a description of a RAST for rat IgE was published (Pauwels et al., 1977b). Also, recently, an immunoenzymatic method, the galactosidase immunosorbent test or GIST (Weltman et ul., 1976), has been developed for the quantitation of total IgE and IgE antibodies in human sera. The GIST differs from the PRIST and the RAST in that the IgE bound to the solid phase is quantitated in terms of its ability to bind conjugates of anti-IgE antibody with galactosidase, determined by subsequent enzyme substrate interaction. In view of the very low concentrations of IgE in human and animal sera, anti-IgE antibodies used in various RIAs have generally been raised against intact IgE myeloma protein. Problems may arise in the quantitation of IgE since antibodies directed against individually specific antigenic determinants, referred to as myelotypic determinants, of the homQiogous* myeloma can give erroneously low values with heterologous IgEs (Johansson et aI., 1972). This complication may be overcome by either using antibodies directed against the Fc fragment of IgE (Ceska & Lundkvist, 1972), on the assumption that all myelotypic determinants are located in the variable regions of the Fab portion, or by the use of 597

SYX

KEN

A. KELLY,

ALEC

H. SEHON

heterologous IgE’s as standards in the assay (Carson cv trl.. 1975; lshizaka rl cl/.. 1976). In this study, an attempt was made to develop a solid phase RIA for rat IgE using antibodies raised against a monoclonal IgE. IgE was quantitated by the competitive

inhibition

of binding

of lZ51-IgE

in a RIST

been used by Salmon et u/. (1969) for the quantitation of human immunoglobulins. It was found that for standardization of both assays, it was essential to use not only antibodies directed against common classspecific IgE determinants. but also heterologous IgE standards. and a modified

‘sandw-ich’

MATERIAIS

assay as had

.AhD

METHODS

Ascetic fluids \rere harvested from LOU/Wsl rats bearing the IgE-secreting immunocytomas. IR-2. 159, 162. 183 and 331: the corresponding immunoglobulins are referred to hereafter by subscripts i.e. IgEz. IgE,,,. IgE,,,. IgE,,, and IgE J3,. The immunoglohulins. IgE,,z and IgE,,,, were isolated from the appropriate ascitic fluids by repeated (3X) precipitation aith an equal volume of saturated ammonium sulfate followed by filtration through a column of Bio-Gel P300 and isoelectric focussing. as previously described (Conrad CI (~1.. 1975). The concentration of IgE ~a$ determined from absorbance measurements at 280 nm, using an extinction coefficient of 13.6 for a solution containing I”,, IgE protein (KulcLycki & Metzger. 1974).

,251 were lahelled with Heights. I I I by using chloraminc-T as the oxidizing agent (Greenwood t~lul., 1964). To I .OmCi “‘1 (10pl) were added 25 111of 0.5 M phosphate buffer, pH 7.5. 75 ~1 (40 pg) IgE (i.e. IgEz. IgE,,z or IgE,,,) and 2.5 1~1(100 Itg) chloramine-T. After 30 sec. at 25 C. 100 ~1 (240 leg) of sodium metahisulfite has added and the reaction mixture diluted with 200 ~1 (7 mg) of carrier KI. All reagents were prepared in 0.05 M phosphate buffer, pH 7.5. Separation of “‘I-labelled IgE from the reaction mixture aas carried out by gel liltration on a Bio-Gel P-60 column (1.6 x 50 cm) equilibrated in phosphate buffered saline, PBS (0.01 M phoiphate. 0.1.5 M NaCI, pH 7.4). Assuming 90”,, recovery. the specific activities of the recovered *‘51-lahelled IgE preparations were 2.29 x 10’ (‘2s1-IgEZ). 2.06 x IO’ (“‘Icounts/mln//lg. These IgE,,,, 1and 2.07 x IO- (“‘I-IgE,,,) lahelled standards \bere subdivided into 2 ml portions and stored at -20 (‘. Purified

(

IgE

and

HERVE

BAZIN

high aftinltj anti-IgE anlibodleh.) The eluted ,mtlbod> fraction was adjusted to pH 7. dlalyred against H,O and Iyophilired. Monospecilic goat anti-IgE, anllscrum \\a\ prepared as dchcribed (Batin 01 II/.. 1974).

The assay buffer conslsted of O.l”,, hobine serum albumln (BSA). 0.5”,, Tween 20. ().I”,, aodium a/ide and 0.05 Xl sodium EDTA in PBS. pH 7.4. The antibody solid phase (Scpharose48) immunosorhenta used m the radioimmunoabsaqs \vere equilibrated and suspended In a volume of assay buffer equal to that of the packed volume (by centrifugation at 800 0) 01 immunosorbent. Serial dilutions of inhibitor and standard IgE solution> were also prepared in assay buffer. In a typical assay. the antibody immunohorbent suspension (0.2 ml) was gently shaken on a gyrotary shaker at 37 (‘ for 2 hr \vlth 0.2 ml 01 solution\ containmg IgE a:, inhibitora under test. The “il-labelled IgE (IO ~tl) was then added and the reaction mixture shaken for a further 2 hr at 37 C. The solid phase was aedlmented by centrifugation at 800 r: for 2 min, washed 3 x with 1.0 ml of cold assay buffer and the bound radloactivitj (B*) measured in a Beckman 300 gamma counter with a countmg efticiency of ht?‘,, for “‘1. Substituting assay buffer (0.2 ml) for inhibitor soIutlon qlelded Bz values for IL’ I-IyE bound in the absence of inhibitor. Non-specifically bound radioactivity (C) ~\as determined by addmg IgE,,, in a large excess (20 jlg10.2 ml) as mhlbttor. All determinations were done in triplicate. Inhibition cur~esaere prcparcd by plotting ‘B*-C‘ against the log,,, of the concentration of Igt or ( B;T--C, ! agamst the log, I1 of the rcclprocal unknown inhibitor solutions.

of the dilution

factor 01

RESULTS

samples

Amersham~Searle.Arlington

For the quantitation of IgE, the ‘sandwich’ solid phase RIA represented schematically in Fig. I(a) was selected. Purified IgE,,2 has coupled to CNBractivated Sepharose-4B (2.8 ng IgE/l ml packed gel) and the solid phase was treated with an excess of monospecific dog anti-IgE,,2 antiserum so that. on the average, the antibodies were univalently attached to the solid phase coupled IgE. leaving the other binding site available for the competitive binding of “‘I-1gE or its unlabelled inhibitors. This is a modification of the ‘sandwich’ RIA used by Salmon et ul. (1969) for the quantitation of human immunoglobulins. Advantages of this assay are (a) the minute quantities of purified IgE on the solid phase select only antibodies of the highest affinity and

.4 171 I\“,“l Antisera to I&E,,, were raibed in rabbits, doga, horses and sheep by biucekly intramuscular in.jections with purified emulsified m Freund’s complete IgE ,hL (2 mg/animal) adjuvant. These antisera wcrc rendered monospecltic for i.chains by absorption with a poly~alent Sepharose-4B immunosorhent (Cuatrecasas, 1970). prepared by coupling to CNBr-activated Sepharose-4B (100 ml packed volume) a mixture of: (a) 500 mg of rat immunoglohulins obtained from a pool of normal rat sera by precipitation u,ith ammonium sulfate at 50”,, saturation, (h) the proteins contained in 30 ml of normal rat serum. and (c) I50 mg of IgM and IgA enriched fractions obtained from IR-163 ascites fluid by tiltration through Blo-Gel P-300 (Lang c1 [rl.. 1977). Purified antihodlcs referred to as anti-I&E, were immunospecitically isolated from the monospecilic dog antl-IgE,,L antiserum by incubating serum with an IgEl-Sepharose-4B the immunosorhent and eluting the antibodies with 0.4 M glycme-HCI buffer. pH 2.8. (Elution with 3 .M KCI as a chaotropic ion (Dandliker et trl.. 1967) failed to dissociate

RADIOIMMUNOASSAY

SYSTEMS

E162BE331

Cl%”

45 ng/ml

1251- E33,




(A--A)

represented by Fig. I(a) with IgE,,,

1

f

0.01

0.001

[&/ml] and IgE,,,

(O---O)

in the competitive

on the solid phase and 12S1-IgE,,,

specificity from the antiserum, (b) the antibody molecule is not covalently coupled to the solid phase so that there is no chemical damage and subsequent loss of its antigen binding capacity and (c) the free distal antigen binding site of the antibody molecule is farther removed from the solid phase than if the antibody molecule were directly attached chemically, thus improving its availability for reaction with antigen. In the RIA protocol represented schematically in Fig. l(a) the antibody solid phase suspension (0.2 ml) was reacted with 0.2 ml of serial dilutions of purified IgE,,z and IgEJ3, solutions for 2 hr at 37°C prior to

I

1

1.0

0.1 IgE Concentration

RIA system

as the labelled standard,

reaction with ’ 251-IgE,,, (200,000 counts/min/lO ~1). As is evident from the results plotted in Fig. 2, only the homologous IgE, 62 was capable of inhibiting the of IgE,,, binding of ‘251-IgE,,2. The concentration required for 507; inhibition (CI,,) was 30 ng/ml. The failure of IgE,,, to inhibit the binding of ‘251-IgE,,, indicates that the antibodies of highest affinity, that were bound to the trace amounts of IgE, 62 on the solid phase, were directed to individually specific antigenic determinants unique to IgE,,2. To eliminate interference by these anti-myelotypic antibodies, the system represented by Fig. 1(b) was used in which the

I

I

0.01 [$/ml]

Fig. 3. Inhibition curves for IgE,,, (triangles) and IgE,,, (circles) in the competitive RIA system represented by Fig. I(b), with I@,,, on thesolidphase and lLSI-IgE,,, as the labelled standard. Two of the anti-IgE,,, antibody preparations used in the sandwich assay are shown: dog anti-IgE,,, (A ---A) and (0~ 0) and sheep anti-IgE,,, (A--A) and (e------0).

M.

1MM

,6’S

I

600

KEN

A. KELLY.

ALEC

H. SEHON

and HERVk

BAZlh

Diiution

Eig. 4. Inhibition curves for IgE,,,: (0 0) and IgE,,, (o- --O) in the competitive RIA system represented by Fig. I(c). with Igkj5.,, on the solid phase and 12il-IgE,,, its the labelled standard. Also included are the inhibition curves for rat IgG (a 0) and serum from a rat hearjng the I@,,,-secreting ~~~~~n~n~~~y~oin~~ (A--- -A). plotted as a l’unction of log,,, [dilution].

purified heterologous IgE,,, has coupled to Sepharose-4B. Examination of the inhibition curves in Fig. 3 reveals that, although both IgE,,, and IgE,,Z were capable of inhibiting the binding of ‘251-IgE,,,, the homologous IgE,,, was a much better inhibitor with a cross-reactivity of 1OO:l based on Cl,, values [Fig. l(b)]. This difference in inhibitory capacity was observed using anti-IgE,,, antibodies from the dog, sheep and horse antisera tested. Since both binding sites on a single antibody molecule are identical with respect to affinity and specificity. the obvious conclusion is that there exist differences in the antigenic determinants common to both lgEtbz and In neither system was IgE in normal or IgIG,,. reaginic rat serum able to inhibit the binding of “‘Iwith the homologous antibody. Only when IgEl 1251-lgE,,, was used as the labelled standard [Fig. t(c)] were IgE,,, and IgE,,, roughly eqlIivalent in inhibiting the binding of ‘251-IgE33, (Fig. 4). Moreover, as shown in Fig. 4, the IgE from the serum of a rat bearing the IR-162 secreting immunocytoma was also capable of marked ~lil~ibition of binding of labelled IgE. By contrast. a rat IgG preparation with an original concentration of I mg/ml failed to show any inhibition. To further compare the relative affinities and specificities of antibodies present in the monospecific dog anti-IgE,,, antiserum (prepared as described earlier). the serum was treated as illustrated schematically in Fig. 5. Antibodies to the class-specific IgE determinants. i.e. anti-IgE,, were isolated with the use of the heterologous IgE,-Sepharose-4B (IgE,-4B) immunosorbent from 150 ml of monospecific dog antiThe i~~lmuno~lobu~ins from the Ig.E,,2 antiserum. effluent containing the anti-myelotype antibodies. as well as from 140 ml of the monospecific anti-IgE,,, dog strum. were isolated by repeated (3 x )

precipitation with ammonium sulfate at a final concentration of 40”,, saturation (3 x 40”,, SAS precipitation). When tested by radial immunodiffLlsion in agar (Fig. 6), the anti-IgE, preparation was found to react with IgE only, showing bands of identity with all myeloma IgE preparations. On the other hand, the anti-myelotype antibody preparation reacted with Subsequently, the antibody only. IgE, 62 immunoglobulin preparations: goat anti-IgE2 (900 (900 pg). dog anti-myelotype,~~ btg), dog anti-IgE,,, (900 pg) and dog anti-IgE, (IO pg). were coupled directly to 30 ml packed volume of CNBr-activated Sepharose-4B; the amount of protein coupled per ml Sepharose-4B was proportional to the amo:mt isolated from the original antisera. Equilibrium studies on the reaction of ’ 2sI-labelled IgE$. IgE, ,,? and ~oNOSPECIFIC DOG fi?Sli-kE&p hTISERW

(290 d

r-- -~~--+ 140 d

15oml

0 IgEz-4fJ

0.4ay/Hc1 n. pli2.8

Effluent

3 x40x SAS ppt?

1.0 gm 1g

ANTI-IGE~~~

3 x 40x shs pptfl

1.0 gm I&

10.4wigAb

MYELOTYPE~_

ANTI-IGE~

Fig. 5. Schematic diagram for the isolation 01’ the antihod) sub-populations, and anti-IgE,. from anti-myelotype monospec!fic dog antt-lgE,,,L antiserum

Crossreactivity

Among

601

Rat IgEs

antiserum [(A) upper trough] and Fig. 6(a). Immunoelectrophoretic analysis of dog anti-IgE,,, antiserum [(B) lower trough] against NRS (9) and IgE,,, ascites tluid (3). monospecific dog anti-IgE,,, Analysis of antibody fractions by double immunodiffusion in 1.22, agar in PBS (pH 7.4). (b) Monospecific (B) shows activity against IgE,,, ascites fluid (3) but not with IgM (6). IgA (7), IgG (8) or dog anti-IgE,,, NRS (9) fractions. (c) Dog anti-IgE, (C)shows bands ofcomplete identity with purified IgE, (I), IgE, ?9 (2), (d) However, doganti-myelotype,,, (D) showsantibody lgE 102 (3) IgE,,, (4) and IgE,,, (5)ascitesfluids. activity only against the homologous IgE,,>.

prepI& 1 with these four antibody-Sepharose-4B arations were performed. Each antibody Sepharose-4B preparation (l/l suspensions by volume in assay buffer)was diluted in a l/l suspension of Sepharose-4B in assay buffer in the range of 1.0-0.05 ml immunosorbent/ml total volume. Volumes of 0.2 ml of each dilution were reacted with 150,000 counts/min ‘251-IgE/0.2 ml for 16 hr at 25 C. The solid phase was then washed 3 x with 1 ml of cold assay buffer before measuring the bound radioactivity. The data were plotted as “/,,total radioactivity bound versus the log,, of the dilution factor (relative antibody concentration) of each immunosorbent. As evident in Fig. 7(a) and (b), both the goat antiIgE, and dog anti-IgE,,, displayed a much higher binding for the homologous than heterologous IgEs. This increased binding most probably represents the reaction of anti-myelotypic antibodies with their respective homologous myeloma proteins. Thus, as is shown in Fig. 7(c), dog anti-myelotype,,, antibody reacted only with 1251-IgE,,z, whereas dog anti-IgE, appeared to react equally with all three myeloma IgEs [Fig. 7(d)]. Relative an tibo&

af$nities

of

anti-myelotype

vs anti-IgE,

Constant volumes (0.2 ml) of suspension of the two solid phase coupled antibody populations, antimyelotype,,,-4B and anti-IgE,-4B, were incubated at 25’C for 16 hr with 0.2 ml of increasing quantities of 1251-IgE,,2 the range of 5 x 104-1 x lo6 in counts/min. Non-specific adsorption of labelled material was determined by preincubating the

100

BO

p

60

; e rs

40

20

100

80

60I2 ID g 40

:

20

‘t \

A\,-I

1.0

fi

1

0.5

0.1

Ralotive

1

,\ .

0.05

1.0

Antibody

Concentration

0.5

0.1

0.05

Fig. 7. Reactivity of ‘251-IgE, (U), “‘I-IgE,,, with goat anti-IgE,(A-A) and *251-IgE,,, (O-_-O) 4B (A), dog anti-IgE,,,4B (B), dog anti-myelotype,,,4B (C) and dog anti-IgE,-IB (D).

602

KEN A. KELLY. ALEC H. SEHON and HERVl? BAZIN

immunosorbents with a large excess (20 pg) of IgE,,, to inhibit the binding by antibody of ‘251-IgE,,, in a control experiment. Antibody-bound ‘251-IgE,,, counts/min, measured by counting the washed solid phase. was corrected for non-specific adsorption. subtracted from total 1251-IgE,,2 counts/min to yield free antigen counts/min. From the specific activity of 12SI-IgE,,2 (2.06 x 10’ counts/min/pg) and assuming a mol. wt of 200,000 for IgE (Johansson & Bennich. 1967). counts/min were converted to molar concentrations of bound (h) and free(c) ‘i”I-lgE, hi at equilibrium. It is likely that some loss in antigen binding capacity may occur when antibodies are directly coupled to a solid phase. Nevertheless, making the usual assumption that the antibody combining sites are equivalent in their reaction with antigen, the residual total number of binding sites (Ah) and the average intrinsic binding constant (K,,) were evaluated according to Nisonoff and Pressman ( 1958) by making use of the equation

1 h

I

I

K”(Ah)~~+(/Ih,

From the plots of I/h against l/c (Fig. 8), the estimated value of K, for the reaction of ‘2S1-lgE,,2 with antimyelotypic antibodies (1.7x 10’” I.M-I) was 10x greater than that for its reaction with anti-IgE, (2 x IOU l.M- I), confirming previous observations made in the sandwich solid phase RIA (Figs. I and 2).

A direct solid phase RIA for IgE uas carried out by preincubating anti-IgE,-4B with serial dilutions of the followmg samples: IgE,, IgE, hZ and IgE,, , standards. normal rat serum (NRS). rat IgG. rat reaginic serum (RRS) and mouse reaginic serum (MRS). The IgE content of each sample was determined in terms of the extent of inhibition of subsequent binding of ““I-l&E. as described earlier. In three separate experiments

Cable 1. Dependence of Cli,, value5 (ng/tnll on “‘I-1glz standards in RIA for IgE Cl,,, (ng/ml) for the Inhibitors” (Mean f E.) Labelled standard

IgE,

lgE,,,

lgE.3, I

‘Mean i i. index of precialon (lo”,,) at (B* -C/b: = SO”,,[Midgley A. R. Jr. 2-4B (0 [Ah]=2.5x IO lo M.I -I, A4.1-1, &=I.7 x IO’O I.,%.-‘) and dog anti-lgER-4B (O-O), K,,=2.Ox 109 I./W’).

Crossreactivity

Among

603

Rat I&Es

Dilution l/2

l/4

l/8

l/lb

1132

l/b4 -100

Rot IgG

B*-C -,,% B,‘-C -80

-60

-40

-20

I

0.1

I .o IgE Concentration

Fig. 9. Inhibiiion I&E, (A---A).

1

0.01

[)9/ml]

of the binding of 1Z51-IgE,,, todog anti-IgE,-4B by I&E,,, (+o), Inhibition curves for RRS. NRS, MRS and rat IgG were plotted [dilution].

I&E,,, (O-m-O), as a function of log,,,

Serum Dilution l/2

l/B

l/32

60

IgE Concentration [pg/m~]

Fig. IO. Inhibition and I&E, (A---A).

of the binding of 1251-I&E,,, to dog anti-I&E,-4B by I&E,, I (O-O), IgE,,2 (O---O) The inhibition curve for NRS (A---/J, was plotted as a function of log,, [serum dilution].

604

KEN A. KELLY,

ALEC

El. SEHON

and HERVI?

BAZlh

80

l/b

Fig. Il. Data for thecompetitive binding of IgE,,, (O-----O). IgEI,Z (o-o)and IgEl(A A, todog anti-IgE,4B in the presence of 1ZSI-IgE,,2. Bound (b) and free (cl IgE at equilibriun~ ~~re~llcui~lted from the inhibition data of Fig. 10. The K,> values for IgE, and I&E,,,, relative to E;,, tbr IgE,,,L. were 0.20 and 0.55, respectively.

8000

RRS

l

\.

6000

\

u

s

s

l

\

4000

8 \ 0 \

2000,

NRS ----L

i 1000

I

I

100

1

PCA liter Fig. 12. Correlation between RAST and to the allergosorbent which had reacted dilutions for standardization (e, PCA titer x 32) and serum D (U, PCA titer

PCA values: the ordinate represents the cpm ‘2SI-anti-lgE, bound with rat reagimc serum (RRS) samples. Serum A used at different titer= 17401; serum B (0. PCA titer=64); serum C ( n . PCA x 4). The background level (counts~min) for NRS is as shown.

Crossreactivity Among Rat IgEs variance due to errors in dilution, absorbance measurements and differences in the extent of impurity in the purified IgE stock solutions. Thus, no judgment can be made on the significance of the differences in CI,, values of the IgE standards using heterologous iz51-IgE. Nevertheless, the estimated IgE content (0.14 pg/ml) of NRS obtained from Sprague-Dawley rats agrees well with the previously reported value of less than 0.4 ng/ml (Ishizaka et al., 1976); the total IgE level in a rat anti-ovalbumin reaginic serum (PCA titer- 128) was 0.30 pg/ml, i.e. not significantly elevated from normal levels (Carson et al., 1975). Assuming the anti-IgE, antibody population to be specific for the common IgE determinant(s), the decreased capacity of IgE, and IgE,,,, relative to IgEi,,, to inhibit the binding of 1251-IgE,,, could only be due to a lower affinity of anti-IgE, for IgE, and From their respective inhibition curves in IgE,,,. Fig. IO, bound (h) and free(c) IgE concentrations were calculated over the range of total concentration of At any particular total inhibitor studied. concentration of IgE inhibitor, the amount bound to the anti-IgE,-4B solid phase was assumed to be equal to the amount of iZSI-IgE,,, displaced by the inhibitor, i.e. Bz-B*. The relative average intrinsic binding constant, K,, was determined for each IgE standard (IgE,, IgE,,, and IgE,,,) from the corresponding plot of l/b vs l/c (Fig. 11). The K, values for IgE, and IgE,,,, relative to X, for IgE,,,, were 0.20 and 0.55, respectively. RASTfor

rat-IgE

The RAST was performed using a modification of the technique of Wide et ~1. (1967). Ovalbumin (1 mg) was coupled to CNBr-activated microcrystalline cellulose. Volumes of 50 ,ul of serial dilutions of test samples of RRS against ovalbumin were added to 0.5 mg ovalbumin-cellulose in 0.5 ml assay buffer, and the tubes were shaken gently at 25°C for 18 hr. After ‘251-anti-IgE, (7 x lo4 washing the cellulose, counts/min, specific activity - 1.35 x lo7 counts/minlpg was added to each tube and the tubes shaken for a further 18 hr at 25°C. A good correlation was obtained between counts/min bound and the IgE antibody content of serial dilutions of the RRS standard (PCA titer- 1740). Although PCA titers were not determined for the dilutions of the standard, counts/min bound was plotted against the log,, of the theoretical values for the PCA titers of these diluted samples (Fig. 12). The estimated sensitivity (i.e. minimum detectable IgE antibody) of the RAST corresponded to a PCA titers of -16. RAST values (counts/min bound) of three other test RRS samples correlated well with their PCA titers. DISCUSSiON

As demonstrated in Fig. 7, antisera raised against the two rat monoclonal IgEs, IgE, and IgEie2, contained a high proportion of antibodies with specificity directed against the unique myelotypic determinants of the homologous myeloma protein. These antibodies, when compared with anti-IgE, antibodies directed against class-specific antigenic determinants (Fig. 8), had a much higher affinity for the homologous

605

myeloma protein. Thus, for example, when an immunosorbent, prepared by coupling trace amounts of purified IgE, 62 to Sepharose-4B, was treated with an anti-IgE,,, antiserum for the ‘sandwich’ RIA [Fig. l(a)], the absorbed antibodies were predominantly directed against the unique determinants of the homologous myeloma protein IgE,,, (Fig. 2). Since in radioimmunoassays it is imperative to avoid the participation of antibodies directed against individually unique antigenic determinants of the myeloma IgE used for immunization, IgE of a different myeloma source to that used for immunization, i.e. heterologous IgE, has been employed either for the isolation of antibodies directed against class-specific determinants (Pauwels et cd., 1977~) or. as in the majority of radioimmunoassays for both human and rat IgE, as the 1Z51-labelled standard. A practical advantage of the ‘sandwich’ RIA depicted in Fig. l(b) is that, by using heterologous coupled to the solid phase, primarily the IgL, antibodies directed against class-specific determinants are expected to bind to the immunosorbent. It was, therefore, surprising to observe the extreme disparity between IgEi6, and IgE,,, in their capacity to inhibit the binding of ‘251-IgE,,2 (Fig. 3). The displacement of the two standard curves was much reduced when heterologous IgE was used as the 1251-labelled standard (Fig. 4). In the ‘sandwich’ RIA employing IgE,e,- monospecific antibodies, directed against both class-specific and myelotypic determinants, and IgE,,,-4B and 1251-IgE,3, [Figs. l(c) and 41 or IgE,4B and 1251-IgE, (figure not included), the slopes ofthe inhibition curves for the homologous IgE,,, and heterologous IgE,,, (or IgE,) deviated significantly, failing to meet the stringent requirement of parallelism in radioimmunoassays (Midgley A. R., Jr. et u1., 1969). Furthermore, the use of a ‘sandwich’ RIA, employing IgE,-4B saturated with an excess of IgE,,,monospecific antibodies as the solid phase and 1251as the labelled standard (manuscript in IgE, preparation), for the quantitation of total rat IgE in normal sera, reaginic sera and in sera of rats whose IgE levels were potentiated markedly by infection with Nippostrongylus brudiensis yielded inhibition curves that were parallel to that of the IgE, standard, represented by the equation y = -52.14x+156.25 where y = (B*-C/B:-C) and x is the log,, [IgE concentration]. On the other hand, all samples contammg IgE,,, yielded inhibition curves parallel to that ofan IgEi6, standard represented by the equation y = -96.13x+282.31. The steeper slope for the IgEle2 curve was interpreted as indicative of a higher affinity of the adsorbed antibodies for the homologous IgE,,,. Experiments are in progress to prepare fractions enriched in IgE from reaginic sera and from sera of rats infected with N. brasiliensis and purified IgE fractions from the ascitic fluid of several IgE-secreting immunocytomas to examine their crossreactivities by RIA. The purpose of these experiments will be to determine if the higher inhibitory capacity of IgE,,, is due to the higher degree ofcomplementarity of,@ between the antigenic

606

KEN A. KELLY. ALEC‘ H. SEHON and HERVi;

determinants of IgE, hZ and the solid phase-adsorbed anti-IgE,,, antibodies directed against class-specific determinants. With the exception of IgE, h2. to date all other IgE preparat~o~ls appear to be equivalent when quantitated by the competitive inhibition ‘sandw,ich’ RI.4 described above, using IgE,-4B saturated with an excess of IgE i bz-monospecik antibodies as the solid phase and ‘2”1-IgE, as the lahellcd standard. These results indicate that one may employ an antibody fraction containing both class-specific and antimyelotype antibodies in a competitive RIA. with the RIST or the ‘sandwich’ technique descrihcd here. provided that one uses a heterologous IgE protein as the labelled standard. Therefore, these techniques appear to be satisfactory for the yuantitatiorl of IgE other than the one used for the production of the antibodies. The quantitation of homologous lgE,,< may be carried out using tither of several competitive RIA techniques. Since the inhibitor! capacity of lgE,,,> is unique, i.c. different from hctcrologous l&Es, the only criterion is that a purified IgE,,, standard bc used in the assay for absolute quatltitati~~n by interpolation. Thus, one may employ tither of the competitive ‘sandwich’ RIA systems depicted in l-‘ig. I or a competitive RIST. using either homologous 01 hetcrologoLls “iI-labclIcd IgE. If l&E,,, is to be quantitated by inhibition of binding of heterologous “‘I-IgE, then contamination by anti-myelotype antibodies must be avoided. Since these antibodies bind IgE,,,,. but not I~et~r~~l~)g~)us‘251-lgE, the assay will mcasurc only that fraction of the IgE,,, sample which binds to antibodies directed against classspecific determinants and can subsequently inhibit the binding of “‘I-lgE, resulting in an L~nderestim~~ti~~llof the concentration of IgE,,, in the sample. When antibodies to class-specific determmants were employed in the RIST (i.e. anti-IgE,) or in the ‘sandwich‘ RIA by ~l~isorpti[~n onto heterol(~~ous I&E, or IgE,,,, the failure of the other IgE preparations to compete equivalently with IgE,,, in the inhibition of (Figs. 10 and 3. respectively) binding of ‘2SI-IgE,,, appears to contradict the ~~ssutnption of identity of class-specific antigenic determinants common to IgE from the various sources studied in this report. This similarity in behaviour in both RIA procedures also rulesout tllcpos5ibility that ~~nti-myelotypealltibodies could have been nonspecifically absorbed onto the solid phase used in the ‘sandwich’ technique. When anti-I&E antibodies were used in the RIST, equivalence in cross-reactivity among the purified standards: IgE,. and IgE,,,, and parellelism of their inhibition I@,,, curves could only be observed when heterologous IgE was used as the labelled standard (Fig. 9). Using the ‘sandwich‘ technique. lgE,,Z ncvcr yielded curves to other IgE standards even when parallel employed. This heterologous ‘2’1-IgE WS discrepancy in the inhibitory behaviour of IgE,,, between the RIST and -sandwich‘ techniques could be due to differences in the two antibody populations involved, on the assumption that the anti-IgE, preparation used in the RIST represents only a fraction of the total adsorbed antibody population involved in the ‘sandwich’ assay. Losses could have arisen from incomplete elution and subsequent denaturation on elution of anti-IgE, from the IgE,-4B

RAZlh

immunosorbent with glycine-HC‘I. pH 2.X (antibodies of highest affinity for IgE, would remain on the immunosorbent), and from its (anti-IgE,) chemical conjugation to the solid phase. These losses would account for the fact that IgE, was the least inhibitory among the three IgE preparations in the RIST (Table I) and yet a stronger inhibitor than IgE,,? m the ‘sandwich’ assay employing “I-lgE2. Different extents of radiation damage to the antigenic determinants of the purified IgE standards as a possible explanation of the observed differences m crossreactivity- may be ruled out since (a) all three IgE preparations were labelled under identical conditions with practically identical specific activitica and were subsequently stored and used under identical conditions; (b) direct binding studies (Fig. 7) showed complete binding of the homologous ‘“‘I-IgE to their respective antibody preparations and practically equivalent binding to the anti-I&!, preparation [Fig. 7(d)]. Since all myeloma IpE preparations were obtained from inbred rats of the same strain. LOU/Wsl. it is improbable that the observed differences could have been due to different allotype anti-allotype interactions. Moreover. immunoglobulins (other than IgE) from IR162 ascitic fluid were used for solid-phase absorption of the antiIgE,,, antisera and, therefore. antiallotypes, if any. would have been absorbed. In the same manner, antibodies to sub-group determinants of the variable regions in the Fab fragment (Bennich & Johannson. 1971) would have also been absorb&. It is obvious that the IgE secreted by the ileocoecal immunocytoma IR 162 possesses unique antigenic determinants characteristic of that immunocytoma. since heterologous IgEs from normal and aliergic rats and from rats of the same LOU/Wsl strain bearing the IR2 and lR331 IgE-secreting immunocytomas all differ from homologous IgE,,, in their reaction with ~~ntib~~dicsagainst IgE,,2. The failure of heterol~~~(~us IgE to inhibit the binding of ‘Z’I-lgE,,2 in the homologous ‘sandwich’ RIA system (Fig. I (a) and Fig. 2) indicates that antibodies were produced against unique myeiotypic determinants not shared by heterologous IgE or by non-IgE isotypcs isolated from to IgE,,z ascitic fluid, since solid phase absorption render the antiserum monospecific for IgE,,, failed to remove these antibodies. The antiserum \+as not absorbed further Bith Fab fragments of IgE,,2 and. hence, the possibility that these unique myelotypic determinants may be contined to the variable Fab region of the molecule cannot be excluded. However, the anti-IgE, antibody preparation, isolated with the IgE,-4B immunosorbent. is considered to be directed against class-specific determinants of the IgE molecule. The observed reduced capacit! of hcterologous binding of

IgE,

relative

to IgE,,2.

to inhlblt

the

‘251-IgE,,Z to anti-I@, antibodies (Fig. 10) could be due to minor differences m the conformational structure of the common class-specific antigenic determinants located in the Fc region, and would reflect the existence of subclasses of I@. In order to detect differences in cross-reactivity among IgE samples one would have to employ a competitive RIA technique. According to the law of mass action for the equilibrium reaction between IgE and its antibody. the amount of IgE- antibody complexes formed in a

Crossreactivity Among radioimmunoassay is a function not only of the concentration of the reactants but also of the affinity constant for the reaction. Therefore, unless all IgE preparations to be assayed reacted with equal affinity with antibody, quantitation by a noncompetitive RIA technique such as the antibody ‘sandwich’ PRIST described by Ceska and Lundkvist (1972) and Pauwels et ~11.(1977~) would produce erroneous results. For the development of a radioimmunoassay for rat IgE employing an antiserum against IgEle2 myeloma protein. this requirement of equivalent cross-reactivity was met only by the use of antibodies directed against class-specific determinants and a heterologous IgE standard. Johansson et al. (1972) acknowledge that quantitation of human IgE is a delicate problem. First, significant differences in the IgE value of a serum can be obtained by the use of several modifications of RIAs, even though the same antiserum and labelled IgE were used. Secondly, the class-specific D,l and D,2 determinants present in the Fc region of human IgE (Ishizaka et ul., 1970; Bennich & Johansson, 1971), differ in conformational stability, introducing difficulties in the quantitation of IgE in samples that have undergone varying degrees of denaturation. Thirdly, if subclasses of IgE do indeed exist, then more difficulties will arise due to differences in specificities of antibodies from one anti-IgE antiserum to another.

CONCLUSION

For the development of a radioimmunoassay for total rat IgE it is necessary to isolate, from anti-rat IgE antisera, antibodies directed against class-specific determinants devoid of antibodies directed against myelotypic determinants of the monoclonal IgE molecule used as the immunizing antigen. In addition, in competitive inhibition RIAs these class-specific antibodies should be used in conjunction with ‘251-labelled purified IgEs other than the IgE used for the production of these antibodies in order that IgE in the test samples should react with equal affinity as the IgE standard; this is a basic requirement for accurate absolute quantitation. A~kno~/edg~merlt.,The authors wish to acknowledge the skillful technical assistance of Ms. Cheryl Barefoot. Ms. Karol McNeil1 for preparation of purified IgE samples and Dr. Valerie Strevens-Stark for performance of the RAST.

Bazin H.. Querinjean P.. Beckers A., Heremans J. F. & Desay F. (1974) Transplantable immunoglobulin-secreting tumours in rats. IV. Sixty-three IgE-secreting immunocytoma turnours. (1974) Ir?l,m4nolog~ 26,713-723. Bennich H. & Johansson S. G. 0. (1971) Structure and function of human immunoglobulin E At/r. Immunol. 13, 1~55. Carson D.. Metzger H. & Bazin H. (1975) A simple radioimmunoassay for the measurement of human and rat IgE levels by ammonium sulfate precipitation. J. /mmu,l. 115. 561 S63.

Rat IgEs

607

Ceska M. & Lundkvist U. (1972) A new and simple radioimmunoassay method for the determination of IgE. fmmunochumistr!~ 9, 1021-1030. Conrad D. H.. Bazin H., Sehon A. H. & Froese A. (1975) Binding parameters of the interaction between rat IgE and rat mast cell receptors. J. Immun. 114. 1688-1691. Cuatrecasas P. (1970) Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J. hiol. Chem. 245, 3059-3065. Dandliker W. B., Alonso R., de Saussure V. A.. Kierszenbaum F.. Levison S. A. & Schapiro H. C. (1967) The effect of chaotropic ions on the dissociation of antigen-antibody complexes. Biochemisfr!, 6, l46@ 1467. Gleich G. J., Averback A. M. & Swedlund H. A. (1970) Concentration of IgE in serum of normal and allergic patients. J. Alkrg_v 45. Greenwood F. C., Hunter W. M. & Glover J. A. (1963) Th,e preparation of ‘“‘I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 1 I4 123. lshiraka K., Ishizaka T. & Lee E. H. (1970) Biologic function of the Fc fragments of E myeloma protein. Immunochemistry

I, 687-702.

lshizaka T.. Urban J. F. & Ishiraka K. (1976) IgE formatlon in the rat following infection with Nippo.strony~/u~ hrusilirtuis. I. Proliferation and differentiation of IgEbearing cells. Cell. Immunol. 22, 248-261. Johansson S. G. 0. & Bennich H. (1967) Immunological studies of an atypical (myeloma) immunoglobulin. Immunolog)~ 13, 381-394. Johansson S. G. O., Bennich H. & Wide L. (1968) A new class of immunoglobulin in human serum. fmnlunolog!~ 14, 265-272. Johansson S. G. O., Bennich H. & Berg T. (1972) The climcal significance of IgE. In Progrcs.\ in Clinktrl Immum~/og~~ (Edited by Schwartz R. S.) Vol. I. p. 157. Grune and Stratton, New York and London. Kulczycki A. & Metzger H. (1974)The interaction of IgE with rat basophilic leukemia cells. II. Quantitative aspects of the binding reaction. J. rxp. Med. 140, 1676 1695. Lang G. M.. Conrad D. H.. Kelly K. A.. Carter B. G.. Froese A. & Sehon A. H. (I 977) Murine and rat IgE: Relationships in terms of bindings to cell receptors and to antibodies against rat epsilon chain. J. Immun. 118, 749-755. Midgley A. R.. Jr.. Niswender G. D. & Rebar R. W. (1969) Principles for the assessment of the reliability of radioimmunoassay methods (precision. accuracy, sensitivity. specificity). Aclu endocr. $3, Suppl. 142. 163-184. Nisonoff A. & Pressman D. (I 958) Heterogeneity and average combining constants of antibodies from individual rabbits. J. Immun. 80, 417428. Pauwels R., Basin H.. Platteau B. & Van Der Straeten M. (1977tr) The measurement of total serum IgE levels in rats J. Ir,l,,runo/. Mrth. 18, 133-140. Pauwels R., Bazin H.. Platteau B. & Van Der Straeten M. (19776) 1,~vitro measurement ofspecific rat IgE antibodies Ann. Immutwl. 128, 675-685. Salmon S. E., Mackey G. & Fudenberg H. H. (1969) “Sandwich” solid phase radioimmunoassay for the quantitative determination of human immunoglobulins. J. Immun. 103, 129 137. Schellenberg R. R. & Adkinson N. F. (1975) Measurement of absolute amounts of antigen-specific human IgE by a radioallergosorbent test (RAST) elution technique. J. Immun. 115, 1577-l 583. Weltman J. K.. Frackelton R. A., Szaro R. P. & Rotman B. (1976) A galactosidase immunosorbent test for human immunoglobuiin E. J. A//erg) c/in. Immunol. 58, 42&431. Wide L., Bennich H. & Johansson S. G. 0. (1967) Diagnosis of allergy by an in ritru test for allergen antibodies. Luncur ii. 1105-l 107.

Standardization of radioimmunoassay: differences in reactivity of rat IgEs with antibodies to a monoclonal IgE.

Molecular Pergamon Immunology, Vol. 16, pp. 597-607 Press Ltd. 1979. Printed in Great Britain STANDARDIZATION OF RADIOIMMUNOASSAY: DIFFERENCES IN R...
1MB Sizes 0 Downloads 0 Views